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Effect of seafood peptones on biomass and metabolic activity by Enterococcus faecalis DM19
Accepted Manuscript Effect of seafood peptones on biomass and metabolic activity by Enterococcus faecalis DM19 Mustapha Djellouli, Oscar Martínez-Álvarez, Mirari Y. Arancibia, Diego FlorezCuadrado, María Ugarte-Ruíz, Lucas Domínguez, Halima Zadi-Karam, Noureddine Karam, Salima Roudj, M. Elvira López-Caballero PII:
S0023-6438(17)30173-1
DOI:
10.1016/j.lwt.2017.03.028
Reference:
YFSTL 6101
To appear in:
LWT - Food Science and Technology
Received Date: 12 December 2016 Revised Date:
23 February 2017
Accepted Date: 14 March 2017
Please cite this article as: Djellouli, M., Martínez-Álvarez, O., Arancibia, M.Y., Florez-Cuadrado, D., Ugarte-Ruíz, M., Domínguez, L., Zadi-Karam, H., Karam, N., Roudj, S., López-Caballero, M.E., Effect of seafood peptones on biomass and metabolic activity by Enterococcus faecalis DM19, LWT - Food Science and Technology (2017), doi: 10.1016/j.lwt.2017.03.028. 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.
ACCEPTED MANUSCRIPT Effect of seafood peptones on biomass and metabolic activity by
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Enterococcus faecalis DM19
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Mustapha Djelloulia,b, Oscar Martínez-Álvarezc, Mirari Y. Arancibiac,d, Diego
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Florez-Cuadradoe, María Ugarte-Ruíze, Lucas Domíngueze, Halima Zadi-
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Karama, Noureddine Karama, Salima Roudja and M. Elvira López-Caballeroc*
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a
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1
Laboratory for Biology of Microorganisms and Biotechnology, Department of
Biotechnology, Faculty of Sciences of Nature and Life, University of Oran 1
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Ahmed Benbella, BP 1524 El-M'Naouer, 31000 Oran, Algeria.
Biotechnology Research Center, Department of Food Biotechnology, Ali
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b
SC
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Mendjeli, Nouvelle Ville, UV 03 BP E73, 25000 Constantine, Algeria
11 12 13 14
Institute of Food Science, Technology and Nutrition (ICTAN-CSIC)**, C/José
Antonio Novais, 28040 Madrid, Spain d
Technical University of Ambato (UTA), Av. Los Chasquis y Río Payamino,
Ambato, Ecuador e
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c
VISAVET Health Surveillance Centre, Complutense University of Madrid,
EP
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Avda. Puerta de Hierro, s/n. 28040 Madrid, Spain. to
whom
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*Author
correspondence
should
be
addressed
[e-mail
[email protected]] 15
**This centre has implemented and maintains a Quality Management System
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which fulfils the requirements of the ISO standard 9001:2008
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ACCEPTED MANUSCRIPT Abbreviations:
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SQES : Squid protein hydrolyzed with Esperase
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SQAP : Squid protein hydrolyzed with Alkaline protease
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SQTR : Squid protein hydrolyzed with Trypsin
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SHAP : Shrimp protein hydrolyzed with Alkaline protease
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SHNP : Shrimp protein hydrolyzed with Neutral protease
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SHEE : Shrimp protein hydrolyzed with endogenous enzymes
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FGTR : Fish gelatin hydrolyzed with Trypsin
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SHPR : Shrimp protein hydrolyzed with Protamex
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ACCEPTED MANUSCRIPT Abstract
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Eight seafood protein hydrolysates (SPHs) obtained from squid, shrimp and fish
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gelatin were incorporated as substitutes of peptones in culture media in order to
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evaluate its effect on survival and metabolic activity (lactic acid, acetic acid and
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bacteriocins production) of Enterococcus faecalis DM19. The substitution of
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commercial peptones in culture media by either a shrimp hydrolysate prepared
33
with Protamex, or by squid protein hydrolysates prepared with Esperase or
34
Alkaline protease, stimulated E. faecalis DM19 growth up to 16%. The
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incorporation of SPHs, mainly from shrimp, in the culture media significantly
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increased production of lactic and acetic acids in more than 60%. Furthermore,
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the media containing SPHs stimulated antimicrobial activity by E. faecalis
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DM19. The inhibitory activity was observed against both Gram-positive and
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Gram-negative microorganisms, but it was remarkably observed against Listeria
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monocytogenes. SPHs incorporated in culture media render properties of bio-
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technological interest, which, together with their low price, make them suitable
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for industrial use.
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Keywords: Enterococcus faecalis DM19, seafood protein hydrolysates,
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bacteriocinogenic activity, antimicrobial activity, renewable materials
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ACCEPTED MANUSCRIPT 1. Introduction
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The genus Enterococcus comprises species considered components of the
49
human and animal intestinal flora (Khan, Flint, & Yu, 2010) and is generally
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found in large numbers in dairy products and fermented foods. Lactic and acetic
51
acid, diacetyl, and bacteriocins are some of the molecules produced by these
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microorganisms that present antimicrobial activity (Gilmore, Lebreton, & van
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Schaik, 2013). Moreover, several E. faecalis strains have been investigated as
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potential probiotics (Franz, Huch, Abriouel, Holzapfel, Gálvez, 2011). Therefore,
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from the viewpoint of their industrial importance, E. faecalis could play a
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significant role in fermentation and preservation of some foods (Khan, Flint, &
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Yu, 2010).
58
Like other lactic acid bacteria (LAB) groups, enterococci can only grow in a rich
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microbial growth medium which contains all the necessary compounds such as
60
amino acids, peptides, fatty acids, vitamins and nucleic acids required for
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optimal growth and metabolite production. These compounds are usually
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provided in the form of complex nitrogen sources such as yeast extract, meat
63
extract, tryptone, peptone or bactopeptone. Those nitrogen sources can reach
64
high prices and be unsuitable for industrial scale production of microorganisms
65
(Zhang & Gresham, 1999). Thus, there is great interest in investigating cheap
66
renewable nitrogenous materials to prepare a low-cost fermentation medium.
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Several authors (Aspmo, Horn, & Eijsink, 2005; Hujanen & Linko, 1996; Li, Han,
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Ji, Wang, & Tan, 2010; Yu, Lei, Ren, Pei, & Feng, 2008) studied various
69
nitrogen sources (yeast extract, malt sprout, peptones, grass extract, corn steep
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liquor, casein hydrolysates, distiller’s waste, ammonium phosphates, wheat
71
bran, fish waste hydrolysates and urea) for lactic acid fermentation. The use of
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studied (Deraz, El-Fawal, Abd-Ellatif, & Khalil, 2011). Nevertheless, to our best
74
knowledge, there is no information about the stimulation of lactic acid and/or
75
production of bacteriocin by E. faecalis in MRS media formulated with seafood
76
(crustacean and cephalopods) protein hydrolysates.
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The aim of the present work was to evaluate the effect of different seafood
78
protein hydrolysates as a peptone source on biomass production of E. faecalis
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DM19. The production of organic acids (lactic, acetic and formic acids) and
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bacteriocins in these culture media were also studied.
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2. Material and methods
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2.1.
83
The E. faecalis DM19 was obtained from the Laboratory for Biology of
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Microorganisms and Biotechnology (University of Oran 1 Ahmed Benbella,
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Algeria). The strain was originally isolated from raw camel milk collected in the
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Mauritania area and identified by PCR amplification using E. faecalis primers
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and 16S rDNA sequencing (Vincent, Roy, Mondou, & Dery, 1998). A search for
88
homology of the DNA sequence was performed using the BLAST algorithm
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available at the National Center for Biotechnology Information (NCBI, USA).
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2.2.
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Squid (Loligo vulgaris) muscle trimmings, by-products (muscle trimmings,
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carapace and heads) derived from processing Pacific white shrimp (Penaeus
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vannamei) and Southern pink shrimp (Penaeus notialis), and high molecular
94
weight (HMW) dried fish skin gelatine (type A) of cold water fish (Sebastes spp.)
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were used as raw material to prepare seafood protein hydrolysates. The by-
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Preparation of seafood protein hydrolysates
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enzymes (Table 1) which were kindly provided by Novozymes (Denmark).
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Enzymatic reactions were controlled by using a pH-stat. After hydrolysis, the
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samples were rapidly heated to 90°C for 15 min to inactivate the enzymes. The
100
hydrolysates were then centrifuged at 10000 x g for 20 minutes at 4ºC
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(Beckman J2-MC, Palo Alto, California, USA). The degree of hydrolysis for each
102
hydrolysate was calculated according to Spellman, McEvoy, O'Cuinn, and
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FitzGerald (2003). The supernatants were freeze-dried under vacuum and kept
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at -20°C until their use in the culture media formulation.
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2.3.
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Peptone in MRS (Man, Rogosa and Sharpe, Merck KGaA, Darmstadt,
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Germany) media was substituted by protein hydrolysates (marine peptones) at
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two different concentrations as depicted in Table 2 (MRS –standard culture
109
media- was considered as control and MRS- -without peptone- as negative
110
control). After adjusting the pH to 7, the media were autoclaved at 121°C for 20
111
min. Inoculum (1%, v/v) consisting of cellular suspension of an 18 h young
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culture of E. faecalis DM19 in MRS broth medium, was adjusted to an OD (600
113
nm) of 0.900 (UV-Visible spectrophotometer model UV-1601 from Shimadzu).
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Then, the broth was centrifuged at 12000 x g (Microspin 24S from Sorvall
115
instruments) for 10 min at 4°C and the sediment was washed twice with
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phosphate buffer (0.1M, pH 7.0) to remove all traces of the culture medium. The
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washed cells were re-suspended in sterile distilled water and then adjusted to
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the initial OD (600nm).They were then used to inoculate modified MRS medium.
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After 24 hours of incubation at 30°C, an aliquot of the medium was taken to
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determine bacterial growth. The rest of the medium was centrifuged at 5000 x g
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for 20 min at 4°C (Sorvall, model RT 6000B). The supernatant was used for the
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assessment of glucose, total nitrogen, organic acids (lactic acid, acetic acid,
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formic acid) and to determine the bacteriocinogenic activity.
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2.4.
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Cell growth was directly monitored by measuring the optical density of the
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culture broth samples at 600 nm using spectrophotometer (UV-Visible
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spectrophotometer model UV-1601 from Shimadzu). The evolution of pH in the
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culture broth was determined with a pH meter (inoLab, D-82362 Weilheim
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Germany).
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The total nitrogen content was determined using the Dumas combustion
131
method following AOAC 992.15 (1995) in a LECO model FP-2000 (Leco
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Instruments, Madrid, Spain) protein/nitrogen calibrated with EDTA. Protein
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content was estimated by multiplying the total nitrogen content by the factor
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6.25, and was expressed as grams of protein/100 grams of sample.
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Glucose and organic acid content (lactic, acetic, formic, pyruvic and citric acids)
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were determined by HPAEC-PAD (High Performance Anion Exchange
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Chromatography with Pulsed Amperometric Detection) using a Metrohm
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Advanced Compact ion chromatography instrument (867 IC. Metrohm)
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equipped with an IC-819 conductivity detector, an IC Pump 818 and an IC-837
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degasser coupled. Firstly, the supernatants (obtained as previously described)
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were diluted in ultrapure water and then filtered through a membrane of 0.45 µm
142
pore size. For glucose determination, 20 µL of samples were injected into a
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Hamilton RC X 30 (250 x 4 mm, 5 µm pore size) column. The samples were
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eluted from the column over 35 min at a flow of 1 mL/min with an isocratic
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gradient of 30 mM NaOH with 3 mM AcNa. For organic acids, the identification
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ACCEPTED MANUSCRIPT and quantification were carried out by injecting 20 µL of the samples into
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Metrosep Organic Acid (250 x 4 mm, 5 µm pore size) column and the elution
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was performed with 1 mM perchloric acid and acetone (10 %) as mobile phase
149
over 20 min at a flow of 0.5 mL/min. All the molecules studied were identified by
150
their retention times and quantified based on calibration curves derived from
151
standards. The results were expressed as mg/L of the culture supernatant. It is
152
evident that concerning the accumulation of acetic acid into the culture medium,
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the initially added sodium acetate concentration (=5.0 g/L) was taken into
154
consideration for the calculations performed. All assays were carried out in
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triplicate.
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2.5.
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Bacteriocinogenic activity and whole genome sequencing (WGS) of E. faecalis DM19
Culture supernatants from the different media tested were filtered through a
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cellulose acetate sterile filter (0.22 µm) to remove bacterial cells. The extracts
160
obtained were then lyophilized and stored at – 20°C. The samples were re-
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suspended in distilled water to a concentration of 5 mg/mL for the culture
162
extracts. Finally, the pH of the prepared solutions was adjusted to 7.0 with
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NaOH (3N).
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The antimicrobial activity of the supernatant was tested with the disc diffusion
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method (Arancibia, Giménez, López-Caballero, Gómez-Guillén, & Montero,
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2014). Briefly, sterile paper discs (5 mm diameter, Whatman No. 1) impregnated
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with 40 µL of the supernatant solutions were placed on the surface of agar
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plates inoculated with several microorganisms from the Spanish Type Culture
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Collection (CECT) selected due to their relevance in human health (either lactic
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acid bacteria or pathogens) or to their role in food spoilage: Aeromonas
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ACCEPTED MANUSCRIPT hydrophila CECT 839T, Aspergillus niger CECT 2088, Bacillus cereus CECT
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148, Bacillus coagulans CECT 56, Bifidobacterium bifidum DSMZ 20215,
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Brochothrix thermosphacta CECT 847, Citrobacter freundii CECT 401,
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Clostridium perfringens CECT 486, Debaryomyces hansenii CECT 11364,
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Enterococcus faecium DSM 20477, Escherichia coli CECT 515, Lactobacillus
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helveticus DSM 20075, Listeria monocytogenes CECT 4032, Penicillium
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expansum DSMZ 62841, Pseudomonas fluorescens CECT 4898, Salmonella
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choleraesuis CECT 4300, Shewanella putrefaciens CECT 5346T, Shigella
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sonnei
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parahaemolyticus CECT 511T, Yersinia enterocolitica CECT 4315. After
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incubation, the diameter of the inhibition zone (considered antimicrobial activity)
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was measured with Corel Draw X6 software. Results were expressed as mm of
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inhibited growth area (magnification ratio 1:10). Each determination was
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performed in triplicate.
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In order to evaluate which type of bacteriocin was present, the genome of E.
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faecalis DM19 was sequenced by whole-genome sequencing (WGS) method
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(Goodwin, McPherson, & McCombie, 2016). The DNA libraries were sequenced
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in the Illumina HiSeq platform, using 100bp paired-end sequencing reads. The
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raw sequence data of the sample was de novo assembled using algorithm
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SPAdes 3.9 (Bankevich et al., 2012). Bacteriocin genes were identified using
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the bacteriocin mining tool Bagel3 (Van Heel, de Jong, Montalbán-López, Kok,
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& Kuipers 2013). Each bacteriocin-like gene identified was checked using
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BLASTX algorithm.
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2.6.
Staphylococcus
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CECT
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Vibrio
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Statistical analysis
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ACCEPTED MANUSCRIPT Statistical tests were performed using the SPSS computer program (SPSS
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Statistical Software, Inc., Chicago, IL, USA). Two-way analysis of variance
197
(ANOVA) was carried out. The difference of means between pairs was resolved
198
by means of confidence intervals using a Tukey-b test at a significance level of
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5%.
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3. Results and discussion
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3.1.
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The substitution of commercial peptone in culture media by hydrolysates from
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squid (SQTR and SQAP) and prawn (SHPR), especially at two-fold
204
concentration, significantly increased the biomass (maxOD600) of E. faecalis
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DM19 (Table 3). In contrast, the growth in media where SHAP or FGTR (one or
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two-fold) were incorporated was significantly lower when compared with that of
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commercial MRS. In addition, the terminal pH in E. faecalis DM19 cultures was
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quite low as expected (Table 3). The data show an inverse correlation between
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biomass production and terminal pH.
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Some studies have been carried out to assess the ability of commercial
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proteolytic
212
microorganisms) to reduce fish protein (whole fish or wastes) into peptides and
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amino acids, which can be an economical source of peptones for bacterial
214
growth media. In fact, protein hydrolysates from seafood by-products can be
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possible substitutes for the usual protein hydrolysate-based substrates in
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microbial cultures (Gao, Hirata, Toorisaka, & Hano, 2006). In this work, seafood
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peptones showed an activity which was in the same range as the commercial
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peptone activity or scarcely higher in some cases, suggesting a well-balanced
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ACCEPTED MANUSCRIPT nutrient concentration of these SPHs media. Stein, Svein, and Vincent (2005a)
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reported that since peptones may differ in buffer capacity and the terminal pH
221
values reached in the Escherichia coli cultures were low, the observed growth
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differences were due to the pH/buffering effect. In the present work, E. faecalis
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showed a different biomass after fermentation in media including SPHs at a
224
similar pH (Table 3). Vázquez, González, and Murado (2004) found similar
225
results when testing the growth of Roseobacter sp on MRS supplemented with
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squid hydrolysate at different concentrations. These authors attributed this
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phenomenon to the presence of compounds (perhaps lipidic peroxides) with a
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slight inhibitory effect on the corresponding microorganism. Moreover, the
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peptidic composition of the SPH added to the MRS media may also affect
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bacterial growth (Table 3) since the results obtained from MRS media including
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hydrolysates derived from the same substrate (squid or shrimp) were different
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(P≤ 0.05). In this connection, Stein, Svein, and Vincent (2005b) cultivated LAB
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on MRS media supplemented with hydrolysates from cod viscera prepared with
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Alcalase, Papain and endogenous enzymes and found that peptone
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performance is highly dependent on composition details as peptide length,
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peptide sequences, and the amount of free amino acids. In the present work, E.
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faecalis DM19 exhibited a clear preference for MRS media including one-fold
238
concentration of squid protein hydrolysates prepared with Esperase or Alkaline
239
protease (SQES and SQAP, respectively), or shrimp protein hydrolysate
240
prepared with Protamex (SHPR). However, the incorporation of a shrimp protein
241
hydrolysate prepared with an alkaline protease (SHAP) in the media affected
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negatively bacterial growth. The amount of SPHs incorporated in MRS media
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also seems to be important in the biomass production (Table 3). The results
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and peptides suitable for the transport of bacteria and proteolytic systems
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(Kunji, Mierau, Hagting, Poolman, & Konings, 1996). Moreover, certain
247
hydrolysates as SHAP or FGTR may contain antimicrobial peptides or
248
compounds that negatively affect bacterial growth (Van’t Hof, Veerman,
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Helmerhorst & Amerongen, 2001).
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The production of organic acids by E. faecalis DM19 could also be affected by
251
the composition of the MRS media. If the culture medium stimulates the
252
production of organic acids by the bacteria, these acids could decrease the pH
253
of the media and regulate bacterial growth significantly. This fact could explain
254
why the media prepared with two-fold concentration of SHEE promoted a low
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production of biomass and a high production of acetic acid. Different
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investigations have been driven in this way to confirm the inhibitory effect of
257
acetic acid on several microorganisms. Recently, Boguta and Jensen (2014)
258
reported that, for Pediococcus acidilactici, 5.3 g/L acetate caused a 4% growth
259
inhibition and showed a synergistic effect when the strain was grown with both
260
acetate and furfural. Therefore, the inhibition phenomenon shown for SHEE
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was probably due to the high concentration of acetate produced by E. faecalis
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(Table 3).
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3.2.
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Effect of SPHs on production of organic acids and consumption of glucose and protein
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The amount of glucose and protein in the different media were measured before
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and after the fermentation process (Table 4). Glucose and protein consumptions
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for bacteria were higher in media with one-fold concentration of SPHs. The
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ACCEPTED MANUSCRIPT highest glucose consumption was found in media containing SQES. Glucose
269
consumption augmented when a higher amount of SPHs was added in the
270
media, while protein consumption decreased significantly. Protein consumption
271
was minimal in media including SPHs prepared with alkaline protease (SQAP
272
and SHAP).
273
Acid production depended on the media and on peptone concentration (Table
274
4). Lactic and acetic acids were the major products of the fermentation process
275
with E. faecalis DM19, while formic acid was produced in low quantities. The
276
incorporation of SPH in the media improved notably the production of lactic acid
277
in all samples, and that of acetic acid especially in SHPR-S and SHEE-S
278
samples. Interestingly, the highest production of lactic acid was achieved in
279
media with the mentioned hydrolysates.
280
As describe before, SHPR is the best supplement for efficient lactic acid
281
production in medium of 1f. Furthermore, the increment for SPH in the media
282
(2f) exerted a positive effect on lactic acid production (Table 4). The quantities
283
produced in SHAP-S and SHPR-S samples were 1.6 times larger compared to
284
those in MRS medium. These differences in production could be due to the
285
marine peptones used in fermentation, since the strain was cultivated under the
286
same experimental conditions. In this regard, lactate production increased in
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Streptococcus zooepidemicus when tryptone was replaced by marine peptones
288
(Vázquez, Montemayor, Fraguas, & Murado, 2009) while the increase in wheat
289
bran concentration promotes lactic acid production by Lactobacillus rhamnosus
290
LA-04-1 (Zheng, Lu, Yizhi, Xiaonan, & Tianwei, 2010). E. faecalis, like other
291
LAB, requires amino acids such as histidine, isoleucine, leucine, methionine,
292
glutamate, glycine, tryptophan, valine (Murray et al., 1993) to achieve optimal
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confirm that the different amino acid and peptide composition of marine
295
peptones could procure different efficiency in the amino acid and peptide uptake
296
systems (Kunji, Mierau, Hagting, Poolman, & Konings, 1996). The increase of
297
nitrogen provided by SPHs, significantly arises glucose uptake, and
298
consequently lactic acid production (Table 4), and hence its assimilation, as has
299
already been reported by Fakas, Papanikolaou,Komaitis, and Aggelis (2008) for
300
Cunninghamella echinulate.
301
3.3.
302
In order to investigate antagonistic profiles of the E.faecalis DM19 strain, cell-
303
free supernatant extracts were tested against 21 microbial strains (Table 5).
304
Differences in the diameter of inhibition halos were particularly notable up to
305
18.73±1.80 mm. Cell-free supernatants from E. faecalis DM19 grown in MRS
306
exhibited bactericidal activity against the majority of studied strains excluding
307
Aspergillus niger CECT 2088, Bacillus coagulans CECT 56, Penicillium
308
expansum DSMZ 62841 and Yersinia enterocolitica CECT 4315. However,
309
these microorganisms were sensitive to the cell-free supernatants from MRS
310
prepared by the hydrolysates (Table 5). Previous works had already reported
311
that E. faecalis produce a broad-spectrum of bacteriocins (enterocins), which
312
are active against Gram-positive and Gram-negative bacteria (Line, Svetoch,
313
Eruslanov, Perelygin et al., 2008; Pieniz, Andreazza, Anghinoni, Camargo, &
314
Brandelli, 2014). This fact could be reasonable since the pH of the culture
315
supernatants used in this study were neutralized to avoid the antimicrobial
316
effect of organic acids (lactate, acetate, etc.) and since anaerobic conditions
317
were imposed to decrease H2O2.
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319
of SPH did not lead to a relevant production of antimicrobial compounds in
320
terms of effectiveness and number of microorganisms inhibited. However, when
321
commercial peptone was substituted by two-fold concentration of SPHs, the
322
supernatants exhibited an interesting inhibitory activity against a large number
323
of indicator strains investigated (Table 5). The highest inhibitory activity was
324
observed mainly against Listeria monocytogenes CECT 4032, which was
325
significantly inhibited by SQES supernatant as compared with MRS (p≤0.05).
326
It has been shown that the composition of the growth medium, particularly the
327
source and concentration of nitrogen, strongly affects the production of
328
bacteriocin (Gänzle, Weber, & Hammes, 1999). As can be seen in Table 5, the
329
production of bacteriocin-like substances by E. faecalis production was
330
enhanced when SPHs concentration increased from 10 to 20 g/L. Accordingly,
331
the increasing concentrations of yeast extract, meat extract or peptone can
332
allow an increase in the production of bacteriocins. It can be possible that fish
333
peptones could have suitable contents, in terms of free amino acids and short
334
peptides, that are precursors for bacteriocin production (Kanmani et al., 2010),
335
whereas some authors have pointed out that bacteriocin production by lactic
336
acid bacteria is a growth-associated process (De Vuyst, Callewaert, & Crabbé,
337
1996).
338
3.4.
339
Once the genome of E. faecalis DM19 was assembled, the presence of
340
bacteriocin genes was tested using the BAGEL3 algorithm. Four sequences
341
related with bacteriocin-like genes were identified but only two showed proper
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Bacteriocin genes identified in the E. faecalis DM19 genome
15
ACCEPTED MANUSCRIPT length and amino acid identity higher than 99%: the enterolysin A and a
343
lantibiotic gene (Table 6). The annotation of the bacteriocins identified was
344
based on amino acid sequences homology (Supplementary data).
345
Enterolysin A is a heat-labile bacteriocin, firstly reported in E. faecalis LMG2333
346
and widely distributed among enterococci strains (Nilsen et al. 2003; Hickey et
347
al. 2003; Nigutova et al. 2007; Almeida et al. 2011). It belongs to the class III of
348
the bacteriocins and has a bacteriolytic mode of action over cell wall (Nilsen et
349
al, 2003). Our results are in line with the data already published about
350
enterolysin A in which it was described that inhibits growth of enterococci,
351
pediococci, lactococci and lactobacilli (Nilsen et al, 2003; Khan et al, 2013).
352
Furthermore, enterolysin A shows a wide spectrum of activity possibly due to
353
the cleavage of the peptide bond in the stem peptide and the interpeptide bridge
354
of the peptidoglycan units (Khan et al, 2013).
355
Besides, the other bacteriocin identified in E. faecalis DM19 is the one known
356
as lantibiotic because of the content of lanthionine and/or methyllanthionine in
357
its primary structure. These substances belong to class I bacteriocins and its
358
activity falls mainly on Gram-positive bacteria (Bierbaum et al, 2009).
359
Lantibiotics are heat-stable peptides and, like enterolysin A, also act on the cell
360
wall of other bacteria (Sahl et al, 1998).
361
Conclusion
362
Protein hydrolysates prepared mainly from squid and shrimp have sufficient
363
nutritional value to support growth and metabolite production of E. faecalis
364
DM19. The increase of nitrogen provided by these hydrolysates in culture media
365
increases glucose uptake, and consequently lactic acid production by this
AC C
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SC
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342
16
ACCEPTED MANUSCRIPT strain. Moreover, the increase of fish peptones enhanced the bacteriocin
367
producing of E. faecalis DM19, which was especially active against Listeria
368
monocytogenes. These strain properties, together with the low price of protein
369
hydrolysates, make them suitable for use in the fermented food industry.
370
Acknowledgements
371
This research was financed by the Spanish Ministry of Economy and
372
Competitiveness (projects AGL2011-27607, AGL2014-52825-R). Author M.
373
Djellouli is funded by The National Centre of Biotechnology Research (CNRBt)
374
of Algeria and ENP (Exceptional National Program) Scholarship provided by the
375
Ministry of Higher Education and Scientific Research of Algeria. Author M.
376
Arancibia is funded by a SENESCYT Scholarship provided by the Ecuadorian
377
government.
378
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ACCEPTED MANUSCRIPT Table 1: Proteolytic enzymes used to hydrolyze fish proteins.
Raw material used
Enzyme
Reaction conditions
pH
T (°C)
Time (min)
8
50
90
Squid (muscle)
Esperase
20.12
SQAP
Squid (muscle)
45.98
8
50
90
SQTR
Squid (muscle)
Alkaline protease Trypsin
21.50
8
37
90
SHAP
Alkaline protease Neutral protease Autohydrolyzed Trypsin
13.47
8
50
90
9.67
7.5
50
90
ND
7.5
40
360
FGTR
P. vannamei (tails, carapace and heads) P. vannamei (tails, carapace and heads) P. vannamei (heads and carapace) Fish gelatin
10.90
9
38
360
SHPR
P. notialis (muscle)
Protamex
6.68
7.5
50
90
SHEE
AC C
EP
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ND: Not determined
M AN U
SHNP
RI PT
SQES
SC
Sample designation
Degree of Hydrolysis (%)
ACCEPTED MANUSCRIPT Table 2: Composition of the culture media tested based on MRS (g/L) b
MRS
MRS
Yeast extract
4
4
4
4
Meat extract
8
8
8
8
Glucose
20
20
20
20
Sodium acetate
5
5
5
5
Tween 80 (mL)
1
1
1
1
Dipotassium hydrogen phosphate
2
2
2
2
Ammonium citrate
2
2
2
2
Mangnesium sulfate
0.2
0.2
0.2
0.2
Manganese sulfate
0.05
0.05
0.05
0.05
10
-
-
-
-
-
10
20
Peptone Seafood peptones
M AN U
MRS
EP
TE D
MRS: Man Rogosa and Sharpe (standard culture media). MRS : Man Rogosa and Sharpe without peptone. a MRS : MRS supplemented with one fold protein hydrolysates b MRS : MRS supplemented with two-fold protein hydrolysates
AC C
RI PT
a
MRS
SC
-
Composition
ACCEPTED MANUSCRIPT
Table 3 : Comparison of growth of E.faecalis DM19 and pH in the media with Seafood Protein Hydrolysates (SPHs). Seafood peptones
MRS
MRS
-
SQES
SQAP
SQTR
1f
4.62
4.55
4.56
4.56
4.57
2f
4.62
4.55
4.69
4.74
4.71
1f
1.58
1.65
1.64
1.64
1.63
2f
1.58
1.65
1.51
1.46
1.49
SHAP
c,A
1.65±0.01
c,A
1.65±0.01
1f
1.81±0.01
2f
1.81±0.01
g,A
2.01±0.02
de,A
1.83±0.02
M AN U
(Growth data) OD600
a,A
1.95±0.01
b,B
1.83±0.02
c,B
2.11±0.01
a,A
1.94±0.00
SHEE
FGTR
SHPR
4.58
4.62
4.57
4.60
4.56
4.66
4.80
4.81
4.79
4.79
1.62
1.58
1.63
1.60
1.64
1.54
1.4
1.39
1.41
1.41
SC
Final pH
∆pH
SHNP
RI PT
Parameters studied
c,B
1.74±0.01
b,A
1.67±0.01 -
e,A
1.81±0.01
c,A
1.80±0.01
e,B
1.82±0.01
d,A
c,A
0.64±0.01
f,B
f,A
2.02±0.01
de,A
1.73±0.01
1.69±0.02 1.70±0.01
a,A
AC C
EP
TE D
Legends for Seafood Protein Hydrolysates (SQES, SQAP, etc.) and composition of culture media (MRS and MRS ) are shown in Table 1 and 2, respectively. 1f: media supplemented with one-fold protein hydrolysates 2f: media supplemented with two-fold protein hydrolysates Different letters (a,b,c…) in the same line indicate significant differences among the different samples at the same concentration (1f or 2f) for the same standard (P≤ 0.05). Different letters (A,B) in the same column indicate significant differences between two concentrations of seafood peptone (1f and 2f) in the same medium for the same standard (P≤ 0.05).
d,B
ACCEPTED MANUSCRIPT
Table 4 : Effect of culture media supplemented with SPHs (cell free supernatants) on consumption of protein and glucose and production of organic acids by E. faecalis DM19.
h,A
MRS -S
6.95±0.25
SQES-S
47.74±0.69
SQAP-S
44.03±0.95
SQTR-S
39.43±0.07
SHAP-S
13.16±0.17
h,A
i,A
6.95±0.25
a,A
47.43±0.07
c,B
67.50±0.54
e,B
64.74±0.69
46.46±0.85
b,B
49.55±0.16
SHNP-S
40.56±0.30
d,B
58.82±0.06
SHEE-S
40.58±0.11
d,B
64.03±0.13
FGTR-S
37.75±0.11
f,B
46.53±0.62
SHPR-S
37.91±0.18
f,B
66.20±0.68
d,A
0.21±0.06
bcd,A
0.35±0.21
f,A
0.51±0.07
a,A
0.58±0.14
c,A
abcd,A
1f 5.14±0.08
a,A
4.58±0.11
0.35±0.21
b,c,B
0.08±0.01
abc,A
0.06±0.01
a,A
0.25±0.11
bcd,A
0.03±0.01
0.84±0.13
e,A
0.38±0.17
d,A
0.24±0.11
c,A
0.65±0.28
g,A
0.49±0.01
b,A
0.50±0.28
c,B
ab,B
c,B
cd,A
0.26±0.03
ab,A
0.10±0.13
bcd,A
abcd,A
2f
abc,A
0.21±0.06
e,A
de,B
5.37±0.40
c,B
6.30±0.28
f,A
5.87±0.37
g,A
4.06±0.18
d,A
4.17±0.24
bcd,A
5.20±0.18
a,A
3.99±0.25
5.14±0.08 4.58±0.11
7.35±0.23
7.68±0.08
8.46±0.61
d,B
8.46±0.61
c,B
7.95±0.06
b,B
8.16±0.23
de,B
6.19±0.01
a,B
8.44±0.18
5.65±0.13
6.56±0.11
bc,B
7.14±0.20
bc,B
5.24±0.20
bc,B
7.70±0.30
0.08±0.01
1f
e,B
5.20±0.11
ab,A
0.16±0.08
f,A
Acetic acid production (g/L)
RI PT
g,A
2f
SC
-
13.16±0.17
1f
Lactic acid production (g/L)
TE D
MRS-S
2f
EP
1f
Protein consumption (%)
M AN U
Glucose consumption (%)
AC C
Supernatant
b,A
f,A
2f
1f 0.05±0.03
f,A
0.04±0.01
de,A
0.07±0.01
c,A
0.06±0.01
de,A
0.06±0.04
b,A
0.07±0.01
c,A
0.07±0.01
a,A
0.08±0.01
e,A
0.07±0.01
b,A
0.07±0.01
4.06±0.18 5.63±0.07
c,B
6.30±0.28
f,B
5.46±0.07
cd,B
6.90±0.28
bc,B
6.26±0.33
a,B
8.64±0.11
de,B
5.16±0.35
a,A
7.37±0.48
5.11±0.16
bc,A
5.43±0.42
ab,A
6.85±0.13
e,A
4.61±0.28
a,A
6.68±0.08
2f
cd,A
5.87±0.37
ef,B
a,A
Formic acid production (g/L)
a,A
0.05±0.03
bc,A
a,A
0.04±0.01
a,A
0.08±0.01
a,B
0.09±0.01
a,A
0.09±0.01
c,A
ab,A
a,A
a,A
a,A
0.07±0.01
abc,A
a,A
0.08±0.01
a,A
0.09±0.01
a,A
0.05±0.01
a,A
0.10±0.03
ab,A
a,A
bc,A
a,A
“-S”: cell free supernatant after growing E. faecalis DM19 in different culture media. Legends for Seafood Protein Hydrolysates origin (SQES, SQAP, etc.) and composition of culture media (MRS and MRS ) are shown in Table 1 and 2, respectively. (1f): media supplemented with one-fold protein hydrolysates and (2f): media supplemented with two-fold protein hydrolysates. Different letters (a,b,c…) in the same column indicate significant differences among different samples at the same concentration (1f or 2f) for the same standard (P≤ 0.05). Different letters (A,B) in the same line indicate significant differences between the two concentrations of seafood peptone (1f and 2f) in the same medium for the same standard (P≤ 0.05).
ACCEPTED MANUSCRIPT
Table 5 : Antimicrobial activity of cell free supernatants from E.faecalis DM19 grown in different modified culture media. Results are expressed as diameters of the inhibition zone in mm .(na : no antimicrobial activity) MRS-S
SHEE-S
FGTR-S
SHPR-S
2f
1f
2f
1f
2f
1f
8.0±0.1b
na
6.3±0.3c
na
6.1±0.1c
na
7.8±0.4b
na
na
na
6.9±0.4bc,BC
na
6.4±0.1cd,CD
na
6.3±0.1cd,CDE
7.4±0.1AB
5.7±0.2d,DE
7.4±1.0b,B
na
na
na
na
na
na
na
5.9±0.6c,C
na
na
11.6±0.2d
na
9.1±0.3e
na
13.0±0.2c
na
5.9±1.0b
na
na
na
na
na
na
15.8±1.7a,A
na
8.6±0.0e,E
6.5±0.3b,F
10.4±0.1d,D
na
15.0±0.4ab,AB
7.1±0.7a
na
5.7±0.1b
na
6.3±0.0b
na
Clostridium perfringens
7.4±0.5bcd
na
6.6±0.1d
na
na
na
Debaryomyces hansenii
18.7±1.8a
na
12.4±0.1bc
na
9.3±2.1de
na
Enterococcus faecium
9.6±1.8a
na
6.5±0.1b
na
7.0±0.7b
na
Escherichia coli
7.0±0.2b
na
na
na
na
Lactobacillus helveticus
9.0±0.1a,A
na
8.4±0.9ab,AB
na
Listeria monocytogenes
9.0±0.8e,F
na
13.0±0.7b,B
na
na
5.7±0.1a,C
6.2±0.4c,C
na
Pseudomonas fluorescens
6.6±1.2a
na
na
Salmonella cholerasuis
9.0±0.2a
na
na
15.0±2.8a,A
na
6.6±0.0c,C
8.1±1.0a
na
17.2±0.2a,A
Bifidobacterium bifidum Brochothrix thermosphacta Citrobacter frenduii
Penicillium expansum
Shewanella putrefaciens Shigella sonnei Staphyloccus aureus Vibrio parahaemolyticus Yersinia enterocolitica
na
6.2±0.2c
11.8±0.3d na
na
10.2±0.1d,D
6.2±0.0b
na
6.3±0.3b
7.3±0.1bcd
na
na
9.6±1.0de
na
11.2±1.0cd
7.4±0.2b
na
8.8±0.3a
na
6.2±0.2c
na
na
7.5±0.6bc,BC
na
6.8±0.1c,C
na
7.3±0.8c,C
8.6±0.8e,F
na
8.4±0.7e,F
na
11.8±0.4cd,CD
5.7±0.0c,C
6.8±0.3a,C
7.1±0.1b,B
6.0±0.3a,C
7.1±0.6b,B
na
na
na
na
na
6.2±0.1ab
na
na
na
na
na
na
na
10.5±0.0b,B
7.6±0.2b,C
11.2±0.1b,B
6.0±0.1b,C
10.7±0.1b,B
6.6±0.1b
na
na
na
8.0±0.9a
na
7.5±0.6ab
na
11.4±0.2f,F
na
11.2±0.4f,F
na
13.9±0.1c,C
na
13.1±0.2d,D
8.2±0.4b
na
6.0±0.2f
na
7.2±0.0cd
na
8.8±0.1a
na
6.6±0.3e
na
na
na
na
8.5±0.3b
na
7.6±0.6cd
na
7.5±0.5cd
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Bacillus coagulans
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Bacillus cereus
EP
Aspergillus niger
AC C
Aeromonas hydrophila
2f
RI PT
1f
SC
Microorganism
SHNP-S
ACCEPTED MANUSCRIPT
MRS-S
SQAP-S
SQTR-S
SHAP-S
2f
1f
2f
1f
2f
1f
2f
8.0±0.1b
na
9.3±0.3a
na
6.2±0.1c
na
6.6±0.3c
na
6.3±0.4c
na
na
6.5±0.1c,C
na
5.7±0.1d,E
na
7.7±1.0a,A
na
7.4±0.2ab,AB
7.4±1.0b,B
na
na
6.5±0.0BC
10.2±0.6a,A
na
5.6±0.3c,C
na
na
na
na
14.4±0.9b
na
17.2±0.7a
na
8.7±0.3e
na
13.9±0.5b
Bifidobacterium bifidum
6.00±1.0b
na
6.2±0.1b
na
8.2±0.1a
na
na
na
na
Brochothrix thermosphacta
15.8±1.7a,A
7.0±0.5a,F
14.2±0.1bc,BC
6.4±0.2F
10.0±0.1d,D
na
13.5±0.9c,C
7.0±0.3b,F
9.6±0.1de,DE
7.1±0.7a
na
na
na
na
na
na
na
na
Clostridium perfringens
7.4±0.5bcd
na
8.7±0.3a
Debaryomyces hansenii
18.7±1.8a
na
14.6±1.9b
Enterococcus faecium
9.6±1.8a
na
6.7±0.4b
Escherichia coli
7.0±0.2b
na
7.1±0.2b
Lactobacillus helveticus
9.0±0.1a,A
na
9.2±0.3a,A
Listeria monocytogenes
9.0±0.8e,F
na
na
5.7±0.3a,C
Pseudomonas fluorescens
6.6±1.2a
na
Salmonella cholerasuis
9.0±0.2a
Citrobacter frenduii
Penicillium expansum
Shewanella putrefaciens Shigella sonnei Staphyloccus aureus Vibrio parahaemolyticus Yersinia enterocolitica
SC
Bacillus coagulans
M AN U
Bacillus cereus
na
8.0±0.0ab
na
7.8±0.1bc
na
7.2±0.9cd
na
12.4±1.0bc
na
11.0±0.8cd
na
8.1±1.4e
na
6.4±0.2b
na
6.7±0.4b
na
7.4±0.2b
na
na
na
8.0±0.3a
na
na
na
7.2±0.3c,C
na
9.0±0.1a,A
6.7±0.7C
6.8±0.5c,C
14.2±0.2a,A
na
11.8±0.2cd,CD
na
12.6±0.1bc,BC
10.2±0.2E
11.4±0.4d,D
5.8±0.3c,C
5.9±0.1a,C
7.4±0.5b,B
na
8.2±0.1a,A
na
na
na
na
na
na
na
na
na
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Aspergillus niger
na
na
na
na
na
5.4±0.2b
na
6.1±0.7b
na
6.0±0.1c,C
na
10.0±0.2b,B
na
10.8±0.2b,B
na
6.5±0.5c,C
8.1±1.0a
na
na
na
10.0±0.2b
na
na
na
6.7±0.2b
17.2±0.2a,A
7.2±0.2G
16.4±0.4b,B
na
12.1±0.6e,E
na
15.8±0.1b,B
na
12.8±0.2d,D
8.2±0.4b
na
7.0±0.2de
na
8.5±0.4ab
na
7.6±0.3c
na
6.5±0.2e
na
na
9.3±0.4a
na
7.3±0.3d
na
8.2±0.2bc
na
8.0±0.3bcd
15.0±2.8a,A
AC C
Aeromonas hydrophila
RI PT
1f
EP
Microorganism
SQES-S
Legends for Seafood Protein Hydrolysates origin (SQES, SQAP, etc.) and composition of culture media (MRS and MRS-) are shown in Table 1 and 2, respectively. (1f): media supplemented with one-fold protein hydrolysates and (2f): media supplemented with two-fold protein hydrolysates. Different letters (a,b,c…) in the same column indicate significant differences among different samples at the same concentration (1f or 2f) for the same standard (P<0.05).Different letters (A,B) in the same line indicate significant differences among samples at different concentrations (1f and 2f) for the same standard (P<0.05). ysates (SQES, SQAP, etc.) origin and composition of culture media (MRS and MRS-) are shown in Table 1 and 2, respectively. 1f): media supplemented with one-
Table 6. Bacteriocin genes identified
ACCEPTED MANUSCRIPT
Length
Amino acid
(amino acids)
identity (%)
Enterolysin A
343
343/343 (100%)
Lantibiotic I
63
63/63 (100%)
E. faecalis pathogenicity island(AF454824)
Lantibiotic II
68
33/33 (100%)
E. faecalis pathogenicity island(AF454824)
Bacteriocin
Annotation E. faecalis (Q9F8B0)
AC C
EP
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UviB-like* 82 54/69 (78%) E. faecalis 20-SD-BW-06 (EPH69978) *UviB-like has not been taken into account as bacteriocin due the amino acid identity is less than 95%
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The influence of SPHs on survival and metabolic activities was investigated
ACCEPTED MANUSCRIPT
Hydrolysates from squid and shrimp stimulated Enterococcus faecalis DM19 growth Enterococcus faecalis DM19 produced lactic acid with excellent efficiency
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Antimicrobial activity increased in media with higher amounts of SPHs
S0023-6438(17)30173-1
DOI:
10.1016/j.lwt.2017.03.028
Reference:
YFSTL 6101
To appear in:
LWT - Food Science and Technology
Received Date: 12 December 2016 Revised Date:
23 February 2017
Accepted Date: 14 March 2017
Please cite this article as: Djellouli, M., Martínez-Álvarez, O., Arancibia, M.Y., Florez-Cuadrado, D., Ugarte-Ruíz, M., Domínguez, L., Zadi-Karam, H., Karam, N., Roudj, S., López-Caballero, M.E., Effect of seafood peptones on biomass and metabolic activity by Enterococcus faecalis DM19, LWT - Food Science and Technology (2017), doi: 10.1016/j.lwt.2017.03.028. 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.
ACCEPTED MANUSCRIPT Effect of seafood peptones on biomass and metabolic activity by
2
Enterococcus faecalis DM19
3
Mustapha Djelloulia,b, Oscar Martínez-Álvarezc, Mirari Y. Arancibiac,d, Diego
4
Florez-Cuadradoe, María Ugarte-Ruíze, Lucas Domíngueze, Halima Zadi-
5
Karama, Noureddine Karama, Salima Roudja and M. Elvira López-Caballeroc*
6
a
RI PT
1
Laboratory for Biology of Microorganisms and Biotechnology, Department of
Biotechnology, Faculty of Sciences of Nature and Life, University of Oran 1
8
Ahmed Benbella, BP 1524 El-M'Naouer, 31000 Oran, Algeria.
Biotechnology Research Center, Department of Food Biotechnology, Ali
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b
SC
7
Mendjeli, Nouvelle Ville, UV 03 BP E73, 25000 Constantine, Algeria
11 12 13 14
Institute of Food Science, Technology and Nutrition (ICTAN-CSIC)**, C/José
Antonio Novais, 28040 Madrid, Spain d
Technical University of Ambato (UTA), Av. Los Chasquis y Río Payamino,
Ambato, Ecuador e
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10
c
VISAVET Health Surveillance Centre, Complutense University of Madrid,
EP
9
Avda. Puerta de Hierro, s/n. 28040 Madrid, Spain. to
whom
AC C
*Author
correspondence
should
be
addressed
[email protected]] 15
**This centre has implemented and maintains a Quality Management System
16
which fulfils the requirements of the ISO standard 9001:2008
17
1
ACCEPTED MANUSCRIPT Abbreviations:
19
SQES : Squid protein hydrolyzed with Esperase
20
SQAP : Squid protein hydrolyzed with Alkaline protease
21
SQTR : Squid protein hydrolyzed with Trypsin
22
SHAP : Shrimp protein hydrolyzed with Alkaline protease
23
SHNP : Shrimp protein hydrolyzed with Neutral protease
24
SHEE : Shrimp protein hydrolyzed with endogenous enzymes
25
FGTR : Fish gelatin hydrolyzed with Trypsin
26
SHPR : Shrimp protein hydrolyzed with Protamex
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2
ACCEPTED MANUSCRIPT Abstract
28
Eight seafood protein hydrolysates (SPHs) obtained from squid, shrimp and fish
29
gelatin were incorporated as substitutes of peptones in culture media in order to
30
evaluate its effect on survival and metabolic activity (lactic acid, acetic acid and
31
bacteriocins production) of Enterococcus faecalis DM19. The substitution of
32
commercial peptones in culture media by either a shrimp hydrolysate prepared
33
with Protamex, or by squid protein hydrolysates prepared with Esperase or
34
Alkaline protease, stimulated E. faecalis DM19 growth up to 16%. The
35
incorporation of SPHs, mainly from shrimp, in the culture media significantly
36
increased production of lactic and acetic acids in more than 60%. Furthermore,
37
the media containing SPHs stimulated antimicrobial activity by E. faecalis
38
DM19. The inhibitory activity was observed against both Gram-positive and
39
Gram-negative microorganisms, but it was remarkably observed against Listeria
40
monocytogenes. SPHs incorporated in culture media render properties of bio-
41
technological interest, which, together with their low price, make them suitable
42
for industrial use.
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Keywords: Enterococcus faecalis DM19, seafood protein hydrolysates,
45
bacteriocinogenic activity, antimicrobial activity, renewable materials
46
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3
ACCEPTED MANUSCRIPT 1. Introduction
48
The genus Enterococcus comprises species considered components of the
49
human and animal intestinal flora (Khan, Flint, & Yu, 2010) and is generally
50
found in large numbers in dairy products and fermented foods. Lactic and acetic
51
acid, diacetyl, and bacteriocins are some of the molecules produced by these
52
microorganisms that present antimicrobial activity (Gilmore, Lebreton, & van
53
Schaik, 2013). Moreover, several E. faecalis strains have been investigated as
54
potential probiotics (Franz, Huch, Abriouel, Holzapfel, Gálvez, 2011). Therefore,
55
from the viewpoint of their industrial importance, E. faecalis could play a
56
significant role in fermentation and preservation of some foods (Khan, Flint, &
57
Yu, 2010).
58
Like other lactic acid bacteria (LAB) groups, enterococci can only grow in a rich
59
microbial growth medium which contains all the necessary compounds such as
60
amino acids, peptides, fatty acids, vitamins and nucleic acids required for
61
optimal growth and metabolite production. These compounds are usually
62
provided in the form of complex nitrogen sources such as yeast extract, meat
63
extract, tryptone, peptone or bactopeptone. Those nitrogen sources can reach
64
high prices and be unsuitable for industrial scale production of microorganisms
65
(Zhang & Gresham, 1999). Thus, there is great interest in investigating cheap
66
renewable nitrogenous materials to prepare a low-cost fermentation medium.
67
Several authors (Aspmo, Horn, & Eijsink, 2005; Hujanen & Linko, 1996; Li, Han,
68
Ji, Wang, & Tan, 2010; Yu, Lei, Ren, Pei, & Feng, 2008) studied various
69
nitrogen sources (yeast extract, malt sprout, peptones, grass extract, corn steep
70
liquor, casein hydrolysates, distiller’s waste, ammonium phosphates, wheat
71
bran, fish waste hydrolysates and urea) for lactic acid fermentation. The use of
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4
ACCEPTED MANUSCRIPT autolysates as a peptone source in bacteriocin production by LAB was also
73
studied (Deraz, El-Fawal, Abd-Ellatif, & Khalil, 2011). Nevertheless, to our best
74
knowledge, there is no information about the stimulation of lactic acid and/or
75
production of bacteriocin by E. faecalis in MRS media formulated with seafood
76
(crustacean and cephalopods) protein hydrolysates.
77
The aim of the present work was to evaluate the effect of different seafood
78
protein hydrolysates as a peptone source on biomass production of E. faecalis
79
DM19. The production of organic acids (lactic, acetic and formic acids) and
80
bacteriocins in these culture media were also studied.
81
2. Material and methods
82
2.1.
83
The E. faecalis DM19 was obtained from the Laboratory for Biology of
84
Microorganisms and Biotechnology (University of Oran 1 Ahmed Benbella,
85
Algeria). The strain was originally isolated from raw camel milk collected in the
86
Mauritania area and identified by PCR amplification using E. faecalis primers
87
and 16S rDNA sequencing (Vincent, Roy, Mondou, & Dery, 1998). A search for
88
homology of the DNA sequence was performed using the BLAST algorithm
89
available at the National Center for Biotechnology Information (NCBI, USA).
90
2.2.
91
Squid (Loligo vulgaris) muscle trimmings, by-products (muscle trimmings,
92
carapace and heads) derived from processing Pacific white shrimp (Penaeus
93
vannamei) and Southern pink shrimp (Penaeus notialis), and high molecular
94
weight (HMW) dried fish skin gelatine (type A) of cold water fish (Sebastes spp.)
95
were used as raw material to prepare seafood protein hydrolysates. The by-
AC C
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Bacterial strain
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Preparation of seafood protein hydrolysates
5
ACCEPTED MANUSCRIPT products were minced and hydrolysed with commercial or endogenous
97
enzymes (Table 1) which were kindly provided by Novozymes (Denmark).
98
Enzymatic reactions were controlled by using a pH-stat. After hydrolysis, the
99
samples were rapidly heated to 90°C for 15 min to inactivate the enzymes. The
100
hydrolysates were then centrifuged at 10000 x g for 20 minutes at 4ºC
101
(Beckman J2-MC, Palo Alto, California, USA). The degree of hydrolysis for each
102
hydrolysate was calculated according to Spellman, McEvoy, O'Cuinn, and
103
FitzGerald (2003). The supernatants were freeze-dried under vacuum and kept
104
at -20°C until their use in the culture media formulation.
105
2.3.
106
Peptone in MRS (Man, Rogosa and Sharpe, Merck KGaA, Darmstadt,
107
Germany) media was substituted by protein hydrolysates (marine peptones) at
108
two different concentrations as depicted in Table 2 (MRS –standard culture
109
media- was considered as control and MRS- -without peptone- as negative
110
control). After adjusting the pH to 7, the media were autoclaved at 121°C for 20
111
min. Inoculum (1%, v/v) consisting of cellular suspension of an 18 h young
112
culture of E. faecalis DM19 in MRS broth medium, was adjusted to an OD (600
113
nm) of 0.900 (UV-Visible spectrophotometer model UV-1601 from Shimadzu).
114
Then, the broth was centrifuged at 12000 x g (Microspin 24S from Sorvall
115
instruments) for 10 min at 4°C and the sediment was washed twice with
116
phosphate buffer (0.1M, pH 7.0) to remove all traces of the culture medium. The
117
washed cells were re-suspended in sterile distilled water and then adjusted to
118
the initial OD (600nm).They were then used to inoculate modified MRS medium.
119
After 24 hours of incubation at 30°C, an aliquot of the medium was taken to
120
determine bacterial growth. The rest of the medium was centrifuged at 5000 x g
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Culture media
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6
ACCEPTED MANUSCRIPT 121
for 20 min at 4°C (Sorvall, model RT 6000B). The supernatant was used for the
122
assessment of glucose, total nitrogen, organic acids (lactic acid, acetic acid,
123
formic acid) and to determine the bacteriocinogenic activity.
124
2.4.
125
Cell growth was directly monitored by measuring the optical density of the
126
culture broth samples at 600 nm using spectrophotometer (UV-Visible
127
spectrophotometer model UV-1601 from Shimadzu). The evolution of pH in the
128
culture broth was determined with a pH meter (inoLab, D-82362 Weilheim
129
Germany).
130
The total nitrogen content was determined using the Dumas combustion
131
method following AOAC 992.15 (1995) in a LECO model FP-2000 (Leco
132
Instruments, Madrid, Spain) protein/nitrogen calibrated with EDTA. Protein
133
content was estimated by multiplying the total nitrogen content by the factor
134
6.25, and was expressed as grams of protein/100 grams of sample.
135
Glucose and organic acid content (lactic, acetic, formic, pyruvic and citric acids)
136
were determined by HPAEC-PAD (High Performance Anion Exchange
137
Chromatography with Pulsed Amperometric Detection) using a Metrohm
138
Advanced Compact ion chromatography instrument (867 IC. Metrohm)
139
equipped with an IC-819 conductivity detector, an IC Pump 818 and an IC-837
140
degasser coupled. Firstly, the supernatants (obtained as previously described)
141
were diluted in ultrapure water and then filtered through a membrane of 0.45 µm
142
pore size. For glucose determination, 20 µL of samples were injected into a
143
Hamilton RC X 30 (250 x 4 mm, 5 µm pore size) column. The samples were
144
eluted from the column over 35 min at a flow of 1 mL/min with an isocratic
145
gradient of 30 mM NaOH with 3 mM AcNa. For organic acids, the identification
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Analytical methods
7
ACCEPTED MANUSCRIPT and quantification were carried out by injecting 20 µL of the samples into
147
Metrosep Organic Acid (250 x 4 mm, 5 µm pore size) column and the elution
148
was performed with 1 mM perchloric acid and acetone (10 %) as mobile phase
149
over 20 min at a flow of 0.5 mL/min. All the molecules studied were identified by
150
their retention times and quantified based on calibration curves derived from
151
standards. The results were expressed as mg/L of the culture supernatant. It is
152
evident that concerning the accumulation of acetic acid into the culture medium,
153
the initially added sodium acetate concentration (=5.0 g/L) was taken into
154
consideration for the calculations performed. All assays were carried out in
155
triplicate.
156
2.5.
SC
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Bacteriocinogenic activity and whole genome sequencing (WGS) of E. faecalis DM19
Culture supernatants from the different media tested were filtered through a
159
cellulose acetate sterile filter (0.22 µm) to remove bacterial cells. The extracts
160
obtained were then lyophilized and stored at – 20°C. The samples were re-
161
suspended in distilled water to a concentration of 5 mg/mL for the culture
162
extracts. Finally, the pH of the prepared solutions was adjusted to 7.0 with
163
NaOH (3N).
164
The antimicrobial activity of the supernatant was tested with the disc diffusion
165
method (Arancibia, Giménez, López-Caballero, Gómez-Guillén, & Montero,
166
2014). Briefly, sterile paper discs (5 mm diameter, Whatman No. 1) impregnated
167
with 40 µL of the supernatant solutions were placed on the surface of agar
168
plates inoculated with several microorganisms from the Spanish Type Culture
169
Collection (CECT) selected due to their relevance in human health (either lactic
170
acid bacteria or pathogens) or to their role in food spoilage: Aeromonas
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8
ACCEPTED MANUSCRIPT hydrophila CECT 839T, Aspergillus niger CECT 2088, Bacillus cereus CECT
172
148, Bacillus coagulans CECT 56, Bifidobacterium bifidum DSMZ 20215,
173
Brochothrix thermosphacta CECT 847, Citrobacter freundii CECT 401,
174
Clostridium perfringens CECT 486, Debaryomyces hansenii CECT 11364,
175
Enterococcus faecium DSM 20477, Escherichia coli CECT 515, Lactobacillus
176
helveticus DSM 20075, Listeria monocytogenes CECT 4032, Penicillium
177
expansum DSMZ 62841, Pseudomonas fluorescens CECT 4898, Salmonella
178
choleraesuis CECT 4300, Shewanella putrefaciens CECT 5346T, Shigella
179
sonnei
180
parahaemolyticus CECT 511T, Yersinia enterocolitica CECT 4315. After
181
incubation, the diameter of the inhibition zone (considered antimicrobial activity)
182
was measured with Corel Draw X6 software. Results were expressed as mm of
183
inhibited growth area (magnification ratio 1:10). Each determination was
184
performed in triplicate.
185
In order to evaluate which type of bacteriocin was present, the genome of E.
186
faecalis DM19 was sequenced by whole-genome sequencing (WGS) method
187
(Goodwin, McPherson, & McCombie, 2016). The DNA libraries were sequenced
188
in the Illumina HiSeq platform, using 100bp paired-end sequencing reads. The
189
raw sequence data of the sample was de novo assembled using algorithm
190
SPAdes 3.9 (Bankevich et al., 2012). Bacteriocin genes were identified using
191
the bacteriocin mining tool Bagel3 (Van Heel, de Jong, Montalbán-López, Kok,
192
& Kuipers 2013). Each bacteriocin-like gene identified was checked using
193
BLASTX algorithm.
194
2.6.
Staphylococcus
aureus
CECT
240,
Vibrio
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Statistical analysis
9
ACCEPTED MANUSCRIPT Statistical tests were performed using the SPSS computer program (SPSS
196
Statistical Software, Inc., Chicago, IL, USA). Two-way analysis of variance
197
(ANOVA) was carried out. The difference of means between pairs was resolved
198
by means of confidence intervals using a Tukey-b test at a significance level of
199
5%.
200
3. Results and discussion
201
3.1.
202
The substitution of commercial peptone in culture media by hydrolysates from
203
squid (SQTR and SQAP) and prawn (SHPR), especially at two-fold
204
concentration, significantly increased the biomass (maxOD600) of E. faecalis
205
DM19 (Table 3). In contrast, the growth in media where SHAP or FGTR (one or
206
two-fold) were incorporated was significantly lower when compared with that of
207
commercial MRS. In addition, the terminal pH in E. faecalis DM19 cultures was
208
quite low as expected (Table 3). The data show an inverse correlation between
209
biomass production and terminal pH.
210
Some studies have been carried out to assess the ability of commercial
211
proteolytic
212
microorganisms) to reduce fish protein (whole fish or wastes) into peptides and
213
amino acids, which can be an economical source of peptones for bacterial
214
growth media. In fact, protein hydrolysates from seafood by-products can be
215
possible substitutes for the usual protein hydrolysate-based substrates in
216
microbial cultures (Gao, Hirata, Toorisaka, & Hano, 2006). In this work, seafood
217
peptones showed an activity which was in the same range as the commercial
218
peptone activity or scarcely higher in some cases, suggesting a well-balanced
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Effect of SPHs on growth
from
different
resources
(plants,
animals
or
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ACCEPTED MANUSCRIPT nutrient concentration of these SPHs media. Stein, Svein, and Vincent (2005a)
220
reported that since peptones may differ in buffer capacity and the terminal pH
221
values reached in the Escherichia coli cultures were low, the observed growth
222
differences were due to the pH/buffering effect. In the present work, E. faecalis
223
showed a different biomass after fermentation in media including SPHs at a
224
similar pH (Table 3). Vázquez, González, and Murado (2004) found similar
225
results when testing the growth of Roseobacter sp on MRS supplemented with
226
squid hydrolysate at different concentrations. These authors attributed this
227
phenomenon to the presence of compounds (perhaps lipidic peroxides) with a
228
slight inhibitory effect on the corresponding microorganism. Moreover, the
229
peptidic composition of the SPH added to the MRS media may also affect
230
bacterial growth (Table 3) since the results obtained from MRS media including
231
hydrolysates derived from the same substrate (squid or shrimp) were different
232
(P≤ 0.05). In this connection, Stein, Svein, and Vincent (2005b) cultivated LAB
233
on MRS media supplemented with hydrolysates from cod viscera prepared with
234
Alcalase, Papain and endogenous enzymes and found that peptone
235
performance is highly dependent on composition details as peptide length,
236
peptide sequences, and the amount of free amino acids. In the present work, E.
237
faecalis DM19 exhibited a clear preference for MRS media including one-fold
238
concentration of squid protein hydrolysates prepared with Esperase or Alkaline
239
protease (SQES and SQAP, respectively), or shrimp protein hydrolysate
240
prepared with Protamex (SHPR). However, the incorporation of a shrimp protein
241
hydrolysate prepared with an alkaline protease (SHAP) in the media affected
242
negatively bacterial growth. The amount of SPHs incorporated in MRS media
243
also seems to be important in the biomass production (Table 3). The results
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11
ACCEPTED MANUSCRIPT may be explained by the fact that certain proteases can release amino acids
245
and peptides suitable for the transport of bacteria and proteolytic systems
246
(Kunji, Mierau, Hagting, Poolman, & Konings, 1996). Moreover, certain
247
hydrolysates as SHAP or FGTR may contain antimicrobial peptides or
248
compounds that negatively affect bacterial growth (Van’t Hof, Veerman,
249
Helmerhorst & Amerongen, 2001).
250
The production of organic acids by E. faecalis DM19 could also be affected by
251
the composition of the MRS media. If the culture medium stimulates the
252
production of organic acids by the bacteria, these acids could decrease the pH
253
of the media and regulate bacterial growth significantly. This fact could explain
254
why the media prepared with two-fold concentration of SHEE promoted a low
255
production of biomass and a high production of acetic acid. Different
256
investigations have been driven in this way to confirm the inhibitory effect of
257
acetic acid on several microorganisms. Recently, Boguta and Jensen (2014)
258
reported that, for Pediococcus acidilactici, 5.3 g/L acetate caused a 4% growth
259
inhibition and showed a synergistic effect when the strain was grown with both
260
acetate and furfural. Therefore, the inhibition phenomenon shown for SHEE
261
was probably due to the high concentration of acetate produced by E. faecalis
262
(Table 3).
263
3.2.
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Effect of SPHs on production of organic acids and consumption of glucose and protein
265
The amount of glucose and protein in the different media were measured before
266
and after the fermentation process (Table 4). Glucose and protein consumptions
267
for bacteria were higher in media with one-fold concentration of SPHs. The
12
ACCEPTED MANUSCRIPT highest glucose consumption was found in media containing SQES. Glucose
269
consumption augmented when a higher amount of SPHs was added in the
270
media, while protein consumption decreased significantly. Protein consumption
271
was minimal in media including SPHs prepared with alkaline protease (SQAP
272
and SHAP).
273
Acid production depended on the media and on peptone concentration (Table
274
4). Lactic and acetic acids were the major products of the fermentation process
275
with E. faecalis DM19, while formic acid was produced in low quantities. The
276
incorporation of SPH in the media improved notably the production of lactic acid
277
in all samples, and that of acetic acid especially in SHPR-S and SHEE-S
278
samples. Interestingly, the highest production of lactic acid was achieved in
279
media with the mentioned hydrolysates.
280
As describe before, SHPR is the best supplement for efficient lactic acid
281
production in medium of 1f. Furthermore, the increment for SPH in the media
282
(2f) exerted a positive effect on lactic acid production (Table 4). The quantities
283
produced in SHAP-S and SHPR-S samples were 1.6 times larger compared to
284
those in MRS medium. These differences in production could be due to the
285
marine peptones used in fermentation, since the strain was cultivated under the
286
same experimental conditions. In this regard, lactate production increased in
287
Streptococcus zooepidemicus when tryptone was replaced by marine peptones
288
(Vázquez, Montemayor, Fraguas, & Murado, 2009) while the increase in wheat
289
bran concentration promotes lactic acid production by Lactobacillus rhamnosus
290
LA-04-1 (Zheng, Lu, Yizhi, Xiaonan, & Tianwei, 2010). E. faecalis, like other
291
LAB, requires amino acids such as histidine, isoleucine, leucine, methionine,
292
glutamate, glycine, tryptophan, valine (Murray et al., 1993) to achieve optimal
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ACCEPTED MANUSCRIPT cultivation conditions. The results obtained with E. faecalis in the present work
294
confirm that the different amino acid and peptide composition of marine
295
peptones could procure different efficiency in the amino acid and peptide uptake
296
systems (Kunji, Mierau, Hagting, Poolman, & Konings, 1996). The increase of
297
nitrogen provided by SPHs, significantly arises glucose uptake, and
298
consequently lactic acid production (Table 4), and hence its assimilation, as has
299
already been reported by Fakas, Papanikolaou,Komaitis, and Aggelis (2008) for
300
Cunninghamella echinulate.
301
3.3.
302
In order to investigate antagonistic profiles of the E.faecalis DM19 strain, cell-
303
free supernatant extracts were tested against 21 microbial strains (Table 5).
304
Differences in the diameter of inhibition halos were particularly notable up to
305
18.73±1.80 mm. Cell-free supernatants from E. faecalis DM19 grown in MRS
306
exhibited bactericidal activity against the majority of studied strains excluding
307
Aspergillus niger CECT 2088, Bacillus coagulans CECT 56, Penicillium
308
expansum DSMZ 62841 and Yersinia enterocolitica CECT 4315. However,
309
these microorganisms were sensitive to the cell-free supernatants from MRS
310
prepared by the hydrolysates (Table 5). Previous works had already reported
311
that E. faecalis produce a broad-spectrum of bacteriocins (enterocins), which
312
are active against Gram-positive and Gram-negative bacteria (Line, Svetoch,
313
Eruslanov, Perelygin et al., 2008; Pieniz, Andreazza, Anghinoni, Camargo, &
314
Brandelli, 2014). This fact could be reasonable since the pH of the culture
315
supernatants used in this study were neutralized to avoid the antimicrobial
316
effect of organic acids (lactate, acetate, etc.) and since anaerobic conditions
317
were imposed to decrease H2O2.
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Effect of SPHs on bacteriocin like inhibitory substances production
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ACCEPTED MANUSCRIPT The substitution of commercial peptone in the media by one-fold concentration
319
of SPH did not lead to a relevant production of antimicrobial compounds in
320
terms of effectiveness and number of microorganisms inhibited. However, when
321
commercial peptone was substituted by two-fold concentration of SPHs, the
322
supernatants exhibited an interesting inhibitory activity against a large number
323
of indicator strains investigated (Table 5). The highest inhibitory activity was
324
observed mainly against Listeria monocytogenes CECT 4032, which was
325
significantly inhibited by SQES supernatant as compared with MRS (p≤0.05).
326
It has been shown that the composition of the growth medium, particularly the
327
source and concentration of nitrogen, strongly affects the production of
328
bacteriocin (Gänzle, Weber, & Hammes, 1999). As can be seen in Table 5, the
329
production of bacteriocin-like substances by E. faecalis production was
330
enhanced when SPHs concentration increased from 10 to 20 g/L. Accordingly,
331
the increasing concentrations of yeast extract, meat extract or peptone can
332
allow an increase in the production of bacteriocins. It can be possible that fish
333
peptones could have suitable contents, in terms of free amino acids and short
334
peptides, that are precursors for bacteriocin production (Kanmani et al., 2010),
335
whereas some authors have pointed out that bacteriocin production by lactic
336
acid bacteria is a growth-associated process (De Vuyst, Callewaert, & Crabbé,
337
1996).
338
3.4.
339
Once the genome of E. faecalis DM19 was assembled, the presence of
340
bacteriocin genes was tested using the BAGEL3 algorithm. Four sequences
341
related with bacteriocin-like genes were identified but only two showed proper
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Bacteriocin genes identified in the E. faecalis DM19 genome
15
ACCEPTED MANUSCRIPT length and amino acid identity higher than 99%: the enterolysin A and a
343
lantibiotic gene (Table 6). The annotation of the bacteriocins identified was
344
based on amino acid sequences homology (Supplementary data).
345
Enterolysin A is a heat-labile bacteriocin, firstly reported in E. faecalis LMG2333
346
and widely distributed among enterococci strains (Nilsen et al. 2003; Hickey et
347
al. 2003; Nigutova et al. 2007; Almeida et al. 2011). It belongs to the class III of
348
the bacteriocins and has a bacteriolytic mode of action over cell wall (Nilsen et
349
al, 2003). Our results are in line with the data already published about
350
enterolysin A in which it was described that inhibits growth of enterococci,
351
pediococci, lactococci and lactobacilli (Nilsen et al, 2003; Khan et al, 2013).
352
Furthermore, enterolysin A shows a wide spectrum of activity possibly due to
353
the cleavage of the peptide bond in the stem peptide and the interpeptide bridge
354
of the peptidoglycan units (Khan et al, 2013).
355
Besides, the other bacteriocin identified in E. faecalis DM19 is the one known
356
as lantibiotic because of the content of lanthionine and/or methyllanthionine in
357
its primary structure. These substances belong to class I bacteriocins and its
358
activity falls mainly on Gram-positive bacteria (Bierbaum et al, 2009).
359
Lantibiotics are heat-stable peptides and, like enterolysin A, also act on the cell
360
wall of other bacteria (Sahl et al, 1998).
361
Conclusion
362
Protein hydrolysates prepared mainly from squid and shrimp have sufficient
363
nutritional value to support growth and metabolite production of E. faecalis
364
DM19. The increase of nitrogen provided by these hydrolysates in culture media
365
increases glucose uptake, and consequently lactic acid production by this
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16
ACCEPTED MANUSCRIPT strain. Moreover, the increase of fish peptones enhanced the bacteriocin
367
producing of E. faecalis DM19, which was especially active against Listeria
368
monocytogenes. These strain properties, together with the low price of protein
369
hydrolysates, make them suitable for use in the fermented food industry.
370
Acknowledgements
371
This research was financed by the Spanish Ministry of Economy and
372
Competitiveness (projects AGL2011-27607, AGL2014-52825-R). Author M.
373
Djellouli is funded by The National Centre of Biotechnology Research (CNRBt)
374
of Algeria and ENP (Exceptional National Program) Scholarship provided by the
375
Ministry of Higher Education and Scientific Research of Algeria. Author M.
376
Arancibia is funded by a SENESCYT Scholarship provided by the Ecuadorian
377
government.
378
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ACCEPTED MANUSCRIPT Table 1: Proteolytic enzymes used to hydrolyze fish proteins.
Raw material used
Enzyme
Reaction conditions
pH
T (°C)
Time (min)
8
50
90
Squid (muscle)
Esperase
20.12
SQAP
Squid (muscle)
45.98
8
50
90
SQTR
Squid (muscle)
Alkaline protease Trypsin
21.50
8
37
90
SHAP
Alkaline protease Neutral protease Autohydrolyzed Trypsin
13.47
8
50
90
9.67
7.5
50
90
ND
7.5
40
360
FGTR
P. vannamei (tails, carapace and heads) P. vannamei (tails, carapace and heads) P. vannamei (heads and carapace) Fish gelatin
10.90
9
38
360
SHPR
P. notialis (muscle)
Protamex
6.68
7.5
50
90
SHEE
AC C
EP
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ND: Not determined
M AN U
SHNP
RI PT
SQES
SC
Sample designation
Degree of Hydrolysis (%)
ACCEPTED MANUSCRIPT Table 2: Composition of the culture media tested based on MRS (g/L) b
MRS
MRS
Yeast extract
4
4
4
4
Meat extract
8
8
8
8
Glucose
20
20
20
20
Sodium acetate
5
5
5
5
Tween 80 (mL)
1
1
1
1
Dipotassium hydrogen phosphate
2
2
2
2
Ammonium citrate
2
2
2
2
Mangnesium sulfate
0.2
0.2
0.2
0.2
Manganese sulfate
0.05
0.05
0.05
0.05
10
-
-
-
-
-
10
20
Peptone Seafood peptones
M AN U
MRS
EP
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MRS: Man Rogosa and Sharpe (standard culture media). MRS : Man Rogosa and Sharpe without peptone. a MRS : MRS supplemented with one fold protein hydrolysates b MRS : MRS supplemented with two-fold protein hydrolysates
AC C
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a
MRS
SC
-
Composition
ACCEPTED MANUSCRIPT
Table 3 : Comparison of growth of E.faecalis DM19 and pH in the media with Seafood Protein Hydrolysates (SPHs). Seafood peptones
MRS
MRS
-
SQES
SQAP
SQTR
1f
4.62
4.55
4.56
4.56
4.57
2f
4.62
4.55
4.69
4.74
4.71
1f
1.58
1.65
1.64
1.64
1.63
2f
1.58
1.65
1.51
1.46
1.49
SHAP
c,A
1.65±0.01
c,A
1.65±0.01
1f
1.81±0.01
2f
1.81±0.01
g,A
2.01±0.02
de,A
1.83±0.02
M AN U
(Growth data) OD600
a,A
1.95±0.01
b,B
1.83±0.02
c,B
2.11±0.01
a,A
1.94±0.00
SHEE
FGTR
SHPR
4.58
4.62
4.57
4.60
4.56
4.66
4.80
4.81
4.79
4.79
1.62
1.58
1.63
1.60
1.64
1.54
1.4
1.39
1.41
1.41
SC
Final pH
∆pH
SHNP
RI PT
Parameters studied
c,B
1.74±0.01
b,A
1.67±0.01 -
e,A
1.81±0.01
c,A
1.80±0.01
e,B
1.82±0.01
d,A
c,A
0.64±0.01
f,B
f,A
2.02±0.01
de,A
1.73±0.01
1.69±0.02 1.70±0.01
a,A
AC C
EP
TE D
Legends for Seafood Protein Hydrolysates (SQES, SQAP, etc.) and composition of culture media (MRS and MRS ) are shown in Table 1 and 2, respectively. 1f: media supplemented with one-fold protein hydrolysates 2f: media supplemented with two-fold protein hydrolysates Different letters (a,b,c…) in the same line indicate significant differences among the different samples at the same concentration (1f or 2f) for the same standard (P≤ 0.05). Different letters (A,B) in the same column indicate significant differences between two concentrations of seafood peptone (1f and 2f) in the same medium for the same standard (P≤ 0.05).
d,B
ACCEPTED MANUSCRIPT
Table 4 : Effect of culture media supplemented with SPHs (cell free supernatants) on consumption of protein and glucose and production of organic acids by E. faecalis DM19.
h,A
MRS -S
6.95±0.25
SQES-S
47.74±0.69
SQAP-S
44.03±0.95
SQTR-S
39.43±0.07
SHAP-S
13.16±0.17
h,A
i,A
6.95±0.25
a,A
47.43±0.07
c,B
67.50±0.54
e,B
64.74±0.69
46.46±0.85
b,B
49.55±0.16
SHNP-S
40.56±0.30
d,B
58.82±0.06
SHEE-S
40.58±0.11
d,B
64.03±0.13
FGTR-S
37.75±0.11
f,B
46.53±0.62
SHPR-S
37.91±0.18
f,B
66.20±0.68
d,A
0.21±0.06
bcd,A
0.35±0.21
f,A
0.51±0.07
a,A
0.58±0.14
c,A
abcd,A
1f 5.14±0.08
a,A
4.58±0.11
0.35±0.21
b,c,B
0.08±0.01
abc,A
0.06±0.01
a,A
0.25±0.11
bcd,A
0.03±0.01
0.84±0.13
e,A
0.38±0.17
d,A
0.24±0.11
c,A
0.65±0.28
g,A
0.49±0.01
b,A
0.50±0.28
c,B
ab,B
c,B
cd,A
0.26±0.03
ab,A
0.10±0.13
bcd,A
abcd,A
2f
abc,A
0.21±0.06
e,A
de,B
5.37±0.40
c,B
6.30±0.28
f,A
5.87±0.37
g,A
4.06±0.18
d,A
4.17±0.24
bcd,A
5.20±0.18
a,A
3.99±0.25
5.14±0.08 4.58±0.11
7.35±0.23
7.68±0.08
8.46±0.61
d,B
8.46±0.61
c,B
7.95±0.06
b,B
8.16±0.23
de,B
6.19±0.01
a,B
8.44±0.18
5.65±0.13
6.56±0.11
bc,B
7.14±0.20
bc,B
5.24±0.20
bc,B
7.70±0.30
0.08±0.01
1f
e,B
5.20±0.11
ab,A
0.16±0.08
f,A
Acetic acid production (g/L)
RI PT
g,A
2f
SC
-
13.16±0.17
1f
Lactic acid production (g/L)
TE D
MRS-S
2f
EP
1f
Protein consumption (%)
M AN U
Glucose consumption (%)
AC C
Supernatant
b,A
f,A
2f
1f 0.05±0.03
f,A
0.04±0.01
de,A
0.07±0.01
c,A
0.06±0.01
de,A
0.06±0.04
b,A
0.07±0.01
c,A
0.07±0.01
a,A
0.08±0.01
e,A
0.07±0.01
b,A
0.07±0.01
4.06±0.18 5.63±0.07
c,B
6.30±0.28
f,B
5.46±0.07
cd,B
6.90±0.28
bc,B
6.26±0.33
a,B
8.64±0.11
de,B
5.16±0.35
a,A
7.37±0.48
5.11±0.16
bc,A
5.43±0.42
ab,A
6.85±0.13
e,A
4.61±0.28
a,A
6.68±0.08
2f
cd,A
5.87±0.37
ef,B
a,A
Formic acid production (g/L)
a,A
0.05±0.03
bc,A
a,A
0.04±0.01
a,A
0.08±0.01
a,B
0.09±0.01
a,A
0.09±0.01
c,A
ab,A
a,A
a,A
a,A
0.07±0.01
abc,A
a,A
0.08±0.01
a,A
0.09±0.01
a,A
0.05±0.01
a,A
0.10±0.03
ab,A
a,A
bc,A
a,A
“-S”: cell free supernatant after growing E. faecalis DM19 in different culture media. Legends for Seafood Protein Hydrolysates origin (SQES, SQAP, etc.) and composition of culture media (MRS and MRS ) are shown in Table 1 and 2, respectively. (1f): media supplemented with one-fold protein hydrolysates and (2f): media supplemented with two-fold protein hydrolysates. Different letters (a,b,c…) in the same column indicate significant differences among different samples at the same concentration (1f or 2f) for the same standard (P≤ 0.05). Different letters (A,B) in the same line indicate significant differences between the two concentrations of seafood peptone (1f and 2f) in the same medium for the same standard (P≤ 0.05).
ACCEPTED MANUSCRIPT
Table 5 : Antimicrobial activity of cell free supernatants from E.faecalis DM19 grown in different modified culture media. Results are expressed as diameters of the inhibition zone in mm .(na : no antimicrobial activity) MRS-S
SHEE-S
FGTR-S
SHPR-S
2f
1f
2f
1f
2f
1f
8.0±0.1b
na
6.3±0.3c
na
6.1±0.1c
na
7.8±0.4b
na
na
na
6.9±0.4bc,BC
na
6.4±0.1cd,CD
na
6.3±0.1cd,CDE
7.4±0.1AB
5.7±0.2d,DE
7.4±1.0b,B
na
na
na
na
na
na
na
5.9±0.6c,C
na
na
11.6±0.2d
na
9.1±0.3e
na
13.0±0.2c
na
5.9±1.0b
na
na
na
na
na
na
15.8±1.7a,A
na
8.6±0.0e,E
6.5±0.3b,F
10.4±0.1d,D
na
15.0±0.4ab,AB
7.1±0.7a
na
5.7±0.1b
na
6.3±0.0b
na
Clostridium perfringens
7.4±0.5bcd
na
6.6±0.1d
na
na
na
Debaryomyces hansenii
18.7±1.8a
na
12.4±0.1bc
na
9.3±2.1de
na
Enterococcus faecium
9.6±1.8a
na
6.5±0.1b
na
7.0±0.7b
na
Escherichia coli
7.0±0.2b
na
na
na
na
Lactobacillus helveticus
9.0±0.1a,A
na
8.4±0.9ab,AB
na
Listeria monocytogenes
9.0±0.8e,F
na
13.0±0.7b,B
na
na
5.7±0.1a,C
6.2±0.4c,C
na
Pseudomonas fluorescens
6.6±1.2a
na
na
Salmonella cholerasuis
9.0±0.2a
na
na
15.0±2.8a,A
na
6.6±0.0c,C
8.1±1.0a
na
17.2±0.2a,A
Bifidobacterium bifidum Brochothrix thermosphacta Citrobacter frenduii
Penicillium expansum
Shewanella putrefaciens Shigella sonnei Staphyloccus aureus Vibrio parahaemolyticus Yersinia enterocolitica
na
6.2±0.2c
11.8±0.3d na
na
10.2±0.1d,D
6.2±0.0b
na
6.3±0.3b
7.3±0.1bcd
na
na
9.6±1.0de
na
11.2±1.0cd
7.4±0.2b
na
8.8±0.3a
na
6.2±0.2c
na
na
7.5±0.6bc,BC
na
6.8±0.1c,C
na
7.3±0.8c,C
8.6±0.8e,F
na
8.4±0.7e,F
na
11.8±0.4cd,CD
5.7±0.0c,C
6.8±0.3a,C
7.1±0.1b,B
6.0±0.3a,C
7.1±0.6b,B
na
na
na
na
na
6.2±0.1ab
na
na
na
na
na
na
na
10.5±0.0b,B
7.6±0.2b,C
11.2±0.1b,B
6.0±0.1b,C
10.7±0.1b,B
6.6±0.1b
na
na
na
8.0±0.9a
na
7.5±0.6ab
na
11.4±0.2f,F
na
11.2±0.4f,F
na
13.9±0.1c,C
na
13.1±0.2d,D
8.2±0.4b
na
6.0±0.2f
na
7.2±0.0cd
na
8.8±0.1a
na
6.6±0.3e
na
na
na
na
8.5±0.3b
na
7.6±0.6cd
na
7.5±0.5cd
M AN U
Bacillus coagulans
TE D
Bacillus cereus
EP
Aspergillus niger
AC C
Aeromonas hydrophila
2f
RI PT
1f
SC
Microorganism
SHNP-S
ACCEPTED MANUSCRIPT
MRS-S
SQAP-S
SQTR-S
SHAP-S
2f
1f
2f
1f
2f
1f
2f
8.0±0.1b
na
9.3±0.3a
na
6.2±0.1c
na
6.6±0.3c
na
6.3±0.4c
na
na
6.5±0.1c,C
na
5.7±0.1d,E
na
7.7±1.0a,A
na
7.4±0.2ab,AB
7.4±1.0b,B
na
na
6.5±0.0BC
10.2±0.6a,A
na
5.6±0.3c,C
na
na
na
na
14.4±0.9b
na
17.2±0.7a
na
8.7±0.3e
na
13.9±0.5b
Bifidobacterium bifidum
6.00±1.0b
na
6.2±0.1b
na
8.2±0.1a
na
na
na
na
Brochothrix thermosphacta
15.8±1.7a,A
7.0±0.5a,F
14.2±0.1bc,BC
6.4±0.2F
10.0±0.1d,D
na
13.5±0.9c,C
7.0±0.3b,F
9.6±0.1de,DE
7.1±0.7a
na
na
na
na
na
na
na
na
Clostridium perfringens
7.4±0.5bcd
na
8.7±0.3a
Debaryomyces hansenii
18.7±1.8a
na
14.6±1.9b
Enterococcus faecium
9.6±1.8a
na
6.7±0.4b
Escherichia coli
7.0±0.2b
na
7.1±0.2b
Lactobacillus helveticus
9.0±0.1a,A
na
9.2±0.3a,A
Listeria monocytogenes
9.0±0.8e,F
na
na
5.7±0.3a,C
Pseudomonas fluorescens
6.6±1.2a
na
Salmonella cholerasuis
9.0±0.2a
Citrobacter frenduii
Penicillium expansum
Shewanella putrefaciens Shigella sonnei Staphyloccus aureus Vibrio parahaemolyticus Yersinia enterocolitica
SC
Bacillus coagulans
M AN U
Bacillus cereus
na
8.0±0.0ab
na
7.8±0.1bc
na
7.2±0.9cd
na
12.4±1.0bc
na
11.0±0.8cd
na
8.1±1.4e
na
6.4±0.2b
na
6.7±0.4b
na
7.4±0.2b
na
na
na
8.0±0.3a
na
na
na
7.2±0.3c,C
na
9.0±0.1a,A
6.7±0.7C
6.8±0.5c,C
14.2±0.2a,A
na
11.8±0.2cd,CD
na
12.6±0.1bc,BC
10.2±0.2E
11.4±0.4d,D
5.8±0.3c,C
5.9±0.1a,C
7.4±0.5b,B
na
8.2±0.1a,A
na
na
na
na
na
na
na
na
na
TE D
Aspergillus niger
na
na
na
na
na
5.4±0.2b
na
6.1±0.7b
na
6.0±0.1c,C
na
10.0±0.2b,B
na
10.8±0.2b,B
na
6.5±0.5c,C
8.1±1.0a
na
na
na
10.0±0.2b
na
na
na
6.7±0.2b
17.2±0.2a,A
7.2±0.2G
16.4±0.4b,B
na
12.1±0.6e,E
na
15.8±0.1b,B
na
12.8±0.2d,D
8.2±0.4b
na
7.0±0.2de
na
8.5±0.4ab
na
7.6±0.3c
na
6.5±0.2e
na
na
9.3±0.4a
na
7.3±0.3d
na
8.2±0.2bc
na
8.0±0.3bcd
15.0±2.8a,A
AC C
Aeromonas hydrophila
RI PT
1f
EP
Microorganism
SQES-S
Legends for Seafood Protein Hydrolysates origin (SQES, SQAP, etc.) and composition of culture media (MRS and MRS-) are shown in Table 1 and 2, respectively. (1f): media supplemented with one-fold protein hydrolysates and (2f): media supplemented with two-fold protein hydrolysates. Different letters (a,b,c…) in the same column indicate significant differences among different samples at the same concentration (1f or 2f) for the same standard (P<0.05).Different letters (A,B) in the same line indicate significant differences among samples at different concentrations (1f and 2f) for the same standard (P<0.05). ysates (SQES, SQAP, etc.) origin and composition of culture media (MRS and MRS-) are shown in Table 1 and 2, respectively. 1f): media supplemented with one-
Table 6. Bacteriocin genes identified
ACCEPTED MANUSCRIPT
Length
Amino acid
(amino acids)
identity (%)
Enterolysin A
343
343/343 (100%)
Lantibiotic I
63
63/63 (100%)
E. faecalis pathogenicity island(AF454824)
Lantibiotic II
68
33/33 (100%)
E. faecalis pathogenicity island(AF454824)
Bacteriocin
Annotation E. faecalis (Q9F8B0)
AC C
EP
TE D
M AN U
SC
RI PT
UviB-like* 82 54/69 (78%) E. faecalis 20-SD-BW-06 (EPH69978) *UviB-like has not been taken into account as bacteriocin due the amino acid identity is less than 95%
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
The influence of SPHs on survival and metabolic activities was investigated
ACCEPTED MANUSCRIPT
Hydrolysates from squid and shrimp stimulated Enterococcus faecalis DM19 growth Enterococcus faecalis DM19 produced lactic acid with excellent efficiency
AC C
EP
TE D
M AN U
SC
RI PT
Antimicrobial activity increased in media with higher amounts of SPHs