- Email: [email protected]
Safety evaluation of two α-amylase enzyme preparations derived from Bacillus licheniformis expressing an α-amylase gene from Cytophaga species
Accepted Manuscript Safety evaluation of two α-amylase enzyme preparations derived from Bacillus licheniformis expressing an α-amylase gene from Cytophaga species Vincent J. Sewalt, Teresa F. Reyes, Quang Bui PII:
S0273-2300(18)30199-5
DOI:
10.1016/j.yrtph.2018.07.015
Reference:
YRTPH 4178
To appear in:
Regulatory Toxicology and Pharmacology
Received Date: 22 December 2017 Revised Date:
15 July 2018
Accepted Date: 22 July 2018
Please cite this article as: Sewalt, V.J., Reyes, T.F., Bui, Q., Safety evaluation of two α-amylase enzyme preparations derived from Bacillus licheniformis expressing an α-amylase gene from Cytophaga species, Regulatory Toxicology and Pharmacology (2018), doi: 10.1016/j.yrtph.2018.07.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Safety evaluation of two α-amylase enzyme preparations derived from Bacillus
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licheniformis expressing an α-amylase gene from Cytophaga species
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Vincent J. Sewalt1,*, Teresa F. Reyes1, and Quang Bui1,#
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DuPont Industrial Biosciences, 925 Page Mill Road, Palo Alto, CA 94304
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Current address: 25 Carriage Lane, North Tustin, CA 92705.
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*Corresponding author: Vincent J. Sewalt, 925 Page Mill Road, Palo Alto, CA 94304. Email:
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[email protected].
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Abstract
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A safety assessment was conducted for a symthetic variant Cytophaga sp. α-amylase enzyme
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expressed in Bacillus licheniformis and formulated into two distinct product formats: whole
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broth (a preparation in which the production organism is completely inactivated, but containing
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residual cell debris) and clarified preparation (from which the production organism is completely
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removed). The enzyme was improved via modern biotechnology techniques for use in the
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endohydrolysis of starch, glycogen, related polysaccharides and oligosaccharides. Applications
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range from carbohydrate processing, including the manufacture of sweeteners, fermentation to
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produce organic acids, amino acids and their salts, and potable or fuel alcohol, with resulting co-
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products (distillers’ grains and corn gluten feed/meal) destined for use in animal feed. The
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toxicological studies summarized in this article (90-day rodent oral gavage and in vitro
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genotoxicity studies) noted no test article-related adverse effects and thus substantiate the safety
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of the α-amylase in not only the clarified form but also as a whole-broth preparation. Consistent
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with the decision tree analysis for enzymes produced with modern biotechnology techniques, this
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paper provides supporting information that this variant amylase with homology to an amylase
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from a potentially pathogenic organism (Cytophaga sp.) can be safely produced in an expression
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host that belongs to a Safe Strain Lineage, for safe use as processing aid to manufacture human
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and animal food.
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Key Words: α-amylase; whole broth; safety studies; carbohydrate processing; fermentation; Cytophaga sp.; Bacillus licheniformis; Safe Strain Lineage
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1. Introduction
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Microbially-derived industrial enzyme preparations are used world-wide in food processing and
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animal feed, and recent scientific advances primarily in the fields of molecular biology and
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protein engineering have led to an emergence of innovative enzyme technologies. Considerations
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for food safety evaluation of enzyme preparations for use in human food originally published by
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Pariza and Foster (1983) were updated during the 1990s for enzymes produced with recombinant
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deoxyribonucleic acid (DNA) technology, aka recombinant DNA (rDNA) technology (IFBC,
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1990; Jonas et al., 1996; SCF, 1992), culminating in an updated decision tree (Pariza and
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Johnson, 2001). The Pariza and Johnson (2001) decision tree was adopted for applications in
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animal feed (Pariza and Cook, 2010) and its use in the GRAS (Generally Recognized As Safe)
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process for enzymes reviewed (Sewalt et al., 2016) and clarified (Sewalt et al., 2017). In short,
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these guidelines provide a peer-reviewed decision tree process for the determination of the safety
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of enzyme preparations used in human and animal food, which are based on history of safe use
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of enzymes, the establishment of Safe Strain Lineages to serve as their production strains, and
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well-known strain improvement methods, all supported with published scientific studies. This
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article presents a safety assessment for two α-amylase enzyme preparations, improved via
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modern biotechnology techniques for use in the endohydrolysis of starch, glycogen, related
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polysaccharides and oligosaccharides; with applications ranging from carbohydrate processing,
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including the manufacture of sweeteners such as high fructose corn syrup (HFCS), and
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fermentation to produce organic acids (e.g., citric and lactic acid), amino acids (e.g., lysine) and
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their salts (e.g., monosodium glutamate), and potable or fuel alcohol, with resulting co-products
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(distillers’ grains and corn gluten feed/meal) destined for use in animal feed. The toxicological
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studies summarized in this article substantiate the safety of two types of enzyme preparations,
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the first being the standard food-grade ‘clarified’ enzyme preparation from which all solids
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including the production organism are removed by centrifugation and/or microfiltration; the
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second a whole-broth preparation containing not only the enzyme but also the inactivated
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organism and all fermentation by-products. The latter product format is used mainly in
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fermentation processes to maximize available substrate for yeast to convert to alcohol or other
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fermentation products. Each preparation contains the same variant α-amylase produced in
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Bacillus licheniformis, the amino acid sequence of which is a synthetic protein-engineered
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variant of the α-amylase from Cytophaga sp. Cytophaga alpha-amylase is capable of hydrolyzing
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raw starch (Jeang et al., 1995) without the need for gelatinization by heating. It is of note that the
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Cytophaga genus contains several species that are fish pathogens. It is the objective of this paper
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to provide supporting information that an enzyme protein inspired after a homolog from a
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pathogenic organism can be safely produced in a safe host for safe use as processing aid to
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manufacture human and animal food.
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The B. licheniformis production strain was genetically engineered to express an optimized
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synthetic variant α-amylase gene most closely related to the α-amylase from Cytophaga sp. The
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host strain is B. licheniformis Bra7, which was developed from its wild-type (wt) parent, by
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classical strain improvement for optimal α-amylase production and lowered protease production.
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The parent strain B. licheniformis Bra7 and strains derived from it have been in use for industrial
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scale production of food-grade α-amylase for starch processing since the late 1980s. The gene
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inserted into the production organism was not physically isolated from the donor strain; instead
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the gene encoding the α-amylase was synthesized in vitro. The gene was modified by introducing
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various amino acid mutations and deletions. This specific variant of Cytophaga sp. α-amylase is
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referred to as C16F.
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The α-amylase with the International Union of Biochemical and Molecular Biology number
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(IUBMB) 3.2.1.1; is a typical glycosyl hydrolase (GH13_5) α-amylase prevalent in Bacillus
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species (van der Kaaij et al., 2007) and commonly used in industry (Guzman-Maldonado &
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Paredes-Lopez, 1995). The sequence of the C16F α-amylase is similar to various other α-
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amylases isolated from commercially relevant bacteria, e.g., it is 81% homologous to Bacillus sp.
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α-amylase 406 and 75% to Bacillus amyloliquefaciens α-amylase (Lee et al., 2006). Given the
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high structural similarity of α-amylase molecules from various sources (e.g. Janeček, 1994,
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1997), and in particular the liquefying Bacillus α-amylases (Yuuki, 1985), significant differences
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in toxicological properties between these homologous enzymes are not expected.
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Bacillus licheniformis has been used for decades in the production of food enzymes with no
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known reports of adverse effects to human health or the environment (de Boer and Diderichsen
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1994). An extensive environmental and human health risk assessment of B. licheniformis,
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including its history of commercial use has been published by the U.S. Environmental Protection
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Agency (EPA, 1997) and it was concluded that B. licheniformis is not a human pathogen nor it is
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toxigenic. Additionally, the U.S. Food and Drug Administration (FDA) has reviewed the safe use
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of food-processing enzymes from well-characterized recombinant microorganisms, including B.
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licheniformis (Olempska-Beer et al., 2006). FDA also concluded that B. licheniformis is neither
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pathogenic nor toxigenic and it is considered as suitable for Good Industrial Large Scale Practice
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(GILSP) worldwide and meets criteria for a safe production microorganism as described by
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Pariza and Johnson (2001).
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The raw starch digesting α-amylase from Cytophaga sp. has been described by Jeang et al.
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(1995, 2002). Cytophaga sp. are unicellular, non-spore forming Gram-negative bacteria
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(Kirchman, 2002) derived from soil. They are part of the Cytophaga-Flavobacteria cluster,
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which are especially proficient in degrading various biopolymers such as cellulose, chitin, and
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pectin (Mayberger, 2011). Cytophaga sp. can be found globally in every habitat in every
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biosphere, including kusaya (a Japanese delicacy consisting of putrid fish), rumens,
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hydrothermal vents, rocks, sea-ice in Antarctica and sediments of lakes and oceans. Cytophaga-
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Flavobacteria is more prevalent in the oceans, making it one of the most abundant of all
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bacterial groups (Kirchman, 2002).
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Some members of Cytophaga sp. genus are reported to be fish pathogens (Carson et al., 1993;
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Dalsgaard, 1993; Soltani et al., 1995) known to cause non-zoonotic Columnaris disease, an
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infection that typically enters through the gills, mouth or small wounds, prevalent where high
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bioloads exist (Cooney et al., 2002), or in stressed environments such as overcrowding or low
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dissolved oxygen levels in the water (Rottman, 1992, Durborow, 1998). However, pathogenicity
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is a complex process that typically involves the expression of specialized invasive elements
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called virulence factors, some of which were discussed by Dalsgaard (1993), and none of which
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are associated with the α-amylase protein or its gene. Many harmless microorganisms (and
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harmful ones, alike) express genes for amylases, which in nature only serve the purpose of starch
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digestion. Amylases are used in numerous industrial applications including food manufacture
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(Gupta et al., 2003, Pandey et al., 2000) and as feed additive, often combined with other
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enzymes, to pigs (e.g., Li et al., 2010) and poultry (e.g., Romero et al., 2013). The only genetic
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information expressed in the B. licheniformis production host is a synthetic α-amylase variant
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gene inspired from the Cytophaga sp. α-amylase sequence, but no actual nucleic acids from
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Cyptophaga sp. were transferred.
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1.1 Development of the production strain of Bacillus licheniformis (C16F)
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The variant α-amylase gene coding sequence was placed under the expression signals of the
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endogenous B. licheniformis amyL gene and the B. subtilis aprE 5’ untranslated region (UTR),
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cloned in a vector derived from Bacillus plasmids pUB110 and pE194 (Gryczan et al., 1980),
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together with the native B. licheniformis cat gene, encoding chloramphenicol acyltransferase.
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The resulting plasmid was integrated into the host chromosome at the cat locus by Campbell
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type recombination. After integration, all vector sequences of the plasmid were deleted by
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recombination between direct repeated cat sequences. The genetic construction was evaluated at
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every step to assess the incorporation of the desired functional genetic information and the
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intended chromosomal modifications were confirmed by PCR and sequence analyses. No new
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antibiotic resistance genes were introduced in the construction of the production organism.
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The safety of C16F α-amylase was evaluated in a series of studies designed according to current
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guidelines on evaluating the safety of microbial enzyme preparations used in human and animal
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food (Pariza and Johnson, 2001; Pariza and Cook, 2010). The studies evaluate the safety of the
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protein-engineered enzyme as well as its toxigenic potential in two manufactured product
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formats: clarified, ultra-filtered concentrate (UFC) and unfiltered whole-broth (WB) preparation.
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The studies support the safety of residual enzyme preparation in the final food, even with the
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worst-case assumption that the enzyme is carried over wholly into the final food even though in
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all cases the enzyme is inactivated and in many cases the inactive enzyme protein is removed in
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the process.
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Pariza and Johnson (2001) establish that a sufficiently high No Observed Adverse Effect Level
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(NOAEL) in an oral toxicity study is one requisite to demonstrate safety. To satisfy this
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requirement, sub-chronic studies in Charles River CD rats using whole-broth (WB) and clarified
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ultra-filtered concentrate (UFC) C16F α-amylase enzyme preparations were conducted by oral
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gavage (DuPont Haskell Global Center, 2014). Additionally, genotoxicity studies were
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performed to address any mutagenicity potential from the C16F α-amylase enzyme preparation.
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Generally, clarified UFC enzyme preparations undergo recovery steps post-fermentation to
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remove cellular material, followed by enzymatic concentration; whereas WB enzyme
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preparations undergo production organism inactivation but skip the cell removal stage. In
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general, the recovery process is a multi-step operation, which starts immediately after the
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fermentation process. The enzyme is recovered from the fermentation broth by 1) Primary
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separation: microfiltration (MF) or flocculation-aided rotary drum vacuum centrifugation
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(RDVC) and separation by filter press, and 2) Concentration by ultrafiltration into UFC.
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Stabilizers and preservatives are added to both UFC and WB fermentation material, followed by
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an additional polish filtration step for stabilized UFC. Typically, clarified UFC-based enzyme
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preparations are employed in food-related applications, while WB enzyme preparations are used
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for non-food applications like ethanol production, although, WB enzyme preparations can be
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safely utilized in food (if appropriate in the application), e.g., fermentation processes.
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The results from the safety studies and our exposure assessment support our conclusion that the
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C16F α-amylase derived from B. licheniformis is safe and suitable for use in food applications
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such as carbohydrate processing and fermentation, with resulting co-products (distillers’ grains
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and corn gluten feed/meal) destined for use in animal feed. The work presented here not only
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adds to the body of evidence of microbial enzyme safety, it also highlights two aspects of
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enzyme safety that hitherto have not been discussed in great detail.
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On the one hand, we provide supporting data that an enzyme from a species that is questionable
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as a direct enzyme source can be safely expressed in a well-established production organism that
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belongs to a Safe Strain Lineage as defined by Pariza and Johnson (2001), in this case DuPont’s
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Bacillus licheniformis Safe Strain Lineage. On the other hand, the comparative toxicological
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information for the α-amylase in both clarified enzyme preparation format and as whole-broth
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enzyme preparation lends support to the safety of the latter format for use in food applications
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where that format is appropriate.
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2. Materials and Methods
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2.1 Test material preparation and characterization
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The C16F α-amylase enzyme preparation manufacturing process follows standard industry
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practice (Kroschwits, 1994; Aunstrup et al., 1979; Aunstrup, 1979), and in accordance with
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FDA’s current Good Manufacturing Practices (cGMP) requirements and ISO (International
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Organization for Standardization) 9001 standards. Raw materials conform to specifications of the
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11th edition of the Food Chemicals Codex (FCC) maintained by the U.S. Pharmacopeia (USP,
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2018). The C16F α-amylase enzyme preparation is manufactured by submerged fermentation of
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a pure culture of the non-pathogenic, non-toxigenic, genetically engineered strain of B.
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licheniformis (strain JML1584). A new lyophilized stock culture vial of the B. licheniformis
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production organism is used to initiate the production of each batch. Each new batch of the stock
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culture is thoroughly controlled for identity, absence of foreign microorganisms, and enzyme-
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generating ability before use. Antibiotics are not used in the fermentation process. Equipment of
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carefully design and construction is operated, cleaned and maintained so as to prevent
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contamination by foreign microorganisms. During all steps of fermentation, physical and
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chemical control measures are taken and microbiological analyses are conducted periodically to
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ensure absence of foreign microorganisms, and upon conclusion of the process, inactivation of
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the production strain. The C16F α-amylase enzyme was prepared in two preparations, one
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whole-broth (WB) and the other as an ultra-filtered concentrate (UFC). The C16F α-amylase,
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regardless of product format, meets purity specifications for enzyme preparations set forth in the
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FCC 11th edition (USP, 2018) and to the General Specifications for Enzyme Preparations used in
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Food Processing as proposed by the Joint FAO/WHO Evaluation Committee for Food Additives
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(JECFA) in the Compendium of Food Additive Specification (JECFA, 2006).
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specifications include absence of live production organism, absence of antimicrobial activity,
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absence of Salmonella and pathogenic E. coli, and specifications for coliforms, total viable
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counts, and heavy metals.
These
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To assess the safety of both C16F α-amylase enzyme preparations, different endpoints of toxicity
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were investigated in separate studies for WB and UFC formats and the results of each evaluated,
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interpreted and assessed in this paper. All toxicology studies presented here were conducted in
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compliance with current Good Laboratory Practice standards and Organization for Economic
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Cooperation and Development (OECD) Guidelines for the Testing of Chemicals., The test
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materials used in all toxicology investigations were the clarified UFC and WB enzyme
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preparations collected from fermentation with the following characteristics (Table 1):
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Table 1. Characteristics of test materials used in toxicology testing
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Study
Subchronic Toxicity 90-Day Oral Gavage Study in Rats In vitro Mammalian Chromosome Aberration Test Bacterial Reverse 10
Study Reference MPI Research (2014b)
MPI Research (2014a)
DuPont Haskell Global Centers, (2014b)
DuPont Haskell Global Centers (2014a)
BioReliance (2014b)
BioReliance (2014a)
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Mutation Assay H-30929 UFC (Ultra-filtered Concentrate) Fermentation liquid,
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H-30928 WB (Whole-Broth)
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Test Material Characteristics
Fermentation Liquid, Brown
Brown
Enzyme Activity
68.3 DLU/L
36.1 DLU/L
pH
6.3
Specific gravity
1.03 g/ml 39.9 mg/ml
Trichloroacetic Acid (TCA) Protein Total Organic Solids (TOS)
1.06 g/ml
91.6 mg/ml
31.2 mg/ml
42.2 mg/ml
7.12%
14.4%
0.11%
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Potassium sorbate Sodium benzoate
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(Dextrose Liberating Units)
0.30%
0.30%
Compliant with JECFA (2006) and FCC (2018)
Compliant with JECFA (2006) and FCC (2018)
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2.2 Experimental Design
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2.2.1. Oral Administration Test (90-day oral gavage study):
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Oral toxicity tests for WB and UFC test articles were based on OECD Test Guideline 408,
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(revised September 1998) and were conducted in compliance with the Guide for the Care and
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Use of Laboratory Animals of the Institute of Laboratory Animal Resources (2011).
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The systemic toxicity of C16F α-amylase (WB or UFC enzyme preparations) was assessed in
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two 13-week oral gavage studies in Charles River (CD) rats of both sexes conducted at MPI
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Specifications for contaminants
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Research, Mattawan, Michigan in accordance with US FDA Good Laboratory Practice (GLP)
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Regulations (21 CFR Part 58) and the OECD Principles of Laboratory Practice
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(ENV/MC/CHEM(98)17), and U.S. Department of Agriculture’s (USDA) Animal Welfare Act
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(9 CFR Parts 1-3).
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Groups of 10 animals per sex were treated by oral gavage with 0 (distilled water), 100, 250 or
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500 mg TOS (Total Organic Solids)/kg bw/day. The vehicle control, 0.11% potassium sorbate
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and 0.38% sodium benzoate in distilled water, was prepared weekly and stored and refrigerated
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at 2-8°C. The dosing volume was 10 ml/kg bw/day. During the study, samples of the dosing
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preparations were collected at different intervals (week 1, 6 and 12) and verified for proper
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dosage concentration.
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Animals of the same sex were housed in groups of two in solid floor polypropylene cages with
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stainless steel mesh lids and softwood bedding (non-aromatic) with access to water via an
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automated system and feed ad libitum. For environmental enrichment, the animals were provided
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a supply of wooden chew blocks and cardboard fun tunnels. All groups were housed under
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controlled temperature, humidity and lighting conditions. Observations for morbidity, mortality,
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injury, and the availability of food and water were conducted twice daily for all animals.
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Cage-side clinical observations were conducted daily. Observations for detailed clinical signs
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were conducted pretest and weekly. Functional Observational Battery (FOB) observations
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(Activity/Arousal, Neuromuscular, and Sensorimotor, Autonomic, and Physiological) and
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locomotor activity tests were conducted pretest and during Week 13. Body weights were
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measured and recorded on Day 1 and 7 and weekly during the study. Food consumption (by
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cage) was measured and recorded weekly and food efficiency was calculated weekly and
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reported as a mean of five cages/sex/treatment. Ophthalmoscopy examinations were conducted
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pretest and prior to the terminal necropsy. Blood and urine samples for clinical pathology and
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chemistry evaluation were collected prior to the terminal necropsy. Blood work included
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hematology analysis: hemoglobin, hematocrit, mean corpuscular volume (MVC), mean
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corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and
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individual cell types (leukocytes, erythrocytes, platelets, absolute reticulocytes, neutrophils,
7
lymphocytes, monocytes, eosinophils, basophils, and ‘other cells’) and coagulation (activated
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partial thromboplastin time (APTT) and prothrombin time. Clinical chemistry included minerals
9
(sodium, potassium, chloride, calcium, phosphorous), total bilirubin, bile acids, enzymes
10
(alkaline phosphatase, gamma glutamyltransferase (GGT), aspartate aminotransferase (AST),
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alanine aminotransferase (ALT), urea nitrogen, creatinine, total protein, albumin, globulin,
12
triglyceride, cholesterol, glucose). Urinanalysis included volume, specific gravity and pH.
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At study termination (Day 91), necropsy examinations were performed and selected organ
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weights were recorded. Tissues from animals in the control and high dose group and gross
15
lesions in all dose groups were microscopically examined.
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2.2.2 Statistical analysis
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Raw data was tabulated within each time interval, and the mean and standard deviation and/or
18
incidence counts (categorical variables) were calculated for each endpoint by sex and group
19
(control group (1) vs. treatment groups (2, 3 and 4). For each continuous endpoint (body weight,
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body weight change, food consumption, functional observational battery observations (FOB,
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continuous endpoints), hematology (except leukocyte counts), coagulation, clinical chemistry,
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and organ weights) treatment groups were compared to the control group using Group Pair-wise
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Comparisons (Levene’s/ANOVA-Dunnett’s/Welch’s). For locomotive activity, Analysis of
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Variance (ANOVA) was used and for leukocyte counts, Log Transformation/Group Pair-Wise
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comparisons (Leven’s/ANOVA-Dunnet’s/Welch’s) were used. For food efficiency, and
4
urinalysis Rank Transformation with Dunnett’s Test was used and for FOB (Categorical
5
Endpoints) the Cochran Haenszel Test was used. Data for some end points were transformed by
6
either a log or rank transformation prior to conducting the specified analysis.
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2.2.3. In vitro Mammalian Chromosomal Aberration Test performed with Human Lymphocytes
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The objective of these assays was to investigate the potential of C16F WB or clarified UFC α-
9
amylase enzyme preparations to induce numerical and/or structural changes in the chromosome
10
of mammalian systems (i.e., human peripheral lymphocytes). These assays were conducted in
11
accordance with OECD guideline No. 473 (OECD, 1997b).
12
In this assay, human lymphocytes were stimulated to divide by the addition of a mitogen (e.g.
13
phytohemagglutinin, PHA). Mitotic activity began at about 40 hours after PHA stimulation and
14
reached a maximum at approximately 3 days.
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Test material (C16F WB or UFC) was mixed with cultures of human peripheral lymphocytes
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both in the presence and absence of metabolic activation (Aroclor 1254-induced rat liver; S-9
17
mix). This study consisted of a preliminary toxicity (dose range finding) and two main assays. In
18
the preliminary assay, all cultures with or without S-9 mix were treated for 4 hours and
19
continuously for 22 hours for cultures without S-9 mix. All cells were harvested 22 hours after
20
treatment initiation. Nine concentrations of α-amylase (50, 100, 250, 500, 750, 1000, 1500, 2500,
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and 5000 µg TP/ml) were used as recommended by the OECD guideline. All dose levels were
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expressed in terms of total protein. From the results of the preliminary toxicity study, the highest
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concentration was selected for the final chromosome aberration assay that induced greater than a
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50% reduction of the mitotic index in the preliminary assay, relative to the vehicle control. Four
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additional dose levels, demonstrating limited toxicity or no toxicity were also evaluated. In cases
4
where there was little or no cytotoxicity (determined by the MI), the highest dose level tested and
5
at least 2 lower dose levels were selected for analysis. In cases where there was little or no
6
cytotoxicity, but a precipitate was observed (with the naked eye) the lowest dose level
7
demonstrating a precipitate and 2 other lower dose levels were selected for analysis. If neither
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cytotoxicity nor precipitation was observed in the preliminary toxicity assay, the highest
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concentration was the limit dose of 5000 µg/mL or 10 mM, whichever was the lower.
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In the definitive assay, cultures with and without S-9 mix were exposed to the test article for 4
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hours, and continuously for 22 hours for those without S-9 mix. Cells were collected 22 hours
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(1.5 normal cell cycles) after initiation of treatment. Two hours prior to harvest, Colcemid was
13
added to the cultures at a final concentration of 01 µg/ml to arrest mitosis.
14
Cells were collected by centrifugation, treated with 0.075 M KCl hypotonic buffer, fixed in
15
methanol:glacial acetic acid (3:1 v/v), capped and stored overnight or longer. To prepare slides,
16
the cells were re-suspended in fixative and then collected by centrifugation. The suspension of
17
fixed cells was applied to glass microscope slides and air-dried. The slides were stained with
18
Giemsa, permanently mounted and scored.
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The mitotic index was recorded as the parentage of cells in mitosis per 500 cells counted.
20
Metaphase analysis (i.e. evaluation of chromosomal aberration) was conducted on at least 200
21
metaphases for each level (100 per duplicate treatment). Cells were scored for both chromatid-
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type and chromosome-type aberrations. Mitomycin C and cyclosphosphamide were used as
2
positive controls for cultures without S-9 mix and cultures with S-9 mix, respectively.
3
Statistical Analysis
4
The clastogenic potential of the test substance was assessed based on the percentage of cells with
5
structural chromosome aberrations. Interpretation of the results also relied on additional
6
considerations including the magnitude of the observed test substance response relative to the
7
vehicle control response and the presence of a dose-responsive trend. Statistical analysis
8
consisted of a Fisher’s exact test with Bonferroni-Holm Adjustment (Fisher, 1985) to compare
9
the percentage of cells with structural or numerical aberrations (or the percentage of cells with
10
more than one aberration, if required) in the test substance groups with the vehicle control
11
response. Before considering any test article effects, the assay was considered acceptable for
12
evaluation of test results only if all of the following criteria were satisfied. For Negative
13
Controls, the frequency of cells with structural chromosome aberrations to be within the negative
14
historical control range. For Positive Controls, the percentage of cells with structural
15
chromosome aberrations to be statistically significantly greater (p < 0.05, Fisher’s exact test)
16
than the vehicle control response. The metabolically activated and non-activated assays of the
17
test are considered independent test results and, if inconclusive, would be repeated separately.
18
A Cochran-Armitage test for test article dose responsiveness (Snedecor and Cochran, 1967) was
19
conducted only in case of statistically significant differences, based on the Fisher’s exact test.
20
The following conditions were used as a guide to determine a positive response: 1) a statistically
21
significant increase (p < 0.05, Fisher’s exact test) in the percentage of cells with structural
22
aberrations was seen in one or more treatment groups relative to the vehicle control response. 2)
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the observed increased frequencies were accompanied by a concentration-related increase, 3) or
2
a statistically significant increase was observed at the highest dose only. Statistically significant
3
values that did not exceed the historical control range for the negative/vehicle control may be
4
judged as not being biologically significant. Equivocal response is defined as a statistically
5
significant elevation in structural chromosome aberrations at more than one test concentration
6
level, except the highest dose, without demonstrating a dose-responsive trend. The test substance
7
was judged negative if the following condition was met: There was no statistically significant
8
increase in the percentage of cells with structural aberrations in any treatment group relative to
9
the vehicle control group.
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2.2.4 Bacterial Reverse Mutation Assays
11
The objective of these assays was to assess the potential of α-amylase (C16F WB or UFC) to
12
induce point mutations (frame-shift and base-pair) in four strains of S. typhimurium TA98,
13
TA100, TA1535 and TA1537 (Ames et al. 1975) and Escherichia coli strain WP2 uvrA (Green
14
and Muriel, 1976) per OECD guideline 471 (OECD, 1997a). The test material was tested both in
15
the presence and absence of a metabolic activation system (Aroclor 1254-induced rat liver; S-9
16
mix). The assay was performed in two phases using the plate incorporation methodology for the
17
positive control, 2-aminoanthracene, with E. coli and the treat and plate methodology for all
18
remaining strains and assays.
19
A screening (dose range) test was performed first to select dose levels for the confirmatory assay.
20
Vehicle control, positive control and 8 doses of the test article (1.5, 5, 15, 50, 150, 500, 1500 and
21
5000 µg per plate were plated, two plates per dose, with overnight cultures of all four strains of
22
S. typhimurium and E. coli WP2 uvrA in the presence and absence of S-9 mix. In the
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confirmatory assay, 6 doses of the test article (15, 50, 150, 500, 1500 and 5000 µg per plate)
2
along with appropriate vehicle and positive controls were plated in triplicate in the presence and
3
absence of S-9 mix. All dose levels were expressed in terms of total protein (TP). The highest
4
dose level (5000 µg TP/plate) is the maximum dose required by the OECD guideline. The
5
positive controls use for assays without S-9 mix were 2-nitrofluorene, N-methyl-N-nitro-N-
6
nitrosoguanadine (MNNG) and ICR-191. For assays with S-9 mix, the positive control was 2-
7
aminoanthracene. Vehicle control plates were treated by the addition of sterile deionized water.
8
Data Analysis
9
For each replicate plating, the mean and standard deviation of the number of revertants per plate
10
were calculated and reported. For the test article to be evaluated as positive, it must cause a dose-
11
related increase in the mean revertants per plate of at least one tester strain over a minimum of
12
two increasing concentrations of test article. Data sets for the tester strains TA1535 and TA1537
13
were judged positive if the increase in mean revertants at the peak of the dose response was
14
greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains
15
TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak
16
of the dose response was greater than or equal to 2.0-times the mean vehicle control value. An
17
equivocal response is a biologically relevant increase in a revertant count that partially meets the
18
criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve
19
the respective threshold cited above or a non-dose responsive increase that is equal to or greater
20
than the respective threshold cited. A response was evaluated as negative, if it was neither
21
positive nor equivocal.
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The following criteria must be met for the initial toxicity-mutation and confirmatory
2
mutagenicity assays to be considered valid. All Salmonella tester strain TA98 and TA100
3
cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the
4
uvrB gene. Cultures of tester strains must demonstrate the presence of the pKM101 plasmid R-
5
factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene. All cultures must
6
demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls as
7
follows (inclusive): TA98, 10 - 50; TA100, 80 - 240; TA1535, 5 - 45; TA1537, 3 - 21; WP2
8
uvrA, 10 - 60. To ensure that appropriate numbers of bacteria are plated, tester strain culture
9
titers must be greater than or equal to 0.3 x 109 cells/mL. The mean of each positive control must
10
exhibit at least a 3.0-fold increase in the number of revertants over the mean value of the
11
respective vehicle control. A minimum of three non-toxic dose levels is required to evaluate
12
assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A
13
>50 % reduction in the mean number of revertants per plate as compared to the mean vehicle
14
control value. This reduction must be accompanied by an abrupt dose-dependent drop in the
15
revertant count. (2) At least a moderate reduction in the background lawn (background lawn
16
code 3, 4 or 5).
17
2.3 Sequence screening for food allergy potential and homology to protein toxins
18
Food Allergens
19
Following the most current allergenicity assessment guidelines developed by the Codex
20
Commission (2009) and Ladics et al. (2011), we used a FASTA (FAST-All) search for matches
21
of 35% identity or more over 80 amino acids of the variant Cytophaga sp. protein against a
22
database of known allergens, further enhanced by the use of an E-value cut-off in the BLAST
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algorithm of 10-7 to suggest a biologically relevant similarity (i.e., potential cross reactivity). We
2
used the Food Allergy Research Resource Program (FARRP) AllergenOnline database
3
(http://www.allergenonline.org/databasefasta.shtml containing 2089 (version 18B released
4
March 23, 2018) peer-reviewed allergen sequences (listed in
5
http://www.allergenonline.org/AllergenOnlineV18B.pdf.
6
Toxins
7
A BLAST search for homology of the mature C16F amino acid protein sequence with known
8
toxins and antinutrients was performed using the UniProtKB annotated Protein Knowledge
9
database (Magrane et al., 2011; http://www.uniprot.org/), UniProt release 2018_04 (April 25,
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2018). This large protein database with hundreds of thousands sequences contains 6,724 proteins
11
that are manually annotated as toxins or venoms (http://www.uniprot.org/program/Toxins). From
12
this search, the top 1,000 hits in the UniProt database were exported to MS Excel, with the
13
appropriate annotation fields (protein name, key words, gene ontology, protein family), allowing
14
for use of search terms “toxin” and “venom”.
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Results
17
3.1. Oral Administration Tests (90-day oral gavage study)
18
The oral gavage tests (WB and UFC) resulted in no treatment-related deaths during the 13-week
19
period. Body weight gain, feed intake, and feed efficiency are summarized in Tables 2-4.
20 21
Table 2. Body weight gain (g) of male and female rats exposed to whole-broth (WB) and clarified (UFC) C16F α-amylase
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Test article
Sex 0
100
250
500
Whole Broth
Male
371.2 ± 31.2*
380.4 ± 27.6
402.6 ± 36.0
389.4 ± 32.4
Whole Broth
Female
121.8 ± 17.9
122.7 ± 15.7
111.9 ± 18.0
125.0 ± 14.6
UFC
Male
296.2 ± 14.7
287.2 ± 26.6
302.9 ± 22.9
292.0 ± 23.0
UFC
Female
132.3 ± 14.0
125.7 ± 9.2
135.5 ± 6.7
142.0 ± 16.9
*± 2 SEM (Standard Error of the Mean, n=10)
Table 3. Daily feed intake (g/animal/day) by male and female rats exposed to whole-broth (WB) and clarified (UFC) C16F α-amylase
5
Sex
100
250
500
Male
27.4 ± 2.08*
27.6 ± 1.49
28.4 ± 2.59
27.4 ± 1.60
Whole Broth
Female
18.5 ± 1.20
17.5 ± 0.94
17.6 ± 1.27
17.6 ± 1.49
UFC
Male
26.4 ± 0.88
26.1 ± 1.71
25.9 ± 0.66
25.7 ± 1.97
UFC
Female
18.6 ± 1.16
18.3 ± 0.72
18.5 ± 0.89
18.9 ± 0.59
*± 2 SEM (Standard Error of the Mean, n=5)
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Table 4. Feed efficiency (%) by male and female rats exposed to whole-broth (WB) and clarified (UFC) C16F α-amylase C16F amylase dose (mg TOS/kg BW/day)
Sex
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0 Whole Broth
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C16F amylase dose (mg TOS/kg BW/day)
0
100
250
500
Whole Broth
Male
15.2 ± 0.87*
15.5 ± 0.87
15.9 ± 0.91
16.0 ± 0.87
Whole Broth
Female
7.36 ± 0.70
7.8 ± 0.52
7.2 ± 0.65
8.0 ± 0.80
UFC
Male
12.7 ± 0.45
12.6 ± 1.06
13.1 ± 0.78
12.8 ± 0.65
UFC
Female
8.0 ± 0.53
7.7 ± 0.51
8.3 ± 0.61
8.5 ± 0.55
*± 2 SEM (Standard Error of the Mean, n=5)
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1
As can be seen in Tables 2-4, there were no significant effects related to the UFC or WB test
2
articles of C16F α-amylase
3
observations, functional observations, locomotor activity, and hematology, coagulation, clinical
4
chemistry, and urinalysis parameters are included as Supplemental Tables A1 through A21 for
5
the UFC test article and B1 through B21 for the WB test article. No differences in functional
6
observations or locomotor activity were noted due to either test article, with the following
7
exceptions:
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Hindlimb grip strength was marginally lower for male rats receiving the highest dose of
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on body weight, feed intake, or feed conversion. Clinical
C16F amylase, regardless of product format (see Suppl. Data Tables A12 and B12). This
10
was not the case for females receiving either UFC or WB test article. Given this
11
inconsistency, and given that all other clinical, functional, and locomotor activity
12
observations were normal, this isolated marginal difference was not considered an
13
adverse effect. -
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Locomotor activity (specifically, rearing count) was reduced for male rats receiving the middle dose of UFC (see Suppl. Data Table A15). This observation was regarded as not
16
attributable to the test article as 1) it was not a dose-dependent, 2) it did not occur for
17
female rats receiving UFC test article, and 3) it was not observed for any concentration of
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WB administered to either sex at any concentration.
18 19
No significant differences were observed in hematology, coagulation, clinical chemistry and
20
urinanalysis panels (Tables A18-A21 and B18-B21).
21
At necropsy, there were no treatment related effects on organ weights, macroscopic observations
22
and histopathologic examination (data not shown). All microscopic findings were considered to
22
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be within the background incidence of findings reported in this age and strain of laboratory
2
animals.
3
In summary, there were no meaningful biological differences between the control and treated
4
groups with respect to all clinical parameters measured. Under the conditions of this study, the
5
No-Observed-Adverse-Effect Level (NOAEL) for both WB and UFC enzyme preparations is
6
500 mg TOS/kg bw/day. This NOAEL is equivalent to approximately 280 mg total protein/kg
7
bw/day (UFC) and 317 mg total protein/kg bw/day (WB).
8
3.2. Genetic Toxicology studies
9
3.2.1 Bacterial Reverse Mutation Assay
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Dosing formulations were adjusted for total protein content based on the concentration as
11
supplied at 91.62 mg/mL (WB) and 39.89 mg/mL (UFC). In the treat and plate method, the
12
volumes of S-9 mix, sham mix, bacterial and test article, vehicle or positive control were
13
increased by a factor of 2.5 or 3.5 to ensure sufficient volume of resuspended bacteria to plate
14
the desired number of replicates. Deionized water was selected as the solvent of choice.
15
In the initial toxicity-mutation assays, the maximum dose tested was 5000 µg per plate; this dose
16
was achieved by diluting the test article at the concentration of 91.62 mg/mL (WB) and 39.89
17
mg/mL (UFC) to a concentration of 50 mg/mL (using a 100 µL aliquot) and 25 mg/mL (using a
18
200 µL aliquot). The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per
19
plate. The WB test article formed a clear solution in water at 0.015 mg/mL, a cloudy solution at
20
0.050 mg/mL and workable suspensions from 0.15 to 50 mg/mL; whereas the UFC test article
21
was a workable suspension at the 39.89 mg/mL concentration, but formed clear solutions in
22
water from 0.0075 to 25 mg/mL.
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In the preliminary assays (Suppl. Data Tables A24-25 and B24-25), no positive mutagenic
2
responses were observed with any of the tester strains in either the absence or presence of S-9
3
mix activation. Neither precipitate nor toxicity was observed for the C16F α-amylase WB
4
enzyme preparation test article. No precipitate was observed for the C16F α-amylase UFC
5
enzyme preparation. Toxicity to the UFC enzyme preparation was observed beginning at 1500
6
µg per plate with tester strain TA98 in the absence of S-9 mix.. Based on the findings of the
7
initial toxicity-mutation assay; the maximum dose plated in the confirmatory mutagenicity assay
8
for both UFC and WB enzyme preparations was 5000 µg per plate, with dose levels 15, 50, 150,
9
500, 1500 and 5000 µg per plate.
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In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any
11
of the tester strains in either the presence or absence of S-9 mix (Suppl. Data Tables A26-27 and
12
B26-27). Increases in revertant counts (1.5- and 1.6-fold maximum increases) were observed for
13
UFC enzyme with only with tester strain WP2 uvrA in the presence and absence of S9
14
activation, respectively. For the WB test article, an increase in revertant counts (2.1-fold
15
maximum increase) was observed only with tester strain TA98 in the presence of S-9 mix
16
(Suppl. Data Table B27). However, these increases were not considered to be indicative of
17
mutagenic activity because 1) the increases were not dose-responsive, 2) the vehicle control
18
levels in the study were on the low end of the acceptable range, and 3) the peak of each response
19
was within historical vehicle control range for this test condition (for WP2uvrA, 32 and 35
20
revertants per plate in the presence and absence of S9; for TA98, 26 revertants/plate in the
21
presence of S-9 mix).
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No precipitate was observed for either WB or UFC test article, whereas toxicity was observed for
2
UFC only, at 5000 µg per plate with tester strain TA98 in the absence of S9 activation. All
3
criteria for valid studies as described in the Materials and Methods were met.
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The results of the bacterial reverse mutation assays (Ames tests) indicate that, under the
6
conditions of these studies, the test articles (WB and UFC) C16F α-amylase enzyme preparation
7
did not exhibit any mutagenic responses in either the presence or absence of Aroclor-induced rat
8
liver S9. Therefore, both WB and UFC test articles were concluded to be negative in this assay.
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3.2.2 In-vitro Chromosomal aberration assay-Human lymphocytes
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In the preliminary toxicity assay, cells were treated for 4 and 22 hours in the non-activated test
12
condition and for 4 hours in the S-9 mix activated test condition. All cells were harvested 22
13
hours after treatment initiation. The cells were exposed to 9 concentrations of each test substance
14
ranging from 50 to 5000 µg/mL, as well as a vehicle control. Test substance precipitation was
15
observed for the WB enzyme preparation at concentrations ≥ 750 µg/mL (see Suppl. Table B22),
16
whereas no test substance precipitation was observed in the treatment media of the UFC enzyme
17
preparation test substance. Interfering precipitation (WB enzyme preparation test substance)
18
prevented the analysis of concentration ≥ 750 µg/mL in the non-activated test conditions and ≥
19
1500 µg/mL in the 4-hour S-9 mix -activated test condition. Substantial toxicity (greater than
20
50% reduction in mitotic index relative to the vehicle control) was observed in the 22-hour non-
21
activated test condition at concentrations ≥ 250 µg/mL. The UFC enzyme preparation test
22
substance showed substantial toxicity (greater than 50% reduction in mitotic index relative to the
23
vehicle control) in the 22-hour non-activated test condition at concentrations ≥ 100 µg/mL (see
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Suppl. Data Table A22). Based on the findings from the preliminary toxicity assay, the highest
2
concentration chosen for the chromosome aberration assay was 1000 µg/mL (WB) and 5000
3
µg/mL (UFC) for the 4-hour conditions and 250 µg/mL (WB) and 100 µg/mL (UFC) for the 22-
4
hour test conditions.
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a) Chromosomal Aberration Assay - WB Test Material:
6
For the C16F α-amylase WB enzyme preparation, the concentrations chosen for the 4-hour
7
test conditions were 50, 100, 250, 500 and 1000 µg/mL and for the 22-hour test condition
8
were 50, 100, 150, 200 and 250 µg/mL. Precipitation was observed in the treatment media at
9
1000 µg/mL. Substantial toxicity was observed in the 22-hour non-activated test condition at
10
100, 200 and 250 µg/mL. Selection of doses for microscopic analysis was based on test
11
substance induced toxicity in the 22-hour test condition. In the 4-hour test conditions,
12
selection of doses for analysis was based on interfering precipitation of the test substance.
13
Cytogenetic evaluations for C16F α-amylase WB enzyme preparation were conducted at 250,
14
500 and 1000 µg/mL in the 4-hour test conditions (see Suppl. Data Table B23). Four-hour
15
exposure to the highest test concentration (5000 µg/mL) did not result in mitotic index
16
changes compared with the vehicle control (indicating no toxicity). In the 22-hour test
17
condition, cytogenetic evaluations were conducted at 50, 150 and 200 µg/mL. The mitotic
18
index for the highest test concentration evaluated microscopically for chromosome
19
aberrations (200 µg/mL) was 7.5%, compared with 16.2% for the vehicle control. This
20
represents a 53.7% mitotic inhibition in relation to the vehicle control. The percentage of
21
cells with numerical or structural aberrations in the test substance-treated groups was not
22
significantly increased above that of the vehicle control at any concentration (p ≥ 0.05,
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Fisher’s exact test). The percentage of cells with structurally damaged chromosomes in the
2
respective positive control groups were statistically significant (p < 0.05, Fisher’s exact test).
3
b) UFC Test Material
4
For the C16F α-amylase UFC enzyme preparation the concentrations were 250, 500, 1000,
5
2500 and 5000 µg/mL for the 4-hour test condition. For the 22-hour test condition, the
6
concentrations were 10, 25, 50, 75 and 100 µg/mL. No test substance precipitation was
7
observed in the treatment media. Substantial toxicity was observed in the 22-hour non-
8
activated test condition at 100 µg/mL. Selection of doses for microscopic analysis was based
9
on test substance induced toxicity in the 22-hour test condition. In the 4-hour test conditions,
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selection of doses for analysis was based on the limit dose for the assay, 5000 µg/mL.
11
Cytogenetic evaluations for C16F α-amylase UFC enzyme preparation were conducted at
12
1000, 2500 and 5000 µg/mL in the 4-hour test conditions (See Suppl. Data Table A23). Four-
13
hour exposure to the highest test concentration (5000 µg/mL) did not result in large changes
14
in mitotic index compared with the vehicle control. In the 22-hour test condition, cytogenetic
15
evaluations were conducted at 25, 50, and 100 µg/mL. The mitotic index for the highest test
16
concentration evaluated microscopically for chromosome aberrations (100 µg/mL), was
17
3.7%, compared with 10.3% for the vehicle control, representing a 64.1% mitotic inhibition
18
in relation to the vehicle control. Under all 3 conditions tested, the percentage of cells with
19
numerical or structural aberrations in the test substance-treated groups was not significantly
20
increased above that of the vehicle control at any concentration (p ≥ 0.05, Fisher’s exact test).
21
The percentage of cells with structurally damaged chromosomes in the respective positive
22
control treatment groups were statistically significant (p < 0.05, Fisher’s exact test).
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Under the conditions of these studies, neither the C16F α-amylase UFC nor the WB enzyme
2
preparation test material were found to induce structural or numerical chromosome aberrations in
3
the in vitro mammalian chromosome aberration test in human peripheral blood lymphocytes in
4
either the non-activated or S-9 mix -activated test systems. The percentages of aberrant cells in
5
the test substance-treated groups were within the historical solvent control range and all criteria
6
for valid assays were met, based on the results for positive and negative controls. It was
7
concluded that both the WB and the UFC test substance were neither mutagenic nor clastogenic
8
in this in vitro mammalian test.
9
3.3 Sequence-based assessment of food allergenicity potential and similarity to toxin proteins
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The search for 80-amino acid stretches with greater than 35% identity to known allergens using
11
the FARRP AllergenOnline database revealed no matches to known food allergens. The UniProt
12
database search showed that the vast majority of sequences were α-amylases, with none of the
13
top 1,000 database hits annotated as either toxin or venom. Based on the sequence homology
14
alone, the C16F variant α-amylase is unlikely a food allergen or oral toxin.
15
4. Discussion
16
4.1 Safety of the production organism
17
Bacillus licheniformis is a proven safe production host for industrial enzymes (e.g., Olempska-
18
Beer et al., 2006; EPA, 1997). It is used as the production organism for several food enzymes
19
that were affirmed GRAS by FDA and for additional food enzymes that were notified to FDA as
20
GRAS (see GRN 22, 24, 72, 79, 265, 277, 472, 560, 564, 572, 587, and 594). Additionally, it is
21
listed in the Official Publication by the American Association of Feed Control Officials
22
(AAFCO, 2018) as a suitable species for the production of numerous enzymes for use in animal
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feed. Moreover, DuPont has previously established a Safe Strain Lineage (SSL) for this species
2
as also discussed in GRAS Notice 664) (see Figure 1) based on repeated evaluation in the Pariza
3
and Johnson (2001) decision tree including toxicological testing. According to the principles of
4
this decision tree, α-amylase (a common food enzyme with a long history of safe use) produced
5
in a production strain pertaining to a Safe Strain Lineage would be acceptable for use in food.
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Figure 1. Bacillus licheniformis BRA7 Safe Strain Lineage (SSL) diagram, showing the position
15
of the production organism of C16F amylase in the lineage (in red) in relation to the other strains
16
in the SSL. The blue colored boxes indicate strains for which toxicology tests were conducted.
17
Most enzymes derived from this SSL were determined to be Generally Recognized as Safe
18
(GRAS), with GRAS Notices submitted for review by the U.S. FDA for enzymes from strains
19
designated with gray horizontal banners indicating the GRN (= GRAS Notice) number, inlcuding
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GRN 664 for C16F amylase. The safety of the C16F α-amylase strain is further supported by its
2
own toxicology testing.
3
The α-amylase enzyme in question is derived from Cytophaga sp., which provides one rationale
4
for further verification of its safety. Most enzymes are sourced from organisms with a history of
5
safe use, which is the case here for the expression host. B. licheniformis. The ‘gene donor’
6
Cytophaga sp. is a Biosafety Level 2 organism, as certain species belonging to this genus are
7
pathogenic to fish. Intuitively one might be wary of expressing DNA from a pathogenic donor
8
organism into an expression host. However, one can safely produce any given common enzyme
9
sequence from a pathogen in a safe host, as long as the expressed enzyme in question is not a
10
virulence factor. The transfer of any potential donor DNA of concern (that is not the target DNA
11
encoding for the otherwise safe enzyme) can be entirely avoided by using synthetic DNA
12
encompassing only the amylase coding sequence.
13
4.2 Safe Strain Lineage- Use of B. licheniformis and Cytophaga – Pariza Decision Tree
14
The safety of the C16F α-amylase in human and animal food has also been established using the
15
decision tree for food processing enzymes as outlined in Pariza and Johnson (2001) and updated
16
for animal feed enzymes by Pariza and Cook (2010), which both address various questions
17
related to enzymes production in organisms developed with rDNA technology. One key question
18
is whether the expressed enzyme in question has a history of safe use in food or feed. Indeed, α-
19
amylase has been used for decades in food processing and in animal feed. Although the
20
Cytophaga sp. α-amylase (C16F) is new as an isolate in commerce, the variant α-amylase
21
expressed in Bacillus licheniformis is still an α-amylase with the designation IUBMB 3.2.1.1.
22
Given the high sequence similarities of CF16 α-amylase to α-amylase molecules from various
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sources (e.g., 81% identity with α-amylase from Bacillus sp. 406 and 75% identity with α-
2
amylase from Bacillus amyloliquefaciens), C16F α-amylase is considered substantially
3
equivalent to these α-amylases with extensive history of safe use. GRAS Notice 664 included a
4
sequence-based similarity assessment of the C16F sequence with known food allergens (Codex,
5
2009; Ladics et al., 2011) and toxins (Magrane et al., 2011), and the safety of C16F α-amylase is
6
further supported by its lack of sequence similarity with any known food allergens or
7
proteinaceous toxins.
8
The rDNA methods used to engineer the production strain included the use of plasmids devoid of
9
mobilization and transfer elements, minimizing the potential presence of transferable antibiotic
10
resistance DNA. In addition, chloramphenicol is not widely used as antibiotic in humans or
11
animals, so its clinical relevance is minimal. With regard to further questions in the decision tree:
12
all introduced DNA was fully sequenced, examined and found to be free of attributes that would
13
render it unsafe for constructing microorganisms to be used to produce food-grade products. The
14
introduced DNA is integrated at the cat locus into chromosome of B. licheniformis. Moreover,
15
the B. licheniformis Bra 7 safe strain lineage is well-established; its safety as a production host
16
and methods of modification are well documented and their safety have been confirmed through
17
repeated toxicology testing (see GRN 664).
18
In short, α-amylase has a history of safe use in food, the sequence of C16F amylase was verified
19
not to display any similarity with known food allergens or toxins, and C16F amylase is safely
20
produced by a member strain of a well-established safe strain lineage for which all essential
21
strain engineering attributes are confirmed, all of which satisfy the requirements in safety
22
evaluation of the enzyme preparation without triggering the need for new toxicology data.
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Finally, although the Pariza and Johnson evaluation resulted in the conclusion to accept the
2
enzyme preparation as “safe without new toxicology testing”, the safety of C16F enzyme
3
preparation was further confirmed through toxicological testing as described above. The
4
toxicology testing was conducted 1) to underpin the substantial equivalence between clarified
5
and whole-broth enzyme α-amylase preparation and 2) to use the results to support regulatory
6
approvals in jurisdictions where toxicology testing is an absolute requirement for food/feed
7
enzyme approval.
8
Not only is B. licheniformis a safe production organism for enzymes, it is also listed as a suitable
9
species to serve as a probiotic Direct Fed Microbial to livestock and poultry (AAFCO, 2018).
10
Hence, given that live B. licheniformis cells are safe for use as probiotics, it is expected that a
11
whole-broth enzyme preparation produced by the same safe organism would be as safe as the
12
clarified (semi-purified) equivalent. However, the lack of comparative data triggered us to
13
include this as a second angle of interest in this study.
14
The whole-broth enzyme format containing solids is not suitable for all food applications, but
15
may be suitable for food applications that include a refinement step such as distillation and/or
16
filtration after the enzymatic action has been completed, such as the manufacture of potable
17
alcohol or organic acids. Whole broth cellulase is used in the manufacture of cellulosic fuel
18
ethanol; similarly, whole-broth α-amylase may be applied in grain fuel ethanol processes with
19
the resulting co-products fed to animals. To the authors’ knowledge, however, the current report
20
provides the first comparative toxicological studies for the two formats (whole-broth and
21
clarified) of an enzyme preparation for use in the processing of human and animal food.
22
The safety of the resulting enzyme preparations was confirmed in the reported genotoxicity and
23
sub-chronic oral toxicity studies. Given the lack of biological and statistical differences between
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the control and treated groups for any of the clinical, systemic or genotoxic parameters
2
measured, the highest dose in two 90-day oral gavage toxicity studies (500 mg TOS/kg bw/day)
3
can serve as the NOAEL for both WB and UFC enzyme preparations.
4
4.3 Uses and Applications
5
The amylase is intended for use in carbohydrate processing, including the manufacture of high
6
fructose corn syrup and fermentation to produce organic acids i.e. lysine, citric and lactic acid,
7
and in the manufacture of potable alcohol and fuel ethanol with resulting co-products (distiller’s
8
grains and corn gluten feed/meal) destined for use in animal feed.
9
In a recent GRAS Notice to FDA’s Center for Food Safety and Applied Nutrition, the human
10
food applications the same α-amylase enzyme were notified as GRAS (GRN 664). The notice to
11
FDA focused on the use of the clarified amylase preparation for human food applications,
12
including the manufacture of sugar syrups, potable alcohol and other biochemicals for use in
13
food (organic acids, MSG). In this paper we further discuss the applications related to the
14
manufacture of animal feed. Additionally, FDA affirmed the GRAS status of mixed
15
carbohydrase/protease enzyme preparation derived from B. licheniformis and α-amylase from B.
16
amyloliquefaciens for use in food with GMP as the only limitation (21CFR 184.1027 and
17
184.1148, respectively). Furthermore, α-amylases from several genetically modified B.
18
licheniformis strains were GRAS notified to FDA, including hybrid B. licheniformis / B.
19
amyloliquefaciens α-amylase (GRN 22), modified B. licheniformis and B. amyloliquefaciens α-
20
amylase (GRN 79), and the agency issued “no questions” letters in response.
21
4.4 Human Exposure to C16F amylase & Margin of Safety
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The safety of Cytophaga sp. variant α-amylase C16F expressed in B. licheniformis as a
2
processing aid in carbohydrate processing and potable alcohol production at the maximum
3
recommended application rates is supported by existing toxicology data. Using the Budget
4
method (Douglass et al., 1997), the determined maximum daily intake for UFC is 0.248 mg
5
TOS/kg bw/day and for WB is 1.01 mg TOS/kg bw/day, which include solid and liquid food
6
application (organic acids, sugar syrups and specialty starches) exposures. The margin of safety
7
for human food applications was calculated as, respectively, 2016 to 495 for UFC and WB,
8
based on a NOAEL of 500 mg TOS/kg bw/day for each, with both exceeding the commonly
9
acceptable margin of safety of 100. Based on a margin of safety several-fold greater than 100
10
even in the worst-case scenarios, the uses of α-amylase as a processing aid in carbohydrate
11
processing and production of organic acids and potable alcohol are not of human health concern,
12
regardless of the product format.
13
4.5 Animal Exposure to C16F α-amylase
14
The C16F α-amylase enzyme preparation (WB and UFC formats) will be used in the dry-grind
15
ethanol (potable or fuel alcohol) with resulting distillers’ grains (DG) fed to animals, as well as
16
in the production of organic acids (e.g., lactic acid), and amino acids (e.g., lysine) for use in
17
animal feed. Additionally, wet milling (WM) co-products such as corn gluten feed and corn
18
gluten meal will also be part of the animal feed application. To calculate the worst-case exposure
19
estimate for animals consuming the DGs and or WM co-products, the following factors were
20
considered: a) 100% conversion efficiency: every kg of dry grain will be converted to
21
approximately equal portions of ethanol, carbon dioxide and DGs; b) 100% transfer: all 100% of
22
the enzyme will be transferred to the DG resulting in a theoretical 3-fold concentration; c) the
23
maximum inclusion rates of DG or WM co-products in animal feed rations will be used, i.e.,
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60% in cattle, 50% in pigs, and 30% in poultry, DM (dry matter) basis and d) the total amount of
2
DG and WM- co-products, when fed together, will not exceed the above stated maximum
3
inclusion rates for DG alone.
4
The maximum use rate in ethanol production trials to-date (6.2 mg total enzyme protein / kg
5
DM) equates to 11 mg TOS/kg DM for the UFC preparation and 42 mg TOS/kg DM for the WB
6
preparation. This results in 33 mg UFC TOS/kg DM or 127 mg WB TOS/kg DM in DG or WM
7
co-products. The worst-case exposure to production animals cattle, pigs and poultry) via their
8
feed is outlined in Tables 5 and 6. Using the NOAEL derived from the 90-day oral toxicity test
9
with either preparation of C16F α-amylase (500 mg TOS/kg bw/day), it was calculated that the
10
exposure to animals via DG or WM co-products is of no safety concern as the margin of safety is
11
far greater than 100.
12
Table 5. Margin of Safety Calculation for C16F α-amylase UFC via DG*
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Pigs
Poultry
Maximum enzyme use rate 11.03 (mg TOS/kg DM) Fold-concentration in DG, DM basis 3 (process dependent) Maximum Enzyme TOS concentration 33.09 in DG (mg/kg DM) Maximum Inclusion rate of DG in 0.6 complete animal ration Maximum Enzyme TOS concentration 19.85 in feed (mg/kg DM) Maximum Daily feed intake 0.03 (kg DM/kg BW) Maximum intake of enzyme TOS 0.59 (mg/kg BW) NOAEL 500 (mg TOS/kg BW/day) Margin of safety 847 *same calculations apply to WM-coproducts
11.03
11.03
3
3
33.09
33.09
0.5
0.3
16.54
9.93
0.045
0.065
0.74
0.645
500
500
676
775
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2
Table 6. Margin of Safety Calculation for C16F α-amylase WB via DG* Poultry
42.4
42.4
42.4
3
3
3
127
127
127
0.6
0.5
0.3
76
64
38
0.03
0.045
0.065
2.3
2.8
2.5
500 219
500 175
500 202
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Maximum enzyme use rate (mg TOS/ kg DM) Fold-concentration in DG, DM basis (process dependent) Maximum Enzyme TOS concentration in DG (mg/kg DM) Maximum Inclusion rate of DG in complete animal ration Maximum Enzyme TOS concentration in feed (mg/kg DM) Maximum Daily feed intake (kg DM/kg BW) Maximum intake of enzyme TOS (mg/kg BW) NOAEL (mg TOS/kg bw/day) Margin of safety *same calculations apply to WM-coproducts
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4.6 C16F α-amylase in the production of lysine for use in animal feed
5
The application of C16F α-amylase in the production of lysine is 6.2 mg total protein/kg starch.
6
Since the yield ratio of dry starch to lysine is 1.2 to 1, then the calculated maximum C16F α-
7
amylase carried over in 1 kg of lysine (assuming no removal) is 7.44 mg. Requirements for
8
lysine, often the first limiting amino acid in animal nutrition, for growing pigs and chickens
9
varies by age as well as dietary energy level. For starting, growing and finishing pigs the range is
10
0.6-1.4% of the diet (NRC, 1998) and for broiler chickens this varies from 0.85 to 1.1% of the
11
diet (90% diet DM basis; NRC, 1994), with the average being around 1%.
12
Although the lysine requirement can often be met by ration formulation with corn and soybean
13
meal, supplementation with crystalline lysine allows for a reduction in total protein content of
14
the diet (usually from soybean meal), and is a cost-effective means to reduce nitrogen excretion.
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The average supplement dosage of lysine is 2 kg of lysine per metric ton of feed for
2
monogastrics. For dairy cattle a benefit of supplementing with lysine up to 0.5% of the diet has
3
been reported (e.g., Wang et al., 2010). Based on the application of C16F α-amylase in the
4
production of lysine, it is calculated that for each animal species the maximum intake of enzyme
5
protein is 0.001 mg/kg BW for UFC and 0.004 mg/kg BW for WB, which is negligible compared
6
with the enzyme use in DG production, thus the margins of safety calculated for DG also cover
7
use of C16F α-amylase in fermentation to manufacture lysine and potentially several other amino
8
acids or organic acids, typically used at 0.5% or less in the animal feed, similar to the lysine
9
example.
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5. Conclusions
11
Results from a battery of toxicology studies investigating the genotoxic and systemic toxicity
12
potential of two C16F α-amylase preparations, demonstrated that regardless of the product form
13
(UFC or WB); the C16F amylase preparation is not a mutagen or clastogen and it doesn’t induce
14
systemic toxicity when administered by gavage to rats for 90 continuous days. The consistent
15
toxicology results between the two enzyme preparations allowed for establishment of the same
16
NOAEL for both products forms at 500 mg TOS/kg bw.
17
Our inclusion of the WB enzyme preparation in the toxicological studies in parallel to the classic
18
food-grade UFC enzyme preparation was to rule out any safety concerns regarding whole-broth
19
enzyme preparations that contain cell debris and rDNA from the production organism. It is
20
widely known that whole-broth enzyme preparations are used in enzyme applications where the
21
finished food product is subjected to further processing that will remove the cell debris, such as it
22
is the case in the distillation process to produce potable alcohol. However, there is nothing
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inherently unsafe about using WB enzyme preparations (containing dell debris and rDNA) in
2
food, as there is nothing inherently unsafe about rDNA unless it encodes for a harmful substance
3
or would easily transfer resistance to an antibiotic of clinical relevance to humans or animals. α-
4
Amylase is not a harmful substance and the chloramphenicol resistance marker (endogenous to
5
B. licheniformis) has been used safely for decades; it is used only in initial strain selection and
6
does not reside on a plasmid that is easily mobilized or transferred. The safety of the resulting
7
enzyme preparation is further illustrated by the current toxicology results. Obviously, such
8
generalization is predicated upon both UFC and WB enzyme preparations being manufactured
9
using the same appropriate raw materials of suitable purity for the intended use per Good
10
Manufacturing Practices, i.e. minimum amounts needed to achieve desired effect (Sewalt et al.,
11
2016). In addition, once the manufacturing process is completed, an evaluation for microbial and
12
chemical contaminants is performed (per the Food Chemical Codex (US Pharmacopeia, 2016)
13
and FAO/WHO’s Joint Evaluation Committee for Food Additives (JECFA, 2006), and only then
14
is the final enzyme preparation product (WB or UFC) released. Furthermore, for both Quality
15
and Intellectual Property (IP) protection reasons, enzyme manufacturers further evaluate the final
16
enzyme product to make sure that they are free of the active production organism.
17
In addition to the food applications previously mentioned, the C16F α-amylase enzyme
18
preparation (WB and UFC) is also intended for use in fermentables e.g., potable alcohol with
19
resulting distillers’ grains (DG) and organic/amino acids for use in animal feed. Similarly, use in
20
wet milling (WM) results in WM co-products, corn gluten feed and corn gluten meal, both of
21
which are used in animal feed as well at inclusion rates no higher than DG. Using the NOAEL
22
derived from the 90-day oral toxicity test with C16F alpha amylase (either UFC or WB), it was
23
calculated that exposure to animals via DG or WM co-products is of no safety concern as the
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safety margin is greater than 100 (see Tables 3-4). The proposed uses of C16F α-amylase in
2
potable alcohol at the proposed application rate of 6.2 mg protein/kg starch for UFC and 27 mg
3
protein/kg starch for WB, respectively, are not a human health concern and are supported by
4
existing toxicological data. Finally, even under the worst-case scenario that C16F α-amylase is
5
applied at the maximum rate for both UFC and WB and the enzyme is not destroyed and/or
6
removed during processing, the use of C16F α-amylase in the manufacture of lysine to be used as
7
a feed additive is toxicologically insignificant.
8
We conclude C16F synthetic variant Cytophaga sp. α-amylase expressed in B. licheniformis in
9
either clarified or whole-broth format is safe in the manufacture of ethanol or wet milling co-
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products and amino acids, without health concern to animals consuming the co-products or
11
amino acids and to humans consuming the potable alcohol, as substantiated by existing
12
toxicology data.
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Acknowledgement
15
We thank Gregory Ladics and Kees Broekhuizen for their critical review of toxicology
16
conclusions and strain details, respectively. In addition, we thank Sanusha Bijj for her skillful
17
assistance in preparing the Supplemental Data Tables.
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References
4
Ames, B.N., McCann, J., and Yamasaki, E. 1975. Methods for detecting carcinogens and
RI PT
3
mutagens with the Salmonella/Mammalian Microsome Mutagenicity Test. Mut. Res. 31,
6
347-364
Association of American Feed Control Officials (AAFCO), 2018. 2018 AAFCO Official Publication, pp. 373-378.
8
9
M AN U
7
SC
5
Aunstrup, K. 1979. Production, isolation, and economics of extracellular enzymes, in: Wingard, L.B., Katchalski-Katzir, E. and Golsdstein, L (Eds.), Applied Biochemistry and
11
Bioengineering, Volume 2, Enzyme Technology. pp. 28-68.
12
TE D
10
Aunstrup, K., Andersen, O., Falch, E. A., and Nielsen, T. K. 1979. Production of Microbial Enzymes in Microbial Technology, 2nd ed., Eds. Peppler, H.J., and Perlman, D., Chapter
14
9, 1. pp. 282-309.
15
EP
13
BioReliance, 2014a. H-30928: Bacterial reverse mutation assay; Report No. AD84GN.507001.BTL.
17
AC C
16
BioReliance, 2014b. H-30929: Bacterial reverse mutation assay; Report No. AD84GP.507001.BTL.
18
19
Carson, J., Schmidtke, L.M., Munday, B.L., 1993. Cytophaga johnsonae: a putative skin pathogen of juvenile farmed barramundi, Lates calcarifer Bloch. J. Fish Dis. 16, 209-218.
20
41
ACCEPTED MANUSCRIPT
1
Codex Alimentarius Commission. 2009. Joint FAO/WHO Food Standards Programme, Codex Alimentarius Commission, Rome, Italy
2
Cooney, R.P., Pantos, O., Le Tissier, M.D.A., Barer, M.R., O’Donnell, A.G., Bythell, J.C., 2002.
RI PT
3 4
Characterization of the bacterial consortium associated with black band disease in coral
5
using molecular microbiological techniques. Environ. Microbiol. 7, 401-413.
Dalsgaard, I., 1993. Virulence mechanisms in Cytophaga psychrophila and other Cytophaga-like
SC
6
bacteria pathogenic for fish. Ann. Rev. Fish Dis. 3, 127-144.
7
De Boer, A.S., Diderichsen, B., 1994. On the industrial use of B. licheniformis: A Review., Appl.
M AN U
8
Microbiol. Biotechol. 40, 595-598.
9
Douglass, J.S., Barraj, L.M., Tennant, D.R., Long, W.R., and Chaisson, C.F., 1997. Evaluation
11
of the budget method for screening food additive intakes. Food Addit. Contam. 14, 791-
12
802.
13
TE D
10
DuPont Haskell Global Centers, 2014a. H-30928: Level 10 C16F Whole Broth: In vitro mammalian chromosome aberration test in human peripheral blood lymphocytes; Report
15
No. 20557-544.
EP
14
Dupont Haskell Global Centers, 2014b.H-30929: Level 10 C16F Clarified: In vitro mammalian
17
chromosome aberration test in human peripheral blood lymphocytes; Report No. 20558-
AC C
16
544.
18
19
Durborow, R.M., Thune, R., Hawke, J.P., Camus, C., 1998. Columnaris disease, a bacterial infection caused by Flavobacterium columnare. Southern Regional Aquaculture Center
20
42
ACCEPTED MANUSCRIPT
1
Publication No. 479, USA. (
2
content/uploads/479.pdf>). Environmental Protection Agency (EPA), 1997. Attachment I—Final Risk Assessment of
4
Bacillus licheniformis. (
5
09/documents/fra005.pdf>).
RI PT
3
Fisher, R.A. 1985. Statistical Methods for Research Workers, 13th edition. Haffner, New York.
7
Green, M.H.L. and Muriel, W.J. 1976. Mutagen testing using trp+ reversion in Escherichia coli.
Gryczan, T., Shivakumar, A.G., Dubnau, D., 1980. Characterization of chimeric plasmid cloning vehicles in Bacillus subtilis. J. of Bacteriol.. 141, 246-253.
10
Gupta, R., Gigras, P., Mohapatra, H., Goswami, V.K., Chauhan, B., 2003. Microbial α-amylase
TE D
11
s: a biotechnological perspective. Proc. Biochem. 38, 1599-1616.
12
13
Guzman-Maldonado, H., Paredes-Lopez, O., 1995. Amylolytic enzymes and products derived from starch: a review. Crit. Rev. Food Sci. Nutr. 35, 373–403.
EP
14
International Food Biotechnology Council (IFBC), 1990. Safety evaluation of foods and food
AC C
15
ingredients derived from microorganisms. In: Biotechnologies and Food: Assuring the
16
Safety of Foods Produced by Genetic Modification. Regul. Toxicol. Pharmacol. 12,
17
S114-S128.
18
19
M AN U
Mut. Res. 38, 3-32.
8
9
SC
6
Janeček, S., 1994. Sequence similarities and evolutionary relationships of microbial, plant and animal α-amylases. Eur. J. Biochem. 2, 519-24.
20
43
ACCEPTED MANUSCRIPT
1
Janeček, S., 1997. α-amylase family: molecular biology and evolution. Prog. Biophys. Mol. Biol. 67, 67-97.
2
JECFA, 2006. General specifications and considerations for enzyme preparations used in food
RI PT
3
processing, in: Compendium of food additive specifications, volume 4. Analytical
5
methods, test procedures and laboratory solutions used by and referenced in the food
6
additive specifications. FAO/WHO JECFA Monographs 1. Rome, pp. xxi-xxv.
7
Jeang, C.L., Y.H. Lee and L.W. Chang., 1995. Purification and characterization of a raw-starch
SC
4
digesting amylase from a soil bacterium Cytophaga sp. Biochem. Mol. Biol. Int. 35, 549-
9
551.
M AN U
8
Jeang, C.L., Chen, L.S., Chen, M.Y. and Shiau, R.J.. 2002. Cloning of a gene encoding raw-
11
starch-digesting amylase from a Cytophaga sp. and its expression in Escherichia coli.
12
Appl. Environ. Microbiol. 68, 3651–3654.
13
TE D
10
Jonas, D.A., Antignac, E., Antoine, J.M., Classen, H.G., Huggett, A., Knudsen, I., Mahler, J., Ockhuizen, T., Smith, M., Teuber, M., Walker, R., de Vogel, P., 1996. The safety
15
assessment of novel foods. Guidelines prepared by ILSI Europe Novel Food Task Force.
16
Food Chem. Toxicol. 34, 931-940.
AC C
17
EP
14
Kirchman, D.L., 2002.The ecology of Cytophaga–Flavobacteria in aquatic environments. FEMS Microbiol. Ecol. 39, 91-100.
18
19
Kroschwitz, J.I., 1994. Enzyme Applications in Encyclopedia of Chemical Technology, 4th Edition. 9, 567-620.
20
44
ACCEPTED MANUSCRIPT
1
Ladics, G.S., R.F. Cressman, C. Herouet-Guicheney, R.A. Herman, L. Privalle, P. Song, J.M. Ward, S. McLain., 2011. Bioinformatics and the allergy assessment of agricultural
3
biotechnology products: Industry practices and recommendations. Regul. Toxicol.
4
Pharmacol. 60, 46-53.
RI PT
2
Lee, S., Mouri, Y., Minoda, M., Oneda, H., Inouye, K., 2006. Comparison of the wild-type α-
6
amylase and its variant enzymes in Bacillus amyloliquefaciens in activity and thermal
7
stability, and insights into engineering the thermal stability of Bacillus α-amylase. J.
8
Biochem. 139, 1007-1015.
M AN U
9
SC
5
Li, Y., Fang, Z. Dai, J., Partridge, G., Ruc, Y., Penga, J., 2010. Corn extrusion and enzyme
10
addition improves digestibility of corn/soy based diets by pigs: In vitro and in vivo
11
studies. Anim. Feed Sci. Technol. 158: 146–154.
Mayberger, J.M., 2011. Studies of genera Cytophaga-Flavobacterium in context of the soil
TE D
12
carbon cycle. Diss. Abstr. Int. 73, 1- 115.
13
14
Magrane, M. and the UniProt consortium., 2011. UniProt Knowledgebase: a hub of integrated protein data. Database, 2011: bar009 2011). ()
16
MPI Research, 2014a. H-30928: Level 10 C16F Whole Broth: Sub-chronic toxicity 90 day oral
AC C
EP
15
gavage study in rats; Report No. 125-181.
17
18
MPI Research, 2014b. H-30929: Subchronic toxicity 90 day oral gavage study in rats; Report No. 125-180..
19
20
NRC (National Research Council), 1994. Nutrient Requirements of Poultry. 9th Revised Edition, National Academy Press
21 45
ACCEPTED MANUSCRIPT
1
NRC, 1998. Nutrient Requirements of Swine. 10th Revised Edition, National Academy Press.
2
OECD, 1998. Test No. 408: Repeated Dose 90-Day Oral Toxicity Study in Rodents. In: OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.
4
OECD, 1997a. Test No. 471: Bacterial Reverse Mutation Test. In: OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.
5
RI PT
3
OECD, 1997b. Test No. 473: In vitro Mammalian Chromosome Aberration Test. In: OECD
7
Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.
M AN U
SC
6
8
Olempska-Beer, Z.S.; Merker, R.I.; Ditto, M.D.; DiNovi, M.J., 2006. Food-processing enzymes
9
from recombinant microorganisms—A review. Regul. Toxicol. Pharmacol. 45,144-158. Pandey, A., Poonam, N., Soccol, C.R., Soccol, V. T., Singh, D., Radjiskumar, M., 2000. Advances in microbial amylases. Biotech. Appl. Biochem. 31, 135-152.
11
12
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10
Pariza, M.W. and Johnson, E.A., 2001. Evaluating the safety of microbial enzyme preparations used in food processing: update for a new century. Regul. Toxicol. Pharmacol.. 33, 173–
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186.
Pariza, M.W. and Foster, E.M., 1983. Determining the Safety of Enzymes Used in Food
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Processing. J. Food Prot. 46, 453-468.
16
17
Pariza, M.W and Cook, M., 2010. Determining the Safety of Enzymes used in Animal Feed. Regul. Toxicol. Pharmacol. 3, 332-42.
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Romero, L.F., Parsons, C.M., Utterback, P.L., Plumstead, P.W., Ravindran, V., 2013. Comparative effects of dietary carbohydrases without or with protease on the ileal
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digestibility of energy and amino acids and AMEn in young broilers. Anim. Feed Sci.
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Technol. 181: 35–44.
Southern Regional Aquaculture Center Publication 474, Mississippi State Univ. MS.
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5
Snedecor, G.W. and Cochran, W.G. 1967. Statistical Methods, 6th edition, pp. 246-248 and 349352. The Iowa State University Press, Ames, Iowa.
SC
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7
Scientific Committee for Food (SCF)., 1992. Guidelines for the Presentation of Data on Food Enzymes, 27th series, Ref. No. EUR 14181 EN, pp. 13-22.
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Rottman, R. W., R. Francis-Floyd, R. Durborow., 1992. The role of stress in fish disease.
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Sewalt, V., Shanahan, D., Gregg, L., La Marta, J., and Carrillo, R., 2016. The Generally Recognized as Safe (GRAS) Process for Industrial Microbial Enzymes. Ind. Biotechnol.
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12(5): 295-302.
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Sewalt, V., LaMarta, J., Shanahan, D., Gregg, L., Carrillo, R., 2017. Letter to the editor regarding “GRAS from the ground up: Review of the Interim Pilot Program for GRAS
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notification” by Hanlon et al., 2017. Food Chem. Toxicol. 105:140-150.
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Soltani, M., Shanker, S., Munday, B.L., 1995. Chemotherapy of Cytophaga/Flexibacter-like bacteria (CFLB) infections in fish: studies validating clinical efficacies of selected
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antimicrobials. J. Fish Dis. 18, 555-565.
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U.S. Pharmacopeia 2018: Enzyme Preparations. In: Food Chemicals Codex, 11th Edition. Washington, D.C., pp. 413-419.
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van der Kaaij, R.M., Janeček, M. J., van der Maarel, E.C., Dijkhuizen, L., 2007. Phylogenetic and biochemical characterization of a novel cluster of intracellular fungal a-amylase
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enzymes. Microbiol. 153, 4003–4015.
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Wang, C., Liu, Y.M, Wang, Z.Q., Yang, J.X., Liu, Y.M., Wu, T., Yan, Ye, H.W. 2010. Effects of dietary supplementation of methionine and lysine on milk production and nitrogen
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utilization in dairy cows. J. Dairy Sci. 93, 3661-3670.
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Yuuki, T., Nomura, T., Tezuka, H., Tsuboi, A., Yamagata, H.,Tsukagoshi, N. & Udaka, S., 1985. Complete nucleotide sequence of a gene coding for heat- and pH-stable α-amylase of
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Bacillus licheniformis: comparison of the amino acid sequences of three bacterial
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liquefying α-amylase s deduced from the DNA sequences. J. Biochem. 98, 1147–1156.
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Highlights
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An α-amylase gene from a pathogen was safely expressed in Bacillus licheniformis. Oral- and genotoxicity studies for the α-amylase enzyme showed no adverse effects. Enzyme safety was equivalent in absence/presence of the inactive production strain. The studies support decision tree analysis for enzymes produced with biotechnology. The α-amylase is safe as processing aid in production of both human and animal food.
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S0273-2300(18)30199-5
DOI:
10.1016/j.yrtph.2018.07.015
Reference:
YRTPH 4178
To appear in:
Regulatory Toxicology and Pharmacology
Received Date: 22 December 2017 Revised Date:
15 July 2018
Accepted Date: 22 July 2018
Please cite this article as: Sewalt, V.J., Reyes, T.F., Bui, Q., Safety evaluation of two α-amylase enzyme preparations derived from Bacillus licheniformis expressing an α-amylase gene from Cytophaga species, Regulatory Toxicology and Pharmacology (2018), doi: 10.1016/j.yrtph.2018.07.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Safety evaluation of two α-amylase enzyme preparations derived from Bacillus
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licheniformis expressing an α-amylase gene from Cytophaga species
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Vincent J. Sewalt1,*, Teresa F. Reyes1, and Quang Bui1,#
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DuPont Industrial Biosciences, 925 Page Mill Road, Palo Alto, CA 94304
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Current address: 25 Carriage Lane, North Tustin, CA 92705.
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*Corresponding author: Vincent J. Sewalt, 925 Page Mill Road, Palo Alto, CA 94304. Email:
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[email protected].
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Abstract
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A safety assessment was conducted for a symthetic variant Cytophaga sp. α-amylase enzyme
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expressed in Bacillus licheniformis and formulated into two distinct product formats: whole
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broth (a preparation in which the production organism is completely inactivated, but containing
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residual cell debris) and clarified preparation (from which the production organism is completely
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removed). The enzyme was improved via modern biotechnology techniques for use in the
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endohydrolysis of starch, glycogen, related polysaccharides and oligosaccharides. Applications
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range from carbohydrate processing, including the manufacture of sweeteners, fermentation to
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produce organic acids, amino acids and their salts, and potable or fuel alcohol, with resulting co-
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products (distillers’ grains and corn gluten feed/meal) destined for use in animal feed. The
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toxicological studies summarized in this article (90-day rodent oral gavage and in vitro
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genotoxicity studies) noted no test article-related adverse effects and thus substantiate the safety
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of the α-amylase in not only the clarified form but also as a whole-broth preparation. Consistent
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with the decision tree analysis for enzymes produced with modern biotechnology techniques, this
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paper provides supporting information that this variant amylase with homology to an amylase
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from a potentially pathogenic organism (Cytophaga sp.) can be safely produced in an expression
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host that belongs to a Safe Strain Lineage, for safe use as processing aid to manufacture human
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and animal food.
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Key Words: α-amylase; whole broth; safety studies; carbohydrate processing; fermentation; Cytophaga sp.; Bacillus licheniformis; Safe Strain Lineage
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1. Introduction
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Microbially-derived industrial enzyme preparations are used world-wide in food processing and
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animal feed, and recent scientific advances primarily in the fields of molecular biology and
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protein engineering have led to an emergence of innovative enzyme technologies. Considerations
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for food safety evaluation of enzyme preparations for use in human food originally published by
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Pariza and Foster (1983) were updated during the 1990s for enzymes produced with recombinant
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deoxyribonucleic acid (DNA) technology, aka recombinant DNA (rDNA) technology (IFBC,
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1990; Jonas et al., 1996; SCF, 1992), culminating in an updated decision tree (Pariza and
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Johnson, 2001). The Pariza and Johnson (2001) decision tree was adopted for applications in
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animal feed (Pariza and Cook, 2010) and its use in the GRAS (Generally Recognized As Safe)
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process for enzymes reviewed (Sewalt et al., 2016) and clarified (Sewalt et al., 2017). In short,
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these guidelines provide a peer-reviewed decision tree process for the determination of the safety
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of enzyme preparations used in human and animal food, which are based on history of safe use
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of enzymes, the establishment of Safe Strain Lineages to serve as their production strains, and
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well-known strain improvement methods, all supported with published scientific studies. This
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article presents a safety assessment for two α-amylase enzyme preparations, improved via
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modern biotechnology techniques for use in the endohydrolysis of starch, glycogen, related
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polysaccharides and oligosaccharides; with applications ranging from carbohydrate processing,
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including the manufacture of sweeteners such as high fructose corn syrup (HFCS), and
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fermentation to produce organic acids (e.g., citric and lactic acid), amino acids (e.g., lysine) and
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their salts (e.g., monosodium glutamate), and potable or fuel alcohol, with resulting co-products
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(distillers’ grains and corn gluten feed/meal) destined for use in animal feed. The toxicological
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studies summarized in this article substantiate the safety of two types of enzyme preparations,
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the first being the standard food-grade ‘clarified’ enzyme preparation from which all solids
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including the production organism are removed by centrifugation and/or microfiltration; the
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second a whole-broth preparation containing not only the enzyme but also the inactivated
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organism and all fermentation by-products. The latter product format is used mainly in
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fermentation processes to maximize available substrate for yeast to convert to alcohol or other
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fermentation products. Each preparation contains the same variant α-amylase produced in
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Bacillus licheniformis, the amino acid sequence of which is a synthetic protein-engineered
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variant of the α-amylase from Cytophaga sp. Cytophaga alpha-amylase is capable of hydrolyzing
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raw starch (Jeang et al., 1995) without the need for gelatinization by heating. It is of note that the
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Cytophaga genus contains several species that are fish pathogens. It is the objective of this paper
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to provide supporting information that an enzyme protein inspired after a homolog from a
12
pathogenic organism can be safely produced in a safe host for safe use as processing aid to
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manufacture human and animal food.
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The B. licheniformis production strain was genetically engineered to express an optimized
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synthetic variant α-amylase gene most closely related to the α-amylase from Cytophaga sp. The
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host strain is B. licheniformis Bra7, which was developed from its wild-type (wt) parent, by
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classical strain improvement for optimal α-amylase production and lowered protease production.
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The parent strain B. licheniformis Bra7 and strains derived from it have been in use for industrial
19
scale production of food-grade α-amylase for starch processing since the late 1980s. The gene
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inserted into the production organism was not physically isolated from the donor strain; instead
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the gene encoding the α-amylase was synthesized in vitro. The gene was modified by introducing
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various amino acid mutations and deletions. This specific variant of Cytophaga sp. α-amylase is
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referred to as C16F.
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The α-amylase with the International Union of Biochemical and Molecular Biology number
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(IUBMB) 3.2.1.1; is a typical glycosyl hydrolase (GH13_5) α-amylase prevalent in Bacillus
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species (van der Kaaij et al., 2007) and commonly used in industry (Guzman-Maldonado &
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Paredes-Lopez, 1995). The sequence of the C16F α-amylase is similar to various other α-
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amylases isolated from commercially relevant bacteria, e.g., it is 81% homologous to Bacillus sp.
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α-amylase 406 and 75% to Bacillus amyloliquefaciens α-amylase (Lee et al., 2006). Given the
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high structural similarity of α-amylase molecules from various sources (e.g. Janeček, 1994,
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1997), and in particular the liquefying Bacillus α-amylases (Yuuki, 1985), significant differences
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in toxicological properties between these homologous enzymes are not expected.
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Bacillus licheniformis has been used for decades in the production of food enzymes with no
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known reports of adverse effects to human health or the environment (de Boer and Diderichsen
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1994). An extensive environmental and human health risk assessment of B. licheniformis,
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including its history of commercial use has been published by the U.S. Environmental Protection
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Agency (EPA, 1997) and it was concluded that B. licheniformis is not a human pathogen nor it is
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toxigenic. Additionally, the U.S. Food and Drug Administration (FDA) has reviewed the safe use
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of food-processing enzymes from well-characterized recombinant microorganisms, including B.
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licheniformis (Olempska-Beer et al., 2006). FDA also concluded that B. licheniformis is neither
18
pathogenic nor toxigenic and it is considered as suitable for Good Industrial Large Scale Practice
19
(GILSP) worldwide and meets criteria for a safe production microorganism as described by
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Pariza and Johnson (2001).
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The raw starch digesting α-amylase from Cytophaga sp. has been described by Jeang et al.
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(1995, 2002). Cytophaga sp. are unicellular, non-spore forming Gram-negative bacteria
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(Kirchman, 2002) derived from soil. They are part of the Cytophaga-Flavobacteria cluster,
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which are especially proficient in degrading various biopolymers such as cellulose, chitin, and
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pectin (Mayberger, 2011). Cytophaga sp. can be found globally in every habitat in every
3
biosphere, including kusaya (a Japanese delicacy consisting of putrid fish), rumens,
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hydrothermal vents, rocks, sea-ice in Antarctica and sediments of lakes and oceans. Cytophaga-
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Flavobacteria is more prevalent in the oceans, making it one of the most abundant of all
6
bacterial groups (Kirchman, 2002).
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Some members of Cytophaga sp. genus are reported to be fish pathogens (Carson et al., 1993;
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Dalsgaard, 1993; Soltani et al., 1995) known to cause non-zoonotic Columnaris disease, an
9
infection that typically enters through the gills, mouth or small wounds, prevalent where high
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bioloads exist (Cooney et al., 2002), or in stressed environments such as overcrowding or low
11
dissolved oxygen levels in the water (Rottman, 1992, Durborow, 1998). However, pathogenicity
12
is a complex process that typically involves the expression of specialized invasive elements
13
called virulence factors, some of which were discussed by Dalsgaard (1993), and none of which
14
are associated with the α-amylase protein or its gene. Many harmless microorganisms (and
15
harmful ones, alike) express genes for amylases, which in nature only serve the purpose of starch
16
digestion. Amylases are used in numerous industrial applications including food manufacture
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(Gupta et al., 2003, Pandey et al., 2000) and as feed additive, often combined with other
18
enzymes, to pigs (e.g., Li et al., 2010) and poultry (e.g., Romero et al., 2013). The only genetic
19
information expressed in the B. licheniformis production host is a synthetic α-amylase variant
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gene inspired from the Cytophaga sp. α-amylase sequence, but no actual nucleic acids from
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Cyptophaga sp. were transferred.
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1.1 Development of the production strain of Bacillus licheniformis (C16F)
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The variant α-amylase gene coding sequence was placed under the expression signals of the
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endogenous B. licheniformis amyL gene and the B. subtilis aprE 5’ untranslated region (UTR),
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cloned in a vector derived from Bacillus plasmids pUB110 and pE194 (Gryczan et al., 1980),
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together with the native B. licheniformis cat gene, encoding chloramphenicol acyltransferase.
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The resulting plasmid was integrated into the host chromosome at the cat locus by Campbell
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type recombination. After integration, all vector sequences of the plasmid were deleted by
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recombination between direct repeated cat sequences. The genetic construction was evaluated at
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every step to assess the incorporation of the desired functional genetic information and the
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intended chromosomal modifications were confirmed by PCR and sequence analyses. No new
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antibiotic resistance genes were introduced in the construction of the production organism.
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The safety of C16F α-amylase was evaluated in a series of studies designed according to current
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guidelines on evaluating the safety of microbial enzyme preparations used in human and animal
13
food (Pariza and Johnson, 2001; Pariza and Cook, 2010). The studies evaluate the safety of the
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protein-engineered enzyme as well as its toxigenic potential in two manufactured product
15
formats: clarified, ultra-filtered concentrate (UFC) and unfiltered whole-broth (WB) preparation.
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The studies support the safety of residual enzyme preparation in the final food, even with the
17
worst-case assumption that the enzyme is carried over wholly into the final food even though in
18
all cases the enzyme is inactivated and in many cases the inactive enzyme protein is removed in
19
the process.
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Pariza and Johnson (2001) establish that a sufficiently high No Observed Adverse Effect Level
21
(NOAEL) in an oral toxicity study is one requisite to demonstrate safety. To satisfy this
22
requirement, sub-chronic studies in Charles River CD rats using whole-broth (WB) and clarified
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ultra-filtered concentrate (UFC) C16F α-amylase enzyme preparations were conducted by oral
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gavage (DuPont Haskell Global Center, 2014). Additionally, genotoxicity studies were
2
performed to address any mutagenicity potential from the C16F α-amylase enzyme preparation.
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Generally, clarified UFC enzyme preparations undergo recovery steps post-fermentation to
4
remove cellular material, followed by enzymatic concentration; whereas WB enzyme
5
preparations undergo production organism inactivation but skip the cell removal stage. In
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general, the recovery process is a multi-step operation, which starts immediately after the
7
fermentation process. The enzyme is recovered from the fermentation broth by 1) Primary
8
separation: microfiltration (MF) or flocculation-aided rotary drum vacuum centrifugation
9
(RDVC) and separation by filter press, and 2) Concentration by ultrafiltration into UFC.
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Stabilizers and preservatives are added to both UFC and WB fermentation material, followed by
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an additional polish filtration step for stabilized UFC. Typically, clarified UFC-based enzyme
12
preparations are employed in food-related applications, while WB enzyme preparations are used
13
for non-food applications like ethanol production, although, WB enzyme preparations can be
14
safely utilized in food (if appropriate in the application), e.g., fermentation processes.
15
The results from the safety studies and our exposure assessment support our conclusion that the
16
C16F α-amylase derived from B. licheniformis is safe and suitable for use in food applications
17
such as carbohydrate processing and fermentation, with resulting co-products (distillers’ grains
18
and corn gluten feed/meal) destined for use in animal feed. The work presented here not only
19
adds to the body of evidence of microbial enzyme safety, it also highlights two aspects of
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enzyme safety that hitherto have not been discussed in great detail.
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On the one hand, we provide supporting data that an enzyme from a species that is questionable
22
as a direct enzyme source can be safely expressed in a well-established production organism that
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belongs to a Safe Strain Lineage as defined by Pariza and Johnson (2001), in this case DuPont’s
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Bacillus licheniformis Safe Strain Lineage. On the other hand, the comparative toxicological
3
information for the α-amylase in both clarified enzyme preparation format and as whole-broth
4
enzyme preparation lends support to the safety of the latter format for use in food applications
5
where that format is appropriate.
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2. Materials and Methods
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2.1 Test material preparation and characterization
8
The C16F α-amylase enzyme preparation manufacturing process follows standard industry
9
practice (Kroschwits, 1994; Aunstrup et al., 1979; Aunstrup, 1979), and in accordance with
10
FDA’s current Good Manufacturing Practices (cGMP) requirements and ISO (International
11
Organization for Standardization) 9001 standards. Raw materials conform to specifications of the
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11th edition of the Food Chemicals Codex (FCC) maintained by the U.S. Pharmacopeia (USP,
13
2018). The C16F α-amylase enzyme preparation is manufactured by submerged fermentation of
14
a pure culture of the non-pathogenic, non-toxigenic, genetically engineered strain of B.
15
licheniformis (strain JML1584). A new lyophilized stock culture vial of the B. licheniformis
16
production organism is used to initiate the production of each batch. Each new batch of the stock
17
culture is thoroughly controlled for identity, absence of foreign microorganisms, and enzyme-
18
generating ability before use. Antibiotics are not used in the fermentation process. Equipment of
19
carefully design and construction is operated, cleaned and maintained so as to prevent
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contamination by foreign microorganisms. During all steps of fermentation, physical and
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chemical control measures are taken and microbiological analyses are conducted periodically to
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ensure absence of foreign microorganisms, and upon conclusion of the process, inactivation of
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the production strain. The C16F α-amylase enzyme was prepared in two preparations, one
2
whole-broth (WB) and the other as an ultra-filtered concentrate (UFC). The C16F α-amylase,
3
regardless of product format, meets purity specifications for enzyme preparations set forth in the
4
FCC 11th edition (USP, 2018) and to the General Specifications for Enzyme Preparations used in
5
Food Processing as proposed by the Joint FAO/WHO Evaluation Committee for Food Additives
6
(JECFA) in the Compendium of Food Additive Specification (JECFA, 2006).
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specifications include absence of live production organism, absence of antimicrobial activity,
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absence of Salmonella and pathogenic E. coli, and specifications for coliforms, total viable
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counts, and heavy metals.
These
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To assess the safety of both C16F α-amylase enzyme preparations, different endpoints of toxicity
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were investigated in separate studies for WB and UFC formats and the results of each evaluated,
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interpreted and assessed in this paper. All toxicology studies presented here were conducted in
13
compliance with current Good Laboratory Practice standards and Organization for Economic
14
Cooperation and Development (OECD) Guidelines for the Testing of Chemicals., The test
15
materials used in all toxicology investigations were the clarified UFC and WB enzyme
16
preparations collected from fermentation with the following characteristics (Table 1):
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Table 1. Characteristics of test materials used in toxicology testing
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Study
Subchronic Toxicity 90-Day Oral Gavage Study in Rats In vitro Mammalian Chromosome Aberration Test Bacterial Reverse 10
Study Reference MPI Research (2014b)
MPI Research (2014a)
DuPont Haskell Global Centers, (2014b)
DuPont Haskell Global Centers (2014a)
BioReliance (2014b)
BioReliance (2014a)
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Mutation Assay H-30929 UFC (Ultra-filtered Concentrate) Fermentation liquid,
Physical
H-30928 WB (Whole-Broth)
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Test Material Characteristics
Fermentation Liquid, Brown
Brown
Enzyme Activity
68.3 DLU/L
36.1 DLU/L
pH
6.3
Specific gravity
1.03 g/ml 39.9 mg/ml
Trichloroacetic Acid (TCA) Protein Total Organic Solids (TOS)
1.06 g/ml
91.6 mg/ml
31.2 mg/ml
42.2 mg/ml
7.12%
14.4%
0.11%
0.11%
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(Dextrose Liberating Units)
0.30%
0.30%
Compliant with JECFA (2006) and FCC (2018)
Compliant with JECFA (2006) and FCC (2018)
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2.2 Experimental Design
3
2.2.1. Oral Administration Test (90-day oral gavage study):
4
Oral toxicity tests for WB and UFC test articles were based on OECD Test Guideline 408,
5
(revised September 1998) and were conducted in compliance with the Guide for the Care and
6
Use of Laboratory Animals of the Institute of Laboratory Animal Resources (2011).
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The systemic toxicity of C16F α-amylase (WB or UFC enzyme preparations) was assessed in
8
two 13-week oral gavage studies in Charles River (CD) rats of both sexes conducted at MPI
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Research, Mattawan, Michigan in accordance with US FDA Good Laboratory Practice (GLP)
2
Regulations (21 CFR Part 58) and the OECD Principles of Laboratory Practice
3
(ENV/MC/CHEM(98)17), and U.S. Department of Agriculture’s (USDA) Animal Welfare Act
4
(9 CFR Parts 1-3).
5
Groups of 10 animals per sex were treated by oral gavage with 0 (distilled water), 100, 250 or
6
500 mg TOS (Total Organic Solids)/kg bw/day. The vehicle control, 0.11% potassium sorbate
7
and 0.38% sodium benzoate in distilled water, was prepared weekly and stored and refrigerated
8
at 2-8°C. The dosing volume was 10 ml/kg bw/day. During the study, samples of the dosing
9
preparations were collected at different intervals (week 1, 6 and 12) and verified for proper
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dosage concentration.
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Animals of the same sex were housed in groups of two in solid floor polypropylene cages with
12
stainless steel mesh lids and softwood bedding (non-aromatic) with access to water via an
13
automated system and feed ad libitum. For environmental enrichment, the animals were provided
14
a supply of wooden chew blocks and cardboard fun tunnels. All groups were housed under
15
controlled temperature, humidity and lighting conditions. Observations for morbidity, mortality,
16
injury, and the availability of food and water were conducted twice daily for all animals.
17
Cage-side clinical observations were conducted daily. Observations for detailed clinical signs
18
were conducted pretest and weekly. Functional Observational Battery (FOB) observations
19
(Activity/Arousal, Neuromuscular, and Sensorimotor, Autonomic, and Physiological) and
20
locomotor activity tests were conducted pretest and during Week 13. Body weights were
21
measured and recorded on Day 1 and 7 and weekly during the study. Food consumption (by
22
cage) was measured and recorded weekly and food efficiency was calculated weekly and
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reported as a mean of five cages/sex/treatment. Ophthalmoscopy examinations were conducted
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pretest and prior to the terminal necropsy. Blood and urine samples for clinical pathology and
3
chemistry evaluation were collected prior to the terminal necropsy. Blood work included
4
hematology analysis: hemoglobin, hematocrit, mean corpuscular volume (MVC), mean
5
corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and
6
individual cell types (leukocytes, erythrocytes, platelets, absolute reticulocytes, neutrophils,
7
lymphocytes, monocytes, eosinophils, basophils, and ‘other cells’) and coagulation (activated
8
partial thromboplastin time (APTT) and prothrombin time. Clinical chemistry included minerals
9
(sodium, potassium, chloride, calcium, phosphorous), total bilirubin, bile acids, enzymes
10
(alkaline phosphatase, gamma glutamyltransferase (GGT), aspartate aminotransferase (AST),
11
alanine aminotransferase (ALT), urea nitrogen, creatinine, total protein, albumin, globulin,
12
triglyceride, cholesterol, glucose). Urinanalysis included volume, specific gravity and pH.
13
At study termination (Day 91), necropsy examinations were performed and selected organ
14
weights were recorded. Tissues from animals in the control and high dose group and gross
15
lesions in all dose groups were microscopically examined.
16
2.2.2 Statistical analysis
17
Raw data was tabulated within each time interval, and the mean and standard deviation and/or
18
incidence counts (categorical variables) were calculated for each endpoint by sex and group
19
(control group (1) vs. treatment groups (2, 3 and 4). For each continuous endpoint (body weight,
20
body weight change, food consumption, functional observational battery observations (FOB,
21
continuous endpoints), hematology (except leukocyte counts), coagulation, clinical chemistry,
22
and organ weights) treatment groups were compared to the control group using Group Pair-wise
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Comparisons (Levene’s/ANOVA-Dunnett’s/Welch’s). For locomotive activity, Analysis of
2
Variance (ANOVA) was used and for leukocyte counts, Log Transformation/Group Pair-Wise
3
comparisons (Leven’s/ANOVA-Dunnet’s/Welch’s) were used. For food efficiency, and
4
urinalysis Rank Transformation with Dunnett’s Test was used and for FOB (Categorical
5
Endpoints) the Cochran Haenszel Test was used. Data for some end points were transformed by
6
either a log or rank transformation prior to conducting the specified analysis.
7
2.2.3. In vitro Mammalian Chromosomal Aberration Test performed with Human Lymphocytes
8
The objective of these assays was to investigate the potential of C16F WB or clarified UFC α-
9
amylase enzyme preparations to induce numerical and/or structural changes in the chromosome
10
of mammalian systems (i.e., human peripheral lymphocytes). These assays were conducted in
11
accordance with OECD guideline No. 473 (OECD, 1997b).
12
In this assay, human lymphocytes were stimulated to divide by the addition of a mitogen (e.g.
13
phytohemagglutinin, PHA). Mitotic activity began at about 40 hours after PHA stimulation and
14
reached a maximum at approximately 3 days.
15
Test material (C16F WB or UFC) was mixed with cultures of human peripheral lymphocytes
16
both in the presence and absence of metabolic activation (Aroclor 1254-induced rat liver; S-9
17
mix). This study consisted of a preliminary toxicity (dose range finding) and two main assays. In
18
the preliminary assay, all cultures with or without S-9 mix were treated for 4 hours and
19
continuously for 22 hours for cultures without S-9 mix. All cells were harvested 22 hours after
20
treatment initiation. Nine concentrations of α-amylase (50, 100, 250, 500, 750, 1000, 1500, 2500,
21
and 5000 µg TP/ml) were used as recommended by the OECD guideline. All dose levels were
22
expressed in terms of total protein. From the results of the preliminary toxicity study, the highest
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concentration was selected for the final chromosome aberration assay that induced greater than a
2
50% reduction of the mitotic index in the preliminary assay, relative to the vehicle control. Four
3
additional dose levels, demonstrating limited toxicity or no toxicity were also evaluated. In cases
4
where there was little or no cytotoxicity (determined by the MI), the highest dose level tested and
5
at least 2 lower dose levels were selected for analysis. In cases where there was little or no
6
cytotoxicity, but a precipitate was observed (with the naked eye) the lowest dose level
7
demonstrating a precipitate and 2 other lower dose levels were selected for analysis. If neither
8
cytotoxicity nor precipitation was observed in the preliminary toxicity assay, the highest
9
concentration was the limit dose of 5000 µg/mL or 10 mM, whichever was the lower.
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In the definitive assay, cultures with and without S-9 mix were exposed to the test article for 4
11
hours, and continuously for 22 hours for those without S-9 mix. Cells were collected 22 hours
12
(1.5 normal cell cycles) after initiation of treatment. Two hours prior to harvest, Colcemid was
13
added to the cultures at a final concentration of 01 µg/ml to arrest mitosis.
14
Cells were collected by centrifugation, treated with 0.075 M KCl hypotonic buffer, fixed in
15
methanol:glacial acetic acid (3:1 v/v), capped and stored overnight or longer. To prepare slides,
16
the cells were re-suspended in fixative and then collected by centrifugation. The suspension of
17
fixed cells was applied to glass microscope slides and air-dried. The slides were stained with
18
Giemsa, permanently mounted and scored.
19
The mitotic index was recorded as the parentage of cells in mitosis per 500 cells counted.
20
Metaphase analysis (i.e. evaluation of chromosomal aberration) was conducted on at least 200
21
metaphases for each level (100 per duplicate treatment). Cells were scored for both chromatid-
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type and chromosome-type aberrations. Mitomycin C and cyclosphosphamide were used as
2
positive controls for cultures without S-9 mix and cultures with S-9 mix, respectively.
3
Statistical Analysis
4
The clastogenic potential of the test substance was assessed based on the percentage of cells with
5
structural chromosome aberrations. Interpretation of the results also relied on additional
6
considerations including the magnitude of the observed test substance response relative to the
7
vehicle control response and the presence of a dose-responsive trend. Statistical analysis
8
consisted of a Fisher’s exact test with Bonferroni-Holm Adjustment (Fisher, 1985) to compare
9
the percentage of cells with structural or numerical aberrations (or the percentage of cells with
10
more than one aberration, if required) in the test substance groups with the vehicle control
11
response. Before considering any test article effects, the assay was considered acceptable for
12
evaluation of test results only if all of the following criteria were satisfied. For Negative
13
Controls, the frequency of cells with structural chromosome aberrations to be within the negative
14
historical control range. For Positive Controls, the percentage of cells with structural
15
chromosome aberrations to be statistically significantly greater (p < 0.05, Fisher’s exact test)
16
than the vehicle control response. The metabolically activated and non-activated assays of the
17
test are considered independent test results and, if inconclusive, would be repeated separately.
18
A Cochran-Armitage test for test article dose responsiveness (Snedecor and Cochran, 1967) was
19
conducted only in case of statistically significant differences, based on the Fisher’s exact test.
20
The following conditions were used as a guide to determine a positive response: 1) a statistically
21
significant increase (p < 0.05, Fisher’s exact test) in the percentage of cells with structural
22
aberrations was seen in one or more treatment groups relative to the vehicle control response. 2)
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the observed increased frequencies were accompanied by a concentration-related increase, 3) or
2
a statistically significant increase was observed at the highest dose only. Statistically significant
3
values that did not exceed the historical control range for the negative/vehicle control may be
4
judged as not being biologically significant. Equivocal response is defined as a statistically
5
significant elevation in structural chromosome aberrations at more than one test concentration
6
level, except the highest dose, without demonstrating a dose-responsive trend. The test substance
7
was judged negative if the following condition was met: There was no statistically significant
8
increase in the percentage of cells with structural aberrations in any treatment group relative to
9
the vehicle control group.
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2.2.4 Bacterial Reverse Mutation Assays
11
The objective of these assays was to assess the potential of α-amylase (C16F WB or UFC) to
12
induce point mutations (frame-shift and base-pair) in four strains of S. typhimurium TA98,
13
TA100, TA1535 and TA1537 (Ames et al. 1975) and Escherichia coli strain WP2 uvrA (Green
14
and Muriel, 1976) per OECD guideline 471 (OECD, 1997a). The test material was tested both in
15
the presence and absence of a metabolic activation system (Aroclor 1254-induced rat liver; S-9
16
mix). The assay was performed in two phases using the plate incorporation methodology for the
17
positive control, 2-aminoanthracene, with E. coli and the treat and plate methodology for all
18
remaining strains and assays.
19
A screening (dose range) test was performed first to select dose levels for the confirmatory assay.
20
Vehicle control, positive control and 8 doses of the test article (1.5, 5, 15, 50, 150, 500, 1500 and
21
5000 µg per plate were plated, two plates per dose, with overnight cultures of all four strains of
22
S. typhimurium and E. coli WP2 uvrA in the presence and absence of S-9 mix. In the
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confirmatory assay, 6 doses of the test article (15, 50, 150, 500, 1500 and 5000 µg per plate)
2
along with appropriate vehicle and positive controls were plated in triplicate in the presence and
3
absence of S-9 mix. All dose levels were expressed in terms of total protein (TP). The highest
4
dose level (5000 µg TP/plate) is the maximum dose required by the OECD guideline. The
5
positive controls use for assays without S-9 mix were 2-nitrofluorene, N-methyl-N-nitro-N-
6
nitrosoguanadine (MNNG) and ICR-191. For assays with S-9 mix, the positive control was 2-
7
aminoanthracene. Vehicle control plates were treated by the addition of sterile deionized water.
8
Data Analysis
9
For each replicate plating, the mean and standard deviation of the number of revertants per plate
10
were calculated and reported. For the test article to be evaluated as positive, it must cause a dose-
11
related increase in the mean revertants per plate of at least one tester strain over a minimum of
12
two increasing concentrations of test article. Data sets for the tester strains TA1535 and TA1537
13
were judged positive if the increase in mean revertants at the peak of the dose response was
14
greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains
15
TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak
16
of the dose response was greater than or equal to 2.0-times the mean vehicle control value. An
17
equivocal response is a biologically relevant increase in a revertant count that partially meets the
18
criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve
19
the respective threshold cited above or a non-dose responsive increase that is equal to or greater
20
than the respective threshold cited. A response was evaluated as negative, if it was neither
21
positive nor equivocal.
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The following criteria must be met for the initial toxicity-mutation and confirmatory
2
mutagenicity assays to be considered valid. All Salmonella tester strain TA98 and TA100
3
cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the
4
uvrB gene. Cultures of tester strains must demonstrate the presence of the pKM101 plasmid R-
5
factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene. All cultures must
6
demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls as
7
follows (inclusive): TA98, 10 - 50; TA100, 80 - 240; TA1535, 5 - 45; TA1537, 3 - 21; WP2
8
uvrA, 10 - 60. To ensure that appropriate numbers of bacteria are plated, tester strain culture
9
titers must be greater than or equal to 0.3 x 109 cells/mL. The mean of each positive control must
10
exhibit at least a 3.0-fold increase in the number of revertants over the mean value of the
11
respective vehicle control. A minimum of three non-toxic dose levels is required to evaluate
12
assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A
13
>50 % reduction in the mean number of revertants per plate as compared to the mean vehicle
14
control value. This reduction must be accompanied by an abrupt dose-dependent drop in the
15
revertant count. (2) At least a moderate reduction in the background lawn (background lawn
16
code 3, 4 or 5).
17
2.3 Sequence screening for food allergy potential and homology to protein toxins
18
Food Allergens
19
Following the most current allergenicity assessment guidelines developed by the Codex
20
Commission (2009) and Ladics et al. (2011), we used a FASTA (FAST-All) search for matches
21
of 35% identity or more over 80 amino acids of the variant Cytophaga sp. protein against a
22
database of known allergens, further enhanced by the use of an E-value cut-off in the BLAST
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algorithm of 10-7 to suggest a biologically relevant similarity (i.e., potential cross reactivity). We
2
used the Food Allergy Research Resource Program (FARRP) AllergenOnline database
3
(http://www.allergenonline.org/databasefasta.shtml containing 2089 (version 18B released
4
March 23, 2018) peer-reviewed allergen sequences (listed in
5
http://www.allergenonline.org/AllergenOnlineV18B.pdf.
6
Toxins
7
A BLAST search for homology of the mature C16F amino acid protein sequence with known
8
toxins and antinutrients was performed using the UniProtKB annotated Protein Knowledge
9
database (Magrane et al., 2011; http://www.uniprot.org/), UniProt release 2018_04 (April 25,
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2018). This large protein database with hundreds of thousands sequences contains 6,724 proteins
11
that are manually annotated as toxins or venoms (http://www.uniprot.org/program/Toxins). From
12
this search, the top 1,000 hits in the UniProt database were exported to MS Excel, with the
13
appropriate annotation fields (protein name, key words, gene ontology, protein family), allowing
14
for use of search terms “toxin” and “venom”.
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Results
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3.1. Oral Administration Tests (90-day oral gavage study)
18
The oral gavage tests (WB and UFC) resulted in no treatment-related deaths during the 13-week
19
period. Body weight gain, feed intake, and feed efficiency are summarized in Tables 2-4.
20 21
Table 2. Body weight gain (g) of male and female rats exposed to whole-broth (WB) and clarified (UFC) C16F α-amylase
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Test article
Sex 0
100
250
500
Whole Broth
Male
371.2 ± 31.2*
380.4 ± 27.6
402.6 ± 36.0
389.4 ± 32.4
Whole Broth
Female
121.8 ± 17.9
122.7 ± 15.7
111.9 ± 18.0
125.0 ± 14.6
UFC
Male
296.2 ± 14.7
287.2 ± 26.6
302.9 ± 22.9
292.0 ± 23.0
UFC
Female
132.3 ± 14.0
125.7 ± 9.2
135.5 ± 6.7
142.0 ± 16.9
*± 2 SEM (Standard Error of the Mean, n=10)
Table 3. Daily feed intake (g/animal/day) by male and female rats exposed to whole-broth (WB) and clarified (UFC) C16F α-amylase
5
Sex
100
250
500
Male
27.4 ± 2.08*
27.6 ± 1.49
28.4 ± 2.59
27.4 ± 1.60
Whole Broth
Female
18.5 ± 1.20
17.5 ± 0.94
17.6 ± 1.27
17.6 ± 1.49
UFC
Male
26.4 ± 0.88
26.1 ± 1.71
25.9 ± 0.66
25.7 ± 1.97
UFC
Female
18.6 ± 1.16
18.3 ± 0.72
18.5 ± 0.89
18.9 ± 0.59
*± 2 SEM (Standard Error of the Mean, n=5)
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Table 4. Feed efficiency (%) by male and female rats exposed to whole-broth (WB) and clarified (UFC) C16F α-amylase C16F amylase dose (mg TOS/kg BW/day)
Sex
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C16F amylase dose (mg TOS/kg BW/day)
0
100
250
500
Whole Broth
Male
15.2 ± 0.87*
15.5 ± 0.87
15.9 ± 0.91
16.0 ± 0.87
Whole Broth
Female
7.36 ± 0.70
7.8 ± 0.52
7.2 ± 0.65
8.0 ± 0.80
UFC
Male
12.7 ± 0.45
12.6 ± 1.06
13.1 ± 0.78
12.8 ± 0.65
UFC
Female
8.0 ± 0.53
7.7 ± 0.51
8.3 ± 0.61
8.5 ± 0.55
*± 2 SEM (Standard Error of the Mean, n=5)
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1
As can be seen in Tables 2-4, there were no significant effects related to the UFC or WB test
2
articles of C16F α-amylase
3
observations, functional observations, locomotor activity, and hematology, coagulation, clinical
4
chemistry, and urinalysis parameters are included as Supplemental Tables A1 through A21 for
5
the UFC test article and B1 through B21 for the WB test article. No differences in functional
6
observations or locomotor activity were noted due to either test article, with the following
7
exceptions:
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Hindlimb grip strength was marginally lower for male rats receiving the highest dose of
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on body weight, feed intake, or feed conversion. Clinical
C16F amylase, regardless of product format (see Suppl. Data Tables A12 and B12). This
10
was not the case for females receiving either UFC or WB test article. Given this
11
inconsistency, and given that all other clinical, functional, and locomotor activity
12
observations were normal, this isolated marginal difference was not considered an
13
adverse effect. -
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Locomotor activity (specifically, rearing count) was reduced for male rats receiving the middle dose of UFC (see Suppl. Data Table A15). This observation was regarded as not
16
attributable to the test article as 1) it was not a dose-dependent, 2) it did not occur for
17
female rats receiving UFC test article, and 3) it was not observed for any concentration of
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WB administered to either sex at any concentration.
18 19
No significant differences were observed in hematology, coagulation, clinical chemistry and
20
urinanalysis panels (Tables A18-A21 and B18-B21).
21
At necropsy, there were no treatment related effects on organ weights, macroscopic observations
22
and histopathologic examination (data not shown). All microscopic findings were considered to
22
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be within the background incidence of findings reported in this age and strain of laboratory
2
animals.
3
In summary, there were no meaningful biological differences between the control and treated
4
groups with respect to all clinical parameters measured. Under the conditions of this study, the
5
No-Observed-Adverse-Effect Level (NOAEL) for both WB and UFC enzyme preparations is
6
500 mg TOS/kg bw/day. This NOAEL is equivalent to approximately 280 mg total protein/kg
7
bw/day (UFC) and 317 mg total protein/kg bw/day (WB).
8
3.2. Genetic Toxicology studies
9
3.2.1 Bacterial Reverse Mutation Assay
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Dosing formulations were adjusted for total protein content based on the concentration as
11
supplied at 91.62 mg/mL (WB) and 39.89 mg/mL (UFC). In the treat and plate method, the
12
volumes of S-9 mix, sham mix, bacterial and test article, vehicle or positive control were
13
increased by a factor of 2.5 or 3.5 to ensure sufficient volume of resuspended bacteria to plate
14
the desired number of replicates. Deionized water was selected as the solvent of choice.
15
In the initial toxicity-mutation assays, the maximum dose tested was 5000 µg per plate; this dose
16
was achieved by diluting the test article at the concentration of 91.62 mg/mL (WB) and 39.89
17
mg/mL (UFC) to a concentration of 50 mg/mL (using a 100 µL aliquot) and 25 mg/mL (using a
18
200 µL aliquot). The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per
19
plate. The WB test article formed a clear solution in water at 0.015 mg/mL, a cloudy solution at
20
0.050 mg/mL and workable suspensions from 0.15 to 50 mg/mL; whereas the UFC test article
21
was a workable suspension at the 39.89 mg/mL concentration, but formed clear solutions in
22
water from 0.0075 to 25 mg/mL.
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In the preliminary assays (Suppl. Data Tables A24-25 and B24-25), no positive mutagenic
2
responses were observed with any of the tester strains in either the absence or presence of S-9
3
mix activation. Neither precipitate nor toxicity was observed for the C16F α-amylase WB
4
enzyme preparation test article. No precipitate was observed for the C16F α-amylase UFC
5
enzyme preparation. Toxicity to the UFC enzyme preparation was observed beginning at 1500
6
µg per plate with tester strain TA98 in the absence of S-9 mix.. Based on the findings of the
7
initial toxicity-mutation assay; the maximum dose plated in the confirmatory mutagenicity assay
8
for both UFC and WB enzyme preparations was 5000 µg per plate, with dose levels 15, 50, 150,
9
500, 1500 and 5000 µg per plate.
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In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any
11
of the tester strains in either the presence or absence of S-9 mix (Suppl. Data Tables A26-27 and
12
B26-27). Increases in revertant counts (1.5- and 1.6-fold maximum increases) were observed for
13
UFC enzyme with only with tester strain WP2 uvrA in the presence and absence of S9
14
activation, respectively. For the WB test article, an increase in revertant counts (2.1-fold
15
maximum increase) was observed only with tester strain TA98 in the presence of S-9 mix
16
(Suppl. Data Table B27). However, these increases were not considered to be indicative of
17
mutagenic activity because 1) the increases were not dose-responsive, 2) the vehicle control
18
levels in the study were on the low end of the acceptable range, and 3) the peak of each response
19
was within historical vehicle control range for this test condition (for WP2uvrA, 32 and 35
20
revertants per plate in the presence and absence of S9; for TA98, 26 revertants/plate in the
21
presence of S-9 mix).
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No precipitate was observed for either WB or UFC test article, whereas toxicity was observed for
2
UFC only, at 5000 µg per plate with tester strain TA98 in the absence of S9 activation. All
3
criteria for valid studies as described in the Materials and Methods were met.
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The results of the bacterial reverse mutation assays (Ames tests) indicate that, under the
6
conditions of these studies, the test articles (WB and UFC) C16F α-amylase enzyme preparation
7
did not exhibit any mutagenic responses in either the presence or absence of Aroclor-induced rat
8
liver S9. Therefore, both WB and UFC test articles were concluded to be negative in this assay.
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3.2.2 In-vitro Chromosomal aberration assay-Human lymphocytes
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In the preliminary toxicity assay, cells were treated for 4 and 22 hours in the non-activated test
12
condition and for 4 hours in the S-9 mix activated test condition. All cells were harvested 22
13
hours after treatment initiation. The cells were exposed to 9 concentrations of each test substance
14
ranging from 50 to 5000 µg/mL, as well as a vehicle control. Test substance precipitation was
15
observed for the WB enzyme preparation at concentrations ≥ 750 µg/mL (see Suppl. Table B22),
16
whereas no test substance precipitation was observed in the treatment media of the UFC enzyme
17
preparation test substance. Interfering precipitation (WB enzyme preparation test substance)
18
prevented the analysis of concentration ≥ 750 µg/mL in the non-activated test conditions and ≥
19
1500 µg/mL in the 4-hour S-9 mix -activated test condition. Substantial toxicity (greater than
20
50% reduction in mitotic index relative to the vehicle control) was observed in the 22-hour non-
21
activated test condition at concentrations ≥ 250 µg/mL. The UFC enzyme preparation test
22
substance showed substantial toxicity (greater than 50% reduction in mitotic index relative to the
23
vehicle control) in the 22-hour non-activated test condition at concentrations ≥ 100 µg/mL (see
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Suppl. Data Table A22). Based on the findings from the preliminary toxicity assay, the highest
2
concentration chosen for the chromosome aberration assay was 1000 µg/mL (WB) and 5000
3
µg/mL (UFC) for the 4-hour conditions and 250 µg/mL (WB) and 100 µg/mL (UFC) for the 22-
4
hour test conditions.
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a) Chromosomal Aberration Assay - WB Test Material:
6
For the C16F α-amylase WB enzyme preparation, the concentrations chosen for the 4-hour
7
test conditions were 50, 100, 250, 500 and 1000 µg/mL and for the 22-hour test condition
8
were 50, 100, 150, 200 and 250 µg/mL. Precipitation was observed in the treatment media at
9
1000 µg/mL. Substantial toxicity was observed in the 22-hour non-activated test condition at
10
100, 200 and 250 µg/mL. Selection of doses for microscopic analysis was based on test
11
substance induced toxicity in the 22-hour test condition. In the 4-hour test conditions,
12
selection of doses for analysis was based on interfering precipitation of the test substance.
13
Cytogenetic evaluations for C16F α-amylase WB enzyme preparation were conducted at 250,
14
500 and 1000 µg/mL in the 4-hour test conditions (see Suppl. Data Table B23). Four-hour
15
exposure to the highest test concentration (5000 µg/mL) did not result in mitotic index
16
changes compared with the vehicle control (indicating no toxicity). In the 22-hour test
17
condition, cytogenetic evaluations were conducted at 50, 150 and 200 µg/mL. The mitotic
18
index for the highest test concentration evaluated microscopically for chromosome
19
aberrations (200 µg/mL) was 7.5%, compared with 16.2% for the vehicle control. This
20
represents a 53.7% mitotic inhibition in relation to the vehicle control. The percentage of
21
cells with numerical or structural aberrations in the test substance-treated groups was not
22
significantly increased above that of the vehicle control at any concentration (p ≥ 0.05,
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Fisher’s exact test). The percentage of cells with structurally damaged chromosomes in the
2
respective positive control groups were statistically significant (p < 0.05, Fisher’s exact test).
3
b) UFC Test Material
4
For the C16F α-amylase UFC enzyme preparation the concentrations were 250, 500, 1000,
5
2500 and 5000 µg/mL for the 4-hour test condition. For the 22-hour test condition, the
6
concentrations were 10, 25, 50, 75 and 100 µg/mL. No test substance precipitation was
7
observed in the treatment media. Substantial toxicity was observed in the 22-hour non-
8
activated test condition at 100 µg/mL. Selection of doses for microscopic analysis was based
9
on test substance induced toxicity in the 22-hour test condition. In the 4-hour test conditions,
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selection of doses for analysis was based on the limit dose for the assay, 5000 µg/mL.
11
Cytogenetic evaluations for C16F α-amylase UFC enzyme preparation were conducted at
12
1000, 2500 and 5000 µg/mL in the 4-hour test conditions (See Suppl. Data Table A23). Four-
13
hour exposure to the highest test concentration (5000 µg/mL) did not result in large changes
14
in mitotic index compared with the vehicle control. In the 22-hour test condition, cytogenetic
15
evaluations were conducted at 25, 50, and 100 µg/mL. The mitotic index for the highest test
16
concentration evaluated microscopically for chromosome aberrations (100 µg/mL), was
17
3.7%, compared with 10.3% for the vehicle control, representing a 64.1% mitotic inhibition
18
in relation to the vehicle control. Under all 3 conditions tested, the percentage of cells with
19
numerical or structural aberrations in the test substance-treated groups was not significantly
20
increased above that of the vehicle control at any concentration (p ≥ 0.05, Fisher’s exact test).
21
The percentage of cells with structurally damaged chromosomes in the respective positive
22
control treatment groups were statistically significant (p < 0.05, Fisher’s exact test).
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Under the conditions of these studies, neither the C16F α-amylase UFC nor the WB enzyme
2
preparation test material were found to induce structural or numerical chromosome aberrations in
3
the in vitro mammalian chromosome aberration test in human peripheral blood lymphocytes in
4
either the non-activated or S-9 mix -activated test systems. The percentages of aberrant cells in
5
the test substance-treated groups were within the historical solvent control range and all criteria
6
for valid assays were met, based on the results for positive and negative controls. It was
7
concluded that both the WB and the UFC test substance were neither mutagenic nor clastogenic
8
in this in vitro mammalian test.
9
3.3 Sequence-based assessment of food allergenicity potential and similarity to toxin proteins
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The search for 80-amino acid stretches with greater than 35% identity to known allergens using
11
the FARRP AllergenOnline database revealed no matches to known food allergens. The UniProt
12
database search showed that the vast majority of sequences were α-amylases, with none of the
13
top 1,000 database hits annotated as either toxin or venom. Based on the sequence homology
14
alone, the C16F variant α-amylase is unlikely a food allergen or oral toxin.
15
4. Discussion
16
4.1 Safety of the production organism
17
Bacillus licheniformis is a proven safe production host for industrial enzymes (e.g., Olempska-
18
Beer et al., 2006; EPA, 1997). It is used as the production organism for several food enzymes
19
that were affirmed GRAS by FDA and for additional food enzymes that were notified to FDA as
20
GRAS (see GRN 22, 24, 72, 79, 265, 277, 472, 560, 564, 572, 587, and 594). Additionally, it is
21
listed in the Official Publication by the American Association of Feed Control Officials
22
(AAFCO, 2018) as a suitable species for the production of numerous enzymes for use in animal
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feed. Moreover, DuPont has previously established a Safe Strain Lineage (SSL) for this species
2
as also discussed in GRAS Notice 664) (see Figure 1) based on repeated evaluation in the Pariza
3
and Johnson (2001) decision tree including toxicological testing. According to the principles of
4
this decision tree, α-amylase (a common food enzyme with a long history of safe use) produced
5
in a production strain pertaining to a Safe Strain Lineage would be acceptable for use in food.
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Figure 1. Bacillus licheniformis BRA7 Safe Strain Lineage (SSL) diagram, showing the position
15
of the production organism of C16F amylase in the lineage (in red) in relation to the other strains
16
in the SSL. The blue colored boxes indicate strains for which toxicology tests were conducted.
17
Most enzymes derived from this SSL were determined to be Generally Recognized as Safe
18
(GRAS), with GRAS Notices submitted for review by the U.S. FDA for enzymes from strains
19
designated with gray horizontal banners indicating the GRN (= GRAS Notice) number, inlcuding
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GRN 664 for C16F amylase. The safety of the C16F α-amylase strain is further supported by its
2
own toxicology testing.
3
The α-amylase enzyme in question is derived from Cytophaga sp., which provides one rationale
4
for further verification of its safety. Most enzymes are sourced from organisms with a history of
5
safe use, which is the case here for the expression host. B. licheniformis. The ‘gene donor’
6
Cytophaga sp. is a Biosafety Level 2 organism, as certain species belonging to this genus are
7
pathogenic to fish. Intuitively one might be wary of expressing DNA from a pathogenic donor
8
organism into an expression host. However, one can safely produce any given common enzyme
9
sequence from a pathogen in a safe host, as long as the expressed enzyme in question is not a
10
virulence factor. The transfer of any potential donor DNA of concern (that is not the target DNA
11
encoding for the otherwise safe enzyme) can be entirely avoided by using synthetic DNA
12
encompassing only the amylase coding sequence.
13
4.2 Safe Strain Lineage- Use of B. licheniformis and Cytophaga – Pariza Decision Tree
14
The safety of the C16F α-amylase in human and animal food has also been established using the
15
decision tree for food processing enzymes as outlined in Pariza and Johnson (2001) and updated
16
for animal feed enzymes by Pariza and Cook (2010), which both address various questions
17
related to enzymes production in organisms developed with rDNA technology. One key question
18
is whether the expressed enzyme in question has a history of safe use in food or feed. Indeed, α-
19
amylase has been used for decades in food processing and in animal feed. Although the
20
Cytophaga sp. α-amylase (C16F) is new as an isolate in commerce, the variant α-amylase
21
expressed in Bacillus licheniformis is still an α-amylase with the designation IUBMB 3.2.1.1.
22
Given the high sequence similarities of CF16 α-amylase to α-amylase molecules from various
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sources (e.g., 81% identity with α-amylase from Bacillus sp. 406 and 75% identity with α-
2
amylase from Bacillus amyloliquefaciens), C16F α-amylase is considered substantially
3
equivalent to these α-amylases with extensive history of safe use. GRAS Notice 664 included a
4
sequence-based similarity assessment of the C16F sequence with known food allergens (Codex,
5
2009; Ladics et al., 2011) and toxins (Magrane et al., 2011), and the safety of C16F α-amylase is
6
further supported by its lack of sequence similarity with any known food allergens or
7
proteinaceous toxins.
8
The rDNA methods used to engineer the production strain included the use of plasmids devoid of
9
mobilization and transfer elements, minimizing the potential presence of transferable antibiotic
10
resistance DNA. In addition, chloramphenicol is not widely used as antibiotic in humans or
11
animals, so its clinical relevance is minimal. With regard to further questions in the decision tree:
12
all introduced DNA was fully sequenced, examined and found to be free of attributes that would
13
render it unsafe for constructing microorganisms to be used to produce food-grade products. The
14
introduced DNA is integrated at the cat locus into chromosome of B. licheniformis. Moreover,
15
the B. licheniformis Bra 7 safe strain lineage is well-established; its safety as a production host
16
and methods of modification are well documented and their safety have been confirmed through
17
repeated toxicology testing (see GRN 664).
18
In short, α-amylase has a history of safe use in food, the sequence of C16F amylase was verified
19
not to display any similarity with known food allergens or toxins, and C16F amylase is safely
20
produced by a member strain of a well-established safe strain lineage for which all essential
21
strain engineering attributes are confirmed, all of which satisfy the requirements in safety
22
evaluation of the enzyme preparation without triggering the need for new toxicology data.
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Finally, although the Pariza and Johnson evaluation resulted in the conclusion to accept the
2
enzyme preparation as “safe without new toxicology testing”, the safety of C16F enzyme
3
preparation was further confirmed through toxicological testing as described above. The
4
toxicology testing was conducted 1) to underpin the substantial equivalence between clarified
5
and whole-broth enzyme α-amylase preparation and 2) to use the results to support regulatory
6
approvals in jurisdictions where toxicology testing is an absolute requirement for food/feed
7
enzyme approval.
8
Not only is B. licheniformis a safe production organism for enzymes, it is also listed as a suitable
9
species to serve as a probiotic Direct Fed Microbial to livestock and poultry (AAFCO, 2018).
10
Hence, given that live B. licheniformis cells are safe for use as probiotics, it is expected that a
11
whole-broth enzyme preparation produced by the same safe organism would be as safe as the
12
clarified (semi-purified) equivalent. However, the lack of comparative data triggered us to
13
include this as a second angle of interest in this study.
14
The whole-broth enzyme format containing solids is not suitable for all food applications, but
15
may be suitable for food applications that include a refinement step such as distillation and/or
16
filtration after the enzymatic action has been completed, such as the manufacture of potable
17
alcohol or organic acids. Whole broth cellulase is used in the manufacture of cellulosic fuel
18
ethanol; similarly, whole-broth α-amylase may be applied in grain fuel ethanol processes with
19
the resulting co-products fed to animals. To the authors’ knowledge, however, the current report
20
provides the first comparative toxicological studies for the two formats (whole-broth and
21
clarified) of an enzyme preparation for use in the processing of human and animal food.
22
The safety of the resulting enzyme preparations was confirmed in the reported genotoxicity and
23
sub-chronic oral toxicity studies. Given the lack of biological and statistical differences between
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the control and treated groups for any of the clinical, systemic or genotoxic parameters
2
measured, the highest dose in two 90-day oral gavage toxicity studies (500 mg TOS/kg bw/day)
3
can serve as the NOAEL for both WB and UFC enzyme preparations.
4
4.3 Uses and Applications
5
The amylase is intended for use in carbohydrate processing, including the manufacture of high
6
fructose corn syrup and fermentation to produce organic acids i.e. lysine, citric and lactic acid,
7
and in the manufacture of potable alcohol and fuel ethanol with resulting co-products (distiller’s
8
grains and corn gluten feed/meal) destined for use in animal feed.
9
In a recent GRAS Notice to FDA’s Center for Food Safety and Applied Nutrition, the human
10
food applications the same α-amylase enzyme were notified as GRAS (GRN 664). The notice to
11
FDA focused on the use of the clarified amylase preparation for human food applications,
12
including the manufacture of sugar syrups, potable alcohol and other biochemicals for use in
13
food (organic acids, MSG). In this paper we further discuss the applications related to the
14
manufacture of animal feed. Additionally, FDA affirmed the GRAS status of mixed
15
carbohydrase/protease enzyme preparation derived from B. licheniformis and α-amylase from B.
16
amyloliquefaciens for use in food with GMP as the only limitation (21CFR 184.1027 and
17
184.1148, respectively). Furthermore, α-amylases from several genetically modified B.
18
licheniformis strains were GRAS notified to FDA, including hybrid B. licheniformis / B.
19
amyloliquefaciens α-amylase (GRN 22), modified B. licheniformis and B. amyloliquefaciens α-
20
amylase (GRN 79), and the agency issued “no questions” letters in response.
21
4.4 Human Exposure to C16F amylase & Margin of Safety
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The safety of Cytophaga sp. variant α-amylase C16F expressed in B. licheniformis as a
2
processing aid in carbohydrate processing and potable alcohol production at the maximum
3
recommended application rates is supported by existing toxicology data. Using the Budget
4
method (Douglass et al., 1997), the determined maximum daily intake for UFC is 0.248 mg
5
TOS/kg bw/day and for WB is 1.01 mg TOS/kg bw/day, which include solid and liquid food
6
application (organic acids, sugar syrups and specialty starches) exposures. The margin of safety
7
for human food applications was calculated as, respectively, 2016 to 495 for UFC and WB,
8
based on a NOAEL of 500 mg TOS/kg bw/day for each, with both exceeding the commonly
9
acceptable margin of safety of 100. Based on a margin of safety several-fold greater than 100
10
even in the worst-case scenarios, the uses of α-amylase as a processing aid in carbohydrate
11
processing and production of organic acids and potable alcohol are not of human health concern,
12
regardless of the product format.
13
4.5 Animal Exposure to C16F α-amylase
14
The C16F α-amylase enzyme preparation (WB and UFC formats) will be used in the dry-grind
15
ethanol (potable or fuel alcohol) with resulting distillers’ grains (DG) fed to animals, as well as
16
in the production of organic acids (e.g., lactic acid), and amino acids (e.g., lysine) for use in
17
animal feed. Additionally, wet milling (WM) co-products such as corn gluten feed and corn
18
gluten meal will also be part of the animal feed application. To calculate the worst-case exposure
19
estimate for animals consuming the DGs and or WM co-products, the following factors were
20
considered: a) 100% conversion efficiency: every kg of dry grain will be converted to
21
approximately equal portions of ethanol, carbon dioxide and DGs; b) 100% transfer: all 100% of
22
the enzyme will be transferred to the DG resulting in a theoretical 3-fold concentration; c) the
23
maximum inclusion rates of DG or WM co-products in animal feed rations will be used, i.e.,
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60% in cattle, 50% in pigs, and 30% in poultry, DM (dry matter) basis and d) the total amount of
2
DG and WM- co-products, when fed together, will not exceed the above stated maximum
3
inclusion rates for DG alone.
4
The maximum use rate in ethanol production trials to-date (6.2 mg total enzyme protein / kg
5
DM) equates to 11 mg TOS/kg DM for the UFC preparation and 42 mg TOS/kg DM for the WB
6
preparation. This results in 33 mg UFC TOS/kg DM or 127 mg WB TOS/kg DM in DG or WM
7
co-products. The worst-case exposure to production animals cattle, pigs and poultry) via their
8
feed is outlined in Tables 5 and 6. Using the NOAEL derived from the 90-day oral toxicity test
9
with either preparation of C16F α-amylase (500 mg TOS/kg bw/day), it was calculated that the
10
exposure to animals via DG or WM co-products is of no safety concern as the margin of safety is
11
far greater than 100.
12
Table 5. Margin of Safety Calculation for C16F α-amylase UFC via DG*
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Pigs
Poultry
Maximum enzyme use rate 11.03 (mg TOS/kg DM) Fold-concentration in DG, DM basis 3 (process dependent) Maximum Enzyme TOS concentration 33.09 in DG (mg/kg DM) Maximum Inclusion rate of DG in 0.6 complete animal ration Maximum Enzyme TOS concentration 19.85 in feed (mg/kg DM) Maximum Daily feed intake 0.03 (kg DM/kg BW) Maximum intake of enzyme TOS 0.59 (mg/kg BW) NOAEL 500 (mg TOS/kg BW/day) Margin of safety 847 *same calculations apply to WM-coproducts
11.03
11.03
3
3
33.09
33.09
0.5
0.3
16.54
9.93
0.045
0.065
0.74
0.645
500
500
676
775
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Table 6. Margin of Safety Calculation for C16F α-amylase WB via DG* Poultry
42.4
42.4
42.4
3
3
3
127
127
127
0.6
0.5
0.3
76
64
38
0.03
0.045
0.065
2.3
2.8
2.5
500 219
500 175
500 202
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Maximum enzyme use rate (mg TOS/ kg DM) Fold-concentration in DG, DM basis (process dependent) Maximum Enzyme TOS concentration in DG (mg/kg DM) Maximum Inclusion rate of DG in complete animal ration Maximum Enzyme TOS concentration in feed (mg/kg DM) Maximum Daily feed intake (kg DM/kg BW) Maximum intake of enzyme TOS (mg/kg BW) NOAEL (mg TOS/kg bw/day) Margin of safety *same calculations apply to WM-coproducts
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4.6 C16F α-amylase in the production of lysine for use in animal feed
5
The application of C16F α-amylase in the production of lysine is 6.2 mg total protein/kg starch.
6
Since the yield ratio of dry starch to lysine is 1.2 to 1, then the calculated maximum C16F α-
7
amylase carried over in 1 kg of lysine (assuming no removal) is 7.44 mg. Requirements for
8
lysine, often the first limiting amino acid in animal nutrition, for growing pigs and chickens
9
varies by age as well as dietary energy level. For starting, growing and finishing pigs the range is
10
0.6-1.4% of the diet (NRC, 1998) and for broiler chickens this varies from 0.85 to 1.1% of the
11
diet (90% diet DM basis; NRC, 1994), with the average being around 1%.
12
Although the lysine requirement can often be met by ration formulation with corn and soybean
13
meal, supplementation with crystalline lysine allows for a reduction in total protein content of
14
the diet (usually from soybean meal), and is a cost-effective means to reduce nitrogen excretion.
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The average supplement dosage of lysine is 2 kg of lysine per metric ton of feed for
2
monogastrics. For dairy cattle a benefit of supplementing with lysine up to 0.5% of the diet has
3
been reported (e.g., Wang et al., 2010). Based on the application of C16F α-amylase in the
4
production of lysine, it is calculated that for each animal species the maximum intake of enzyme
5
protein is 0.001 mg/kg BW for UFC and 0.004 mg/kg BW for WB, which is negligible compared
6
with the enzyme use in DG production, thus the margins of safety calculated for DG also cover
7
use of C16F α-amylase in fermentation to manufacture lysine and potentially several other amino
8
acids or organic acids, typically used at 0.5% or less in the animal feed, similar to the lysine
9
example.
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5. Conclusions
11
Results from a battery of toxicology studies investigating the genotoxic and systemic toxicity
12
potential of two C16F α-amylase preparations, demonstrated that regardless of the product form
13
(UFC or WB); the C16F amylase preparation is not a mutagen or clastogen and it doesn’t induce
14
systemic toxicity when administered by gavage to rats for 90 continuous days. The consistent
15
toxicology results between the two enzyme preparations allowed for establishment of the same
16
NOAEL for both products forms at 500 mg TOS/kg bw.
17
Our inclusion of the WB enzyme preparation in the toxicological studies in parallel to the classic
18
food-grade UFC enzyme preparation was to rule out any safety concerns regarding whole-broth
19
enzyme preparations that contain cell debris and rDNA from the production organism. It is
20
widely known that whole-broth enzyme preparations are used in enzyme applications where the
21
finished food product is subjected to further processing that will remove the cell debris, such as it
22
is the case in the distillation process to produce potable alcohol. However, there is nothing
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inherently unsafe about using WB enzyme preparations (containing dell debris and rDNA) in
2
food, as there is nothing inherently unsafe about rDNA unless it encodes for a harmful substance
3
or would easily transfer resistance to an antibiotic of clinical relevance to humans or animals. α-
4
Amylase is not a harmful substance and the chloramphenicol resistance marker (endogenous to
5
B. licheniformis) has been used safely for decades; it is used only in initial strain selection and
6
does not reside on a plasmid that is easily mobilized or transferred. The safety of the resulting
7
enzyme preparation is further illustrated by the current toxicology results. Obviously, such
8
generalization is predicated upon both UFC and WB enzyme preparations being manufactured
9
using the same appropriate raw materials of suitable purity for the intended use per Good
10
Manufacturing Practices, i.e. minimum amounts needed to achieve desired effect (Sewalt et al.,
11
2016). In addition, once the manufacturing process is completed, an evaluation for microbial and
12
chemical contaminants is performed (per the Food Chemical Codex (US Pharmacopeia, 2016)
13
and FAO/WHO’s Joint Evaluation Committee for Food Additives (JECFA, 2006), and only then
14
is the final enzyme preparation product (WB or UFC) released. Furthermore, for both Quality
15
and Intellectual Property (IP) protection reasons, enzyme manufacturers further evaluate the final
16
enzyme product to make sure that they are free of the active production organism.
17
In addition to the food applications previously mentioned, the C16F α-amylase enzyme
18
preparation (WB and UFC) is also intended for use in fermentables e.g., potable alcohol with
19
resulting distillers’ grains (DG) and organic/amino acids for use in animal feed. Similarly, use in
20
wet milling (WM) results in WM co-products, corn gluten feed and corn gluten meal, both of
21
which are used in animal feed as well at inclusion rates no higher than DG. Using the NOAEL
22
derived from the 90-day oral toxicity test with C16F alpha amylase (either UFC or WB), it was
23
calculated that exposure to animals via DG or WM co-products is of no safety concern as the
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safety margin is greater than 100 (see Tables 3-4). The proposed uses of C16F α-amylase in
2
potable alcohol at the proposed application rate of 6.2 mg protein/kg starch for UFC and 27 mg
3
protein/kg starch for WB, respectively, are not a human health concern and are supported by
4
existing toxicological data. Finally, even under the worst-case scenario that C16F α-amylase is
5
applied at the maximum rate for both UFC and WB and the enzyme is not destroyed and/or
6
removed during processing, the use of C16F α-amylase in the manufacture of lysine to be used as
7
a feed additive is toxicologically insignificant.
8
We conclude C16F synthetic variant Cytophaga sp. α-amylase expressed in B. licheniformis in
9
either clarified or whole-broth format is safe in the manufacture of ethanol or wet milling co-
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products and amino acids, without health concern to animals consuming the co-products or
11
amino acids and to humans consuming the potable alcohol, as substantiated by existing
12
toxicology data.
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Acknowledgement
15
We thank Gregory Ladics and Kees Broekhuizen for their critical review of toxicology
16
conclusions and strain details, respectively. In addition, we thank Sanusha Bijj for her skillful
17
assistance in preparing the Supplemental Data Tables.
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References
4
Ames, B.N., McCann, J., and Yamasaki, E. 1975. Methods for detecting carcinogens and
RI PT
3
mutagens with the Salmonella/Mammalian Microsome Mutagenicity Test. Mut. Res. 31,
6
347-364
Association of American Feed Control Officials (AAFCO), 2018. 2018 AAFCO Official Publication, pp. 373-378.
8
9
M AN U
7
SC
5
Aunstrup, K. 1979. Production, isolation, and economics of extracellular enzymes, in: Wingard, L.B., Katchalski-Katzir, E. and Golsdstein, L (Eds.), Applied Biochemistry and
11
Bioengineering, Volume 2, Enzyme Technology. pp. 28-68.
12
TE D
10
Aunstrup, K., Andersen, O., Falch, E. A., and Nielsen, T. K. 1979. Production of Microbial Enzymes in Microbial Technology, 2nd ed., Eds. Peppler, H.J., and Perlman, D., Chapter
14
9, 1. pp. 282-309.
15
EP
13
BioReliance, 2014a. H-30928: Bacterial reverse mutation assay; Report No. AD84GN.507001.BTL.
17
AC C
16
BioReliance, 2014b. H-30929: Bacterial reverse mutation assay; Report No. AD84GP.507001.BTL.
18
19
Carson, J., Schmidtke, L.M., Munday, B.L., 1993. Cytophaga johnsonae: a putative skin pathogen of juvenile farmed barramundi, Lates calcarifer Bloch. J. Fish Dis. 16, 209-218.
20
41
ACCEPTED MANUSCRIPT
1
Codex Alimentarius Commission. 2009. Joint FAO/WHO Food Standards Programme, Codex Alimentarius Commission, Rome, Italy
2
Cooney, R.P., Pantos, O., Le Tissier, M.D.A., Barer, M.R., O’Donnell, A.G., Bythell, J.C., 2002.
RI PT
3 4
Characterization of the bacterial consortium associated with black band disease in coral
5
using molecular microbiological techniques. Environ. Microbiol. 7, 401-413.
Dalsgaard, I., 1993. Virulence mechanisms in Cytophaga psychrophila and other Cytophaga-like
SC
6
bacteria pathogenic for fish. Ann. Rev. Fish Dis. 3, 127-144.
7
De Boer, A.S., Diderichsen, B., 1994. On the industrial use of B. licheniformis: A Review., Appl.
M AN U
8
Microbiol. Biotechol. 40, 595-598.
9
Douglass, J.S., Barraj, L.M., Tennant, D.R., Long, W.R., and Chaisson, C.F., 1997. Evaluation
11
of the budget method for screening food additive intakes. Food Addit. Contam. 14, 791-
12
802.
13
TE D
10
DuPont Haskell Global Centers, 2014a. H-30928: Level 10 C16F Whole Broth: In vitro mammalian chromosome aberration test in human peripheral blood lymphocytes; Report
15
No. 20557-544.
EP
14
Dupont Haskell Global Centers, 2014b.H-30929: Level 10 C16F Clarified: In vitro mammalian
17
chromosome aberration test in human peripheral blood lymphocytes; Report No. 20558-
AC C
16
544.
18
19
Durborow, R.M., Thune, R., Hawke, J.P., Camus, C., 1998. Columnaris disease, a bacterial infection caused by Flavobacterium columnare. Southern Regional Aquaculture Center
20
42
ACCEPTED MANUSCRIPT
1
Publication No. 479, USA. (
2
content/uploads/479.pdf>). Environmental Protection Agency (EPA), 1997. Attachment I—Final Risk Assessment of
4
Bacillus licheniformis. (
5
09/documents/fra005.pdf>).
RI PT
3
Fisher, R.A. 1985. Statistical Methods for Research Workers, 13th edition. Haffner, New York.
7
Green, M.H.L. and Muriel, W.J. 1976. Mutagen testing using trp+ reversion in Escherichia coli.
Gryczan, T., Shivakumar, A.G., Dubnau, D., 1980. Characterization of chimeric plasmid cloning vehicles in Bacillus subtilis. J. of Bacteriol.. 141, 246-253.
10
Gupta, R., Gigras, P., Mohapatra, H., Goswami, V.K., Chauhan, B., 2003. Microbial α-amylase
TE D
11
s: a biotechnological perspective. Proc. Biochem. 38, 1599-1616.
12
13
Guzman-Maldonado, H., Paredes-Lopez, O., 1995. Amylolytic enzymes and products derived from starch: a review. Crit. Rev. Food Sci. Nutr. 35, 373–403.
EP
14
International Food Biotechnology Council (IFBC), 1990. Safety evaluation of foods and food
AC C
15
ingredients derived from microorganisms. In: Biotechnologies and Food: Assuring the
16
Safety of Foods Produced by Genetic Modification. Regul. Toxicol. Pharmacol. 12,
17
S114-S128.
18
19
M AN U
Mut. Res. 38, 3-32.
8
9
SC
6
Janeček, S., 1994. Sequence similarities and evolutionary relationships of microbial, plant and animal α-amylases. Eur. J. Biochem. 2, 519-24.
20
43
ACCEPTED MANUSCRIPT
1
Janeček, S., 1997. α-amylase family: molecular biology and evolution. Prog. Biophys. Mol. Biol. 67, 67-97.
2
JECFA, 2006. General specifications and considerations for enzyme preparations used in food
RI PT
3
processing, in: Compendium of food additive specifications, volume 4. Analytical
5
methods, test procedures and laboratory solutions used by and referenced in the food
6
additive specifications. FAO/WHO JECFA Monographs 1. Rome, pp. xxi-xxv.
7
Jeang, C.L., Y.H. Lee and L.W. Chang., 1995. Purification and characterization of a raw-starch
SC
4
digesting amylase from a soil bacterium Cytophaga sp. Biochem. Mol. Biol. Int. 35, 549-
9
551.
M AN U
8
Jeang, C.L., Chen, L.S., Chen, M.Y. and Shiau, R.J.. 2002. Cloning of a gene encoding raw-
11
starch-digesting amylase from a Cytophaga sp. and its expression in Escherichia coli.
12
Appl. Environ. Microbiol. 68, 3651–3654.
13
TE D
10
Jonas, D.A., Antignac, E., Antoine, J.M., Classen, H.G., Huggett, A., Knudsen, I., Mahler, J., Ockhuizen, T., Smith, M., Teuber, M., Walker, R., de Vogel, P., 1996. The safety
15
assessment of novel foods. Guidelines prepared by ILSI Europe Novel Food Task Force.
16
Food Chem. Toxicol. 34, 931-940.
AC C
17
EP
14
Kirchman, D.L., 2002.The ecology of Cytophaga–Flavobacteria in aquatic environments. FEMS Microbiol. Ecol. 39, 91-100.
18
19
Kroschwitz, J.I., 1994. Enzyme Applications in Encyclopedia of Chemical Technology, 4th Edition. 9, 567-620.
20
44
ACCEPTED MANUSCRIPT
1
Ladics, G.S., R.F. Cressman, C. Herouet-Guicheney, R.A. Herman, L. Privalle, P. Song, J.M. Ward, S. McLain., 2011. Bioinformatics and the allergy assessment of agricultural
3
biotechnology products: Industry practices and recommendations. Regul. Toxicol.
4
Pharmacol. 60, 46-53.
RI PT
2
Lee, S., Mouri, Y., Minoda, M., Oneda, H., Inouye, K., 2006. Comparison of the wild-type α-
6
amylase and its variant enzymes in Bacillus amyloliquefaciens in activity and thermal
7
stability, and insights into engineering the thermal stability of Bacillus α-amylase. J.
8
Biochem. 139, 1007-1015.
M AN U
9
SC
5
Li, Y., Fang, Z. Dai, J., Partridge, G., Ruc, Y., Penga, J., 2010. Corn extrusion and enzyme
10
addition improves digestibility of corn/soy based diets by pigs: In vitro and in vivo
11
studies. Anim. Feed Sci. Technol. 158: 146–154.
Mayberger, J.M., 2011. Studies of genera Cytophaga-Flavobacterium in context of the soil
TE D
12
carbon cycle. Diss. Abstr. Int. 73, 1- 115.
13
14
Magrane, M. and the UniProt consortium., 2011. UniProt Knowledgebase: a hub of integrated protein data. Database, 2011: bar009 2011). (
16
MPI Research, 2014a. H-30928: Level 10 C16F Whole Broth: Sub-chronic toxicity 90 day oral
AC C
EP
15
gavage study in rats; Report No. 125-181.
17
18
MPI Research, 2014b. H-30929: Subchronic toxicity 90 day oral gavage study in rats; Report No. 125-180..
19
20
NRC (National Research Council), 1994. Nutrient Requirements of Poultry. 9th Revised Edition, National Academy Press
21 45
ACCEPTED MANUSCRIPT
1
NRC, 1998. Nutrient Requirements of Swine. 10th Revised Edition, National Academy Press.
2
OECD, 1998. Test No. 408: Repeated Dose 90-Day Oral Toxicity Study in Rodents. In: OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.
4
OECD, 1997a. Test No. 471: Bacterial Reverse Mutation Test. In: OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.
5
RI PT
3
OECD, 1997b. Test No. 473: In vitro Mammalian Chromosome Aberration Test. In: OECD
7
Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.
M AN U
SC
6
8
Olempska-Beer, Z.S.; Merker, R.I.; Ditto, M.D.; DiNovi, M.J., 2006. Food-processing enzymes
9
from recombinant microorganisms—A review. Regul. Toxicol. Pharmacol. 45,144-158. Pandey, A., Poonam, N., Soccol, C.R., Soccol, V. T., Singh, D., Radjiskumar, M., 2000. Advances in microbial amylases. Biotech. Appl. Biochem. 31, 135-152.
11
12
TE D
10
Pariza, M.W. and Johnson, E.A., 2001. Evaluating the safety of microbial enzyme preparations used in food processing: update for a new century. Regul. Toxicol. Pharmacol.. 33, 173–
14
186.
Pariza, M.W. and Foster, E.M., 1983. Determining the Safety of Enzymes Used in Food
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15
Processing. J. Food Prot. 46, 453-468.
16
17
Pariza, M.W and Cook, M., 2010. Determining the Safety of Enzymes used in Animal Feed. Regul. Toxicol. Pharmacol. 3, 332-42.
18
19
EP
13
Romero, L.F., Parsons, C.M., Utterback, P.L., Plumstead, P.W., Ravindran, V., 2013. Comparative effects of dietary carbohydrases without or with protease on the ileal
20
46
ACCEPTED MANUSCRIPT
1
digestibility of energy and amino acids and AMEn in young broilers. Anim. Feed Sci.
2
Technol. 181: 35–44.
Southern Regional Aquaculture Center Publication 474, Mississippi State Univ. MS.
4
5
Snedecor, G.W. and Cochran, W.G. 1967. Statistical Methods, 6th edition, pp. 246-248 and 349352. The Iowa State University Press, Ames, Iowa.
SC
6
7
Scientific Committee for Food (SCF)., 1992. Guidelines for the Presentation of Data on Food Enzymes, 27th series, Ref. No. EUR 14181 EN, pp. 13-22.
8
9
RI PT
Rottman, R. W., R. Francis-Floyd, R. Durborow., 1992. The role of stress in fish disease.
M AN U
3
Sewalt, V., Shanahan, D., Gregg, L., La Marta, J., and Carrillo, R., 2016. The Generally Recognized as Safe (GRAS) Process for Industrial Microbial Enzymes. Ind. Biotechnol.
11
12(5): 295-302.
12
TE D
10
Sewalt, V., LaMarta, J., Shanahan, D., Gregg, L., Carrillo, R., 2017. Letter to the editor regarding “GRAS from the ground up: Review of the Interim Pilot Program for GRAS
14
notification” by Hanlon et al., 2017. Food Chem. Toxicol. 105:140-150.
15
EP
13
Soltani, M., Shanker, S., Munday, B.L., 1995. Chemotherapy of Cytophaga/Flexibacter-like bacteria (CFLB) infections in fish: studies validating clinical efficacies of selected
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16
antimicrobials. J. Fish Dis. 18, 555-565.
17
18
U.S. Pharmacopeia 2018: Enzyme Preparations. In: Food Chemicals Codex, 11th Edition. Washington, D.C., pp. 413-419.
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van der Kaaij, R.M., Janeček, M. J., van der Maarel, E.C., Dijkhuizen, L., 2007. Phylogenetic and biochemical characterization of a novel cluster of intracellular fungal a-amylase
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enzymes. Microbiol. 153, 4003–4015.
4
RI PT
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Wang, C., Liu, Y.M, Wang, Z.Q., Yang, J.X., Liu, Y.M., Wu, T., Yan, Ye, H.W. 2010. Effects of dietary supplementation of methionine and lysine on milk production and nitrogen
6
utilization in dairy cows. J. Dairy Sci. 93, 3661-3670.
7
SC
5
Yuuki, T., Nomura, T., Tezuka, H., Tsuboi, A., Yamagata, H.,Tsukagoshi, N. & Udaka, S., 1985. Complete nucleotide sequence of a gene coding for heat- and pH-stable α-amylase of
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Bacillus licheniformis: comparison of the amino acid sequences of three bacterial
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liquefying α-amylase s deduced from the DNA sequences. J. Biochem. 98, 1147–1156.
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An α-amylase gene from a pathogen was safely expressed in Bacillus licheniformis. Oral- and genotoxicity studies for the α-amylase enzyme showed no adverse effects. Enzyme safety was equivalent in absence/presence of the inactive production strain. The studies support decision tree analysis for enzymes produced with biotechnology. The α-amylase is safe as processing aid in production of both human and animal food.
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