Characterization and application of newly isolated nisin producing Lactococcus lactis strains for control of Listeria monocytogenes growth in fresh cheese

Accepted Manuscript Characterization and application of newly isolated nisin producing Lactococcus lactis strains for control of Listeria monocytogenes growth in fresh cheese Kristina Mulkyte, Neringa Kasnauskyte, Loreta Serniene, Greta Gölz, Thomas Alter, Vilma Kaskoniene, Audrius Sigitas Maruska, Mindaugas Malakauskas PII:

S0023-6438(17)30696-5

DOI:

10.1016/j.lwt.2017.09.021

Reference:

YFSTL 6536

To appear in:

LWT - Food Science and Technology

Received Date: 10 April 2017 Revised Date:

13 September 2017

Accepted Date: 15 September 2017

Please cite this article as: Mulkyte, K., Kasnauskyte, N., Serniene, L., Gölz, G., Alter, T., Kaskoniene, V., Maruska, A.S., Malakauskas, M., Characterization and application of newly isolated nisin producing Lactococcus lactis strains for control of Listeria monocytogenes growth in fresh cheese, LWT - Food Science and Technology (2017), doi: 10.1016/j.lwt.2017.09.021. 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 Characterization and application of newly isolated nisin producing Lactococcus lactis strains

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for control of Listeria monocytogenes growth in fresh cheese

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Kristina Mulkytea, Neringa Kasnauskytea, Loreta Sernienea, Greta Gölzb, Thomas Alterb, Vilma

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Kaskonienec, Audrius Sigitas Maruskac and Mindaugas Malakauskasa

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Author Affiliation

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Sciences, Tilzes st. 18, LT-47181, Kaunas, Lithuania.

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Department of Food Safety and Quality, Veterinary Academy, Lithuanian University of Health

Department of Veterinary Medicine, Institute of Food Safety and Food Hygiene, Freie Universität

Berlin, Königsweg 69, 14163 Berlin, Germany.

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Lithuania.

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Faculty of Natural Sciences, Vytautas Magnus University, Vileikos st. 8-212, LT-44404, Kaunas,

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Contact information for Corresponding Author

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Kristina Mulkytė, Tilzes st. 18, LT-47181, Kaunas, Lithuania, [email protected], Tel.:

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+37069648550

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ACCEPTED MANUSCRIPT ABSTRACT

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This study was aimed to screen for nisin producing Lactococcus lactis strains with prominent

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antimicrobial and technological characteristics applicable for the dairy industry. Twelve out of 181

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Lactococcus spp. strains isolated from goat and cow milk, fermented wheat and buckwheat samples

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were identified as L. lactis strains harbouring nisin A, Z or novel nisin variant GLc03 genes. For the

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first time technological characteristics of L. lactis strains harbouring nisin variant GLc03 were

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evaluated. All 12 isolated strains showed clear antagonistic activity against tested food spoilage and

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pathogenic bacteria. Moreover, strains encoding nisin Z presented favourable enzymatic activities of

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acid phosphatase, esterase lipase and phosphohydrolase. However, strains encoding novel nisin

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variant GLc03 were lacking appropriate technological characteristics being resistant to tetracycline

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and producing harmful β-glucosidase, therefore these strains are not applicable for the dairy food

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production. Two nisin A producing L. lactis strains were poor acidifiers and one strain showed

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resistance to tetracycline. Three strains were selected according to safety and technological criteria

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and were examined in fresh cheese production for control of Listeria monocytogenes growth.

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Listeria numbers were significantly reduced (P < 0.001) in model cheese suggesting the application

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of these strains for fresh cheese safety improvement.

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Keywords: nisin; Lactococcus lactis; Listeria monocytogenes; cheese

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ACCEPTED MANUSCRIPT 1. Introduction

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Dairy industry faces many challenges and inhibition of food spoilage and pathogenic bacteria in

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order to extend shelf life of dairy products is one of the main goals. Achieving this goal through

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biopreservation using natural antimicrobial compounds is of big interest not only for dairy but also

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for the whole food industry and attracts special attention of consumers. In recent years one of the

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biggest considerations in biopreservation is dedicated to lactic acid bacteria (LAB) (Grosu-Tudor,

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Stancu, Pelinescu, & Zamfir, 2014; Ponce, Moreira, del Valle, & Roura, 2008).

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LAB could be found naturally in various food sources like dairy products, meat and vegetables. LAB

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produces several antimicrobial compounds that make these bacteria suitable for food

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biopreservation. These antimicrobial compounds include organic acids, reuterin, diacetyl, acetoin,

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hydrogen peroxide, antifungal peptides and bacteriocins (Reis, Paula, Casarotti, & Penna, 2012).

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Production of bacteriocins enhances the ability of LAB to control the growth of pathogens and food

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spoilage bacteria in food products (Dal Bello et al., 2012) and makes them of particular interest to

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food industry offering natural alternatives for chemical additives to improve the safety and quality of

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food products. Bacteriocins are considered to be safe food biopreservatives and can be degraded by

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gastrointestinal proteases (Jeevaratnam, Jamuna, & Bawa, 2005). Moreover, bacteriocins are natural

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means that could control or reduce listeria numbers in food, especially in dairy products. Special

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attention is drawn to fresh cheese as it could be contaminated with L. monocytogenes – pathogenic

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bacteria, that is hard to control in this dairy product (Coelho, Silva, Ribeiro, Dapkevicius, & Rosa,

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2014). Among the LAB group, Lactococcus lactis is well known and often used in the food industry

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due to well-expressed production of bacteriocin nisin. Nisin has suitable characteristics for food

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preservation like high activity, a broad spectrum of antibacterial activity, rapid action, high stability

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against high temperature and acid (Zendo, 2013). The incorporation of nisin producing L. lactis

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strain as starter, adjunct or protective culture provides an alternative not only to chemical additives

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but could also contribute to better sensory characteristics of foods. While nisin A is the best studied

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ACCEPTED MANUSCRIPT bacteriocin produced by LAB, newly isolated nisin producing bacteria or novel nisin variants with

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different antagonistic activity could help to control undesirable bacteria more efficiently.

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Newly isolated bacteriocin producing bacteria have to be properly characterized in order to be

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applied for food production. It is important to determine the type of bacteriocin produced, evaluate

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antimicrobial spectra and efficiency, technologically relevant abilities as well as other important

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characteristics. In dairy industry for cheese production most important characteristics of L. lactis are

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the ability to produce acid rapidly (Cogan et al., 1997), salt tolerance, proteolytic activity (Hannon et

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al., 2003), diacetyl production (Beshkova, Simova, Frengova, Simov, & Dimitrov, 2003) and

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antibiotic resistance (Nieto-Arribas, Seseña, Poveda, Palop, & Cabezas, 2009).

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The aim of this study was to select nisin producing L. lactis strains with exceptional technological

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characteristics and antibacterial activity isolated from local goat and cow milk, fermented wheat and

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buckwheat samples in order to apply them for more effective control of Listeria monocytogenes in

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fresh cheese manufacturing.

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2. Materials and methods

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2.1. Isolation of LAB

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Raw goat and cow milk samples were collected from local markets and kept refrigerated (4°C) until

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analysis. Grain samples were fermented by traditional fermentation and also fermented wheat and

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buckwheat drinks were used for isolation of LAB. Fermented drinks were made by soaking grains

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with water for 12 h then washed and left to ferment with warm water. After 48 h of fermentation the

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grains were drained and liquid was used as fermented beverage.

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Samples were subjected to ten-fold dilution series using maximum recovery diluent (Oxoid,

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Basingstoke, UK). Selected dilutions were spread in duplicates on plate count agar (PCA)

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(Liofilchem, Roseto degli Abruzzi, Italy) supplemented with 10% sterile skim milk (Oxoid), 20

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mg/L bromcresol purple (Sigma Aldrich, St. Louis, U.S.), 40 mg/L nalidixic acid, 10 mg/L

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natamycin (both Sigma Aldrich) and incubated at 30°C for 48 h under aerobic conditions (Corroler,

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ACCEPTED MANUSCRIPT Mangin, Desmasures, & Gueguen, 1998). After incubation, representative colonies from each

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sample were selected and purified on M17 agar plates (Merck, Darmstadt, Germany) by several

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transfers. Incubation was done for 48 h at 37°C. Pure colonies were selected and subjected to Gram

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staining and catalase tests. LAB characteristic colonies (Gram positive, catalase negative) were

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stored at -80°C in M17 broth (Merck) in the presence of 30% glycerol until further analysis. Before

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conducting any experiments, strains were revitalized in MRS broth (Biolife, Milano, Italy) by

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growing for 18 h at 30°C.

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2.2. Identification of nisin encoding gene

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PCR amplification was carried out for species verification of L. lactis and to detect the presence of

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nisin genes. DNA extraction was performed using GenEluteBacterial Genomic DNA kit (Sigma-

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Aldrich) and following the manufacturer’s instructions for gram positive bacteria. The primers used

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to identify L. lactis were G1 (5’-GAAGTCGTAACAAGG-3’) and L1 (5’-CAAGGCATCCACCGT-

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3’), while nisin genes were detected by the primers NISL (5’-CGAGCATAATAAACGGC-3’) and

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NISR (5’-GGATAGTATCCATGTCTGAAC-3’). PCR conditions were the same as described

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previously by Moschetti and others (Moschetti, Blaiotta, Villani, & Coppola, 2001).

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PCR products were separated in 2% agarose gel using 100 bp DNA ladder (Thermo Fisher

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Scientific, Vilnius, Lithuania) as the molecular weight standard and visualized by ethidium bromide

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staining (Sigma-Aldrich). A 380 bp fragment verified L. lactis and 320 bp fragment represented

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nisin structural gene.

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From nis+ isolates the nisin gene was amplified by PCR using primers NisP5 (5’-

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GGTTTGGTATCTGTTTCGAAG-3’) and NisP3 (5’-TCTTTCCCATTAACTTGTACTGTG-3’)

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and conditions described by Pisano et al. (2015). PCR products were subsequently purified using the

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GeneJET PCR Purification Kit (Thermo Fisher Scientific) and sequenced. To determine nisin

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variants, the sequences of PCR products for the nisin gene were in silico translated to amino acid

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sequences using BioNumerics 7.1 (Applied Maths, Sint-Maart, Belgium) and compared with the

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ACCEPTED MANUSCRIPT sequences of other nisin variants from GeneBank database (http://www.ncbi.nlm.nih.gov/). For

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identification of lactococcin A, B or M genes in nisin producing L. lactis strains, primers LactABM-

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F (5’-GAAGAGGGCAATCAGTAGAG-3’), LactA-R (5’-GTGTTCTATTTATAGCTAATG-3’),

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LactB-R (5’-CCAGGATTTTCTTGATTTACTTC-3’), LactM-R (5’-

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GTGTACTGGTCTAGCATAAG-3’) and PCR conditions described by Pisano et al. (2015) were

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used. Amplification products were visualized on ethidium bromide-stained (Sigma-Aldrich) 1.8%

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agarose gel using 100 bp DNA ladder (Thermo Fisher Scientific) as the molecular weight standard.

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2.3. Antibacterial activity of nisin positive LAB strains

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Antibacterial activity was evaluated using agar spot test (Schillinger & Lücke, 1989). Of each

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revitalized strain, 3 µl were spotted on the surface of MRS agar (Biolife) and incubated

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anaerobically in a jar with Anaerogen (Oxoid) for the generation of anaerobic conditions for 24 h at

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30°C. Plates were then overlaid with 7 mL soft agar (0.7%) inoculated with 100 µl of the indicator

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strain and incubated for 24 h at optimal growth temperature and atmosphere for the indicator strain.

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All indicator strains used in the study (Fig. 2) were revitalized before the experiment in the

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appropriate medium (brain-heart infusion broth (Oxoid) was used for all of the tested pathogenic

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strains and MRS broth (Biolife, Milano, Italy) was used for Lactobacillus delbruecki revitalization)

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and temperature (37°C temperature was used for all of the strains, except Bacillus cereus and

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Pseudomonas florescens were revitalized at 30°C, Brochotix thermosphacta at 25°C). Antibacterial

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activity was evaluated by measuring clear inhibition zone diameter around the colony of the tested

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strain.

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2.4. Antibacterial activity of cell-free supernatant of nisin producing LAB strains

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LAB isolates were revitalized and the cells were harvested by centrifugation at 14,000 rpm for 10

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min and the pH of the CFS was adjusted to 6.0 with 1 M NaOH and heat treated for 10 min at 80°C.

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10 µl of CFS was spotted on the surface of LB agar (Liofilchem) previously inoculated with the

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ACCEPTED MANUSCRIPT indicator strain. Plates were incubated at 30°C for 24 h and the presence of an inhibition zone around

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the spotted CFS indicated a positive result.

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2.5. Technological characterization of nisin producing L. lactis strains

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2.5.1. Extracellular proteolytic activity

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Two µl of revitalized strains were spotted on the surface of skim milk agar composed of 20% sterile

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skim milk and 80% water agar. After incubation at 30°C for 4 days, clear zones around the colonies

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indicated proteolytic activity.

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2.5.2. Caseinolytic and lipolytic activities

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Caseinolytic and lipolytic activities were determined according to the methods of Pisano et al.

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(2015). To evaluate caseinolytic activity, plates containing PCA (Liofilchem) supplemented with

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10% sterile reconstituted skim milk (Oxoid) were used. After 18 h incubation at 30°C in aerobic

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conditions, clear zones around the colony indicated caseinolytic activity. Lipolytic activity was

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tested on M17 agar (Merck) supplemented with 0.01% calcium chloride and 0.1% Tween 80

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(Liofilchem). Lipolytic colonies were surrounded with cloudy zones.

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2.5.3 Diacetyl production

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10 mL of UHT milk was inoculated with 1% (v/v) of revitalized strains and incubated at 30°C for

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24 h. 1 mL of each culture was mixed with 0.5 mL of 1% (v/v) α-naphtol (Sigma-Aldrich) and 16%

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(w/v) KOH and incubated at 30°C for 10 min. Diacetyl production was indicated by the formation of

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a red ring at the top of the tubes (Dal Bello et al., 2012).

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2.5.4. Acidifying activity

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UHT low-fat milk (1.5%) was inoculated with 1% of revitalized strains and pH values were

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measured after 6 and 24 h. Non-inoculated milk was used as control.

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2.5.5. Growth in different salt concentrations

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Strains were grown in M17 broth supplemented with 4% and 6.5% NaCl. Salt tolerance was

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evaluated after 48 h incubation at 30°C and evaluated by visual observation.

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ACCEPTED MANUSCRIPT 2.5.6. Antibiotic resistance evaluation

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Antibiotic susceptibility was evaluated using MIC Test Strips (Liofilchem) and following the

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manufacturer’s instructions. The antibiotics tested were chloramphenicol, clindamycin,

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streptomycin, gentamicin, tetracycline, erythromycin and ampicillin. Minimum Inhibitory

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Concentrations (MIC) were determined from the MIC reading scale and expressed in µg/mL.

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2.5.7. Enzymatic activity evaluation

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Enzymatic activity was evaluated using the API ZYM kit (bioMerieux, Marcy-l’Étoile, France). The

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API ZYM strips were inoculated, incubated, and interpreted according to the manufacturer’s

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instructions. Changes of color were scored from 0 to 5. Color reaction grade 0 was interpreted to

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correspond to a negative reaction, grades 1 and 2 corresponded to a weak reaction (5 to <20 nmol)

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and grades 3, 4, and 5 corresponded to a strong reaction (>20 nmol).

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2.6.

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Fresh cheeses were experimantally prepared with pasteurised cow‘s milk obtained from local farm

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using strains 56, 59 and 63 respectively. Three individual batches of each cheese were prepared.

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After warming milk to 30°C, calcium chloride (0.2 g/L, Merck) was added to the milk. Milk was

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distributed in three 3.0 L vats and individually inoculated with 2% L. monocytogenes suspension for

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a final concentration of 104-106 cfu/mL. 2 % of each LAB culture was then added to the milk and

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incubated for 2 h. After incubation rennet (Hansen Sticks, pure chymosin (EC 3.4.23.4), 1400

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IMCU/stick (>= 1.300 IMCU/S), 0.02 g/L) was added to the milk and incubated at 30 °C for 6 h.

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Control cheese from pasteurised milk was made without any LAB culture, just with L.

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monocytogenes inoculum. Once the coagulum was sufficiently firm, it was cut into 1-2 cm cubes and

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agitated slowly for 30 min at 21 °C. The curd was transferred to synthetic cheese bags and

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maintained at 21 °C for 1 h for dripping. Fresh cheeses were unmoulded (emptied from the bags),

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packed into plastic bags, and stored at 4°C for 0, 6, 24, 48, 72 (3 days) and 168 (7 days) h. For

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enumeration of L. monocytogenes, cheese samples were diluted (1:10, w/v) in buffered peptone

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Application of nisin producng L. lactis strains in fresh cheese

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ACCEPTED MANUSCRIPT water (Liofilchem) and mixed. The mixture was serially diluted, plated on Agar Listeria Ottavany &

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Agosti (Biolife, Italy) with supplements and incubated at 37°C for 48 h.

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3. Results and discussion

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3.1. Isolation and identification of nisin producing L. lactis strains

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LAB are usually found in nutrient-rich environments and are able to grow in most raw foods,

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however requires fermentable carbohydrates, amino acids, fatty acids, salts and vitamins for growth

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(Abbasiliasi et al., 2012). Most commonly bacteriocin producing L. lactis strains are isolated from

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dairy products like cheese (Alegría, Delgado, Roces, López, & Mayo, 2010; Kumari, Akkoç, &

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Akçelik, 2012) and milk (Bravo, Rodríguez, & Medina, 2009), also from fermented foods like

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fermented vegetables (Grosu-Tudor et al., 2014) and meat products (Biscola et al., 2013). Therefore,

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we chose local cow and goat milk, fermented wheat and buckwheat samples to screen for bacteriocin

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producing L. lactis strains. PCR based identification of selected presumptive 745 Lactoccocus spp.

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colonies confirmed 181 isolates as L. lactis (Table 1). Twelve of these strains were nisin producers

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based on the niz+ gene identification. We also screened for the bacteriocin lactococcin B gene in

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these strains as it was previously reported that L. lactis strains, coding for nisin genes, could also

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encode for lactococcin B (Bello et al., 2010; Pisano et al., 2015). Four out of 12 strains encoded for

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both a nisin and the lactococcin B gene (strains 22, 10R, 23R and 24R). Lactococcin B exclusively

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inhibits the growth of sensitive lactococci. Strains that produces this bacteriocin could be used as

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starters in cheese making process to mediate lysis of natural starter strains in order to accelerate

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ripening and increase flavor development (Pisano et al., 2015).

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L. lactis are able to produce various nisin variants, therefore amplified nisin genes of all 12 L. lactis

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strains were sequenced and in silico translated to compare the amino acid sequences. Sequencing of

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structural nis-genes revealed that two L. lactis strains encode for nisin A, four strains for nisin Z, and

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six strains for the novel nisin variant GLc03 (KF146295) (Fig. 1), recently described by other

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researchers (Perin & Nero, 2014).

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ACCEPTED MANUSCRIPT 3.2. Antibacterial activity evaluation

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Antibacterial activity evaluation of nisin producing L. lactis strains using agar spot test method

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revealed different inhibitory activities against foodborne pathogenic and spoilage bacteria (Fig. 2).

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Non-pathogenic Lactobacillus delbruecki ATCC 12315 strain was used to test the antimicrobial

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effect of bacteriocins and combination of other antimicrobial compounds produced like organic acid,

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hydrogen peroxide and others. Strains isolated from raw goat milk encoding nisin Z gene (22, 56,

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59) showed highest antibacterial activity against L. monocytogenes and E. coli, whereas inhibition of

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spoilage bacteria such as Brochothrix thermosphacta was more prominent in strains isolated from

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raw cow milk encoding nisin A gene (strains 15, 20). Overall, strains 10R, 23R, 24R, 25R, 28R and

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31R isolated from fermented wheat or buckwheat samples encoding novel nisin variant GLc03 had

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an exceptional antagonistic activity against Bacillus cereus, bacteria which could cause food

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poisoning (Crovadore et al., 2016). The biggest zone of inhibition (33 mm) against B. cereus was

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generated by L. lactis strain 28R, approximately four fold higher compared to control strain L. lactis

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ATCC 11454. In addition these strains showed high antibacterial activity against Salmonella

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Typhimurium, a bacterium which is the most important cause of bacterial human foodborne

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outbreaks in the EU (EFSA, 2015). These results correlate with findings of Perin and Nero, who first

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identified and described the antibacterial activity of the novel nisin variant GLc03 and stated that

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inhibitory activity against Gram negative bacteria must be due to non-specific antimicrobial

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substances produced by L. lactis like organic acids or peroxide (Perin & Nero, 2014). However,

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antibacterial activity of cell free supernatants (CFS) showed different antibacterial activity results

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(data not shown). Only supernatants of strains 15, 20, 22, 56, 59 and 63 isolated from raw cow and

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goat milk samples showed antibacterial activity against all eight tested indicator bacteria. However,

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none of the supernatants of the L. lactis strains, isolated from fermented wheat or buckwheat

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samples, showed antibacterial activity. It was previously reported, that not always antibacterial

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activity determined by agar spot test method is confirmed with CFS (Alegría et al., 2010; Hernández,

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ACCEPTED MANUSCRIPT Cardell, & Zárate, 2005; Larsen, Vogensen, & Josephsen, 1993). This could be explained by the

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presence of other compounds produced by LAB (like hydrogen peroxide and fatty acids) that enables

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observation of antibacterial activity using agar spot test method (De Vuyst & Leroy, 2007).

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3.3. Technological characterization

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Main technological characteristics of nisin producing L. lactis strains were evaluated in order to

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assess the suitability of isolated strains to be used in the dairy industry (Table 2). To our knowledge,

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this is the first study evaluating technological characteristics of L. lactis strains encoding novel nisin

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variant GLc03. Six strains 15, 20, 10R, 23R, 24R and 25R showed strong and three strains ATCC

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11454, 59 and 31R showed weak caseinolytic activity. However, none of the tested strains

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demonstrated lipolytic activity or had the ability to produce diacetyl. Three strains demonstrated

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extracellular proteolytic activity. All strains tolerated up to 4% NaCl concentration, whereas the four

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strains 15, 10R, 23R and 24R, among which three were isolated from fermented grain and one from

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cow milk sample, showed only weak tolerance to stronger salt concentration, demonstrated by weak

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growth at 6.5% NaCl. In production of dairy products, NaCl could be applied up to 6% (Schirru et

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al., 2012). That makes the ability of strains to tolerate salt concentrations higher than 6% very

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important. Eight isolated nisin producing L. lactis demonstrated this ability. L. lactis strain 22,

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encoding nisin Z gene, showed high acidifying activity (pH dropped by 2.39 units after 24 h)

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whereas other strains induced pH drop from 1.3 to 1.94 units. Three strains 15, 20 and 23R were

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only weak acidifiers showing pH drop of 0.35, 0.63 and 0.75 respectively. However, six strains were

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found to be more efficient acidifiers than L. lactis control strain.

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3.4. Safety evaluation

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In biopreservation, safety of LAB is another important prerequisite. Therefore antibiotic resistance

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of all nisin producing L. lactis strains was assessed (Table 3). Antibiotic resistant strains can be

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harmful to the health of both humans and animals because they are capable of transferring antibiotic

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resistance genes to pathogenic bacteria, which can contaminate raw food products like milk and

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ACCEPTED MANUSCRIPT meat (Abbasiliasi et al., 2012). Four nisin producing L. lactis strains showed antibiotic resistance

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above the breakpoint provided by European Food Safety Authority (EFSA, 2012) to tetracyclin. The

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breakpoint for L. lactis to tetracyclin suggested by EFSA is 4 µg/mL whereas strain 20 isolated from

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raw cow milk (encoding nisin A gene) had minimum inhibitory concentration of 6 µg/mL, strains

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10R, 23R and 24R isolated from fermented wheat and buckwheat samples (encoding GLc03 gene)

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had minimum inhibitory concentrations of 128 µg/mL, 16 µg/mL and 8 µg/mL, respectively.

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Resistance to tetracycline may be caused by the huge amount of this antibiotic that have been used

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for years in agriculture livestock farming (Zycka-Krzesinska, Boguslawska, Aleksandrzak-

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Piekarczyk, Jopek, & Bardowski, 2015). Apart from the tetracycline resistance, all strains were

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susceptible to clindamycin, gentamicin, chloramphenicol, erythromycin and ampicillin. Ampicillin,

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erythromycin, chloramphenicol and tetracycline are antibiotics belonging to the β-lactam group. It

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was previously reported that Lactobacillus, Lactococcus and Pediococcus acidilactici strains were

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sensitive to β-lactam antibiotics (Abbasiliasi et al., 2012; Liasi et al., 2009) and our results indicate

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similar findings.

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Although L. lactis is included in the Qualified Presumption of Safety (QPS) list (Andreoletti et al.,

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2012) and authorized for use in the food and feed chain within the EU, some properties and

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enzymatic activity of these strains can produce hazardous compounds that should be avoided in food

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products. From the enzymatic point of view, strains should not produce harmful enzymes like β-

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glucosidase or β-glucuronidase (Ji, Jang, & Kim, 2015). To test any activities of these enzymes, the

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API ZYM kit was used (Table 4). No activity of β-glucuronidase was detected, though four strains

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(23R, 24R, 28R and 31R) of plant origin were found to produce strong β-glucosidase activity. Other

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enzymatic activities evaluation revealed no or weak activities for alkaline phosphatase, esterase

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(C4), esterase lipase (C8), lipase (C14), trypsin, α-galactosidase, α-glucosidase, N-acetyl-β-

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glucosaminidase, α-mannosidase, α-fucosidase. Weak phosphohydrolase activities were determined

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for all of the strains except strain 59, isolated from raw goat milk (encoding nisin Z gene). Activity

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ACCEPTED MANUSCRIPT of this enzyme is important for the hydrolysis of phosphopeptides during cheese ripening (Fox,

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Lucey, & Cogan, 1990). Strains 22, 56, 59 and 63 presented the best favorable enzymatic activities

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of acid phosphatase (an enzyme that is essential for the hydrolysis of phosphopeptides prevalent in

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cheese ripening), leucine (strong activity), valine and cysteine arylamidase (weak activity except

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strain 63 that showed no activity of these enzymes that are linked to flavour formation).

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3.5.

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cheese

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To detect the strain most suitable for practical application in the dairy industry as an additional

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measure against L. monocytogenes, we tested the inhibitory effect on L. monocytogenes of our three

308

nisin producing L. lactis strains (56, 59 and 63). These strains were found to be safe and had

309

interesting technological characteristics. The results are presented in Fig. 3.

310

The decrease of L. monocytogenes numbers (P<0.001) was observed immediately after 1 h and

311

remained significant compared to control (P<0.001) during storage in all cheeses containing all three

312

nisin producing L. lactis strains. However, after 7 days of storage the biggest impact compared to

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other nisin producing L. lactis strains (P<0.05) on listeria numbers was observed in cheese

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inoculated with strain 63. This strain showed weaker antibacterial activity than strains 56 and 59

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against L. monocytogenes using agar spot test, but was able to reduce listeria counts in experimental

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cheese more effectively. Overall, all three nisin producing L. lactis strains reduced listeria numbers

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almost by 2 log units during seven days of storage and this decrease was significant (P<0.001)

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compared to control sample. These results comply with other authors, who used bacteriocin

319

producing L. lactis strains to control L. monocytogenes growth in fresh cheese (Coelho et al., 2014).

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4. Conclusions

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In this study, 12 nisin producing L. lactis strains were identified from the collection of 181 L. lactis

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isolates. Two nisin A, four nisin Z and six novel nisin variant GLc03 producing L. lactis strains were

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evaluated according to safety and technological criteria. Only four strains encoding nisin Z (22, 56,

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Application of nisin producing L. lactis strains for control of L. monocytogenes in fresh

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different broad range antagonistic activity and technologically interesting characteristics. These

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strains were good acidifiers, had the ability to tolerate salt, moreover all these strains had acid

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phosphatase activity, strains 22 and 56 had esterase lipase (C4 and C8) and strain 59 had good

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phosphohydrolase activity. These enzymatic activities are important technological characteristics for

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dairy industry in cheese production. In order to prevent the growth of L. monocytogenes in fresh

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cheese, three strains (56, 59 and 63) were selected for fresh cheese manufacturing (Table 5 shows

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summarized elimination of certain strains from the cheese experiment) and were able to significantly

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reduce (P < 0.001) listeria numbers within 7 days of cheese storage. Although the reduction of

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listeria numbers by 5 log units is preferred, the reduction by 2 log units after 7 days of storage could

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be improved using hurdle technology and combining the effect of nisin producing L. lactis strains

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with other hurdles like modified atmosphere packaging. Further experiments should be done in this

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field, but at this time our isolated L. lactis strains demonstrated a great potential for application as an

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additional measure to control the growth of Listeria monocytogenes in fresh cheese production.

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Acknowledgments

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This work was supported by the Research Council of Lithuania (grant number MIP-63/2015).

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Tables list: Table 1. Isolation of L. lactis strains from milk and fermented grain samples

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Table 2. Technological characteristics of nisin producing L. lactis strains Table 3. Antibiotic susceptibility of nisin producing L. lactis strains

Table 4. Enzymatic activities of nisin producing L. lactis strains evaluated by the API-ZYM test

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Table 5. Properties of nisin producing L. lactis strains that influenced the elimination of certain strains from cheese experiments

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Figures list:

Fig. 1. Amino acid sequences of nisin variants showing amino acid changes deducted by the sequencing of nisin region from 12 Lactococcus lactis strains

Fig. 2. Antibacterial activity of nisin producing Lactococcus lactis strains

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model cheeses during 7 days of storage at 4°C

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Table 1. Isolation of L. lactis strains from milk and fermented grain samples

11

130

50 (38)

47 (36)

18

50

29 (58)

29 (58)

17

90

14 (16)

11 (12)

5

50

4 (8)

4 (50)

5

48

10 (21)

5

50

6 (12)

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Raw cow milk Fermented cow milk Raw goat milk Fermented goat milk Fermented buckwheat1 Fermented buckwheat2 Fermented wheat1 Fermented wheat2

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Number of colonies identified as L. lactis (%) 72 (26)

Number of selected colonies

6 (13)

6 (12)

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Sample name

Number of colonies with Lactococcus phenotype (%) 75 (27)

Number of samples

10 (20)

1

fermented by traditional fermentation

2

fermented beverage

6 (12)

Lipolytic activity

Diacetyl production

+/-

-

-

+

-

+ + + + + + + + + + + +

+ + +/+ + + + +/-

-

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L. lactis ATCC 11454 15 20 22 56 59 63 10R 23R 24R 25R 28R 31R a +/-

Salt tolerance 4% 6,5% NaCl NaCl

Caseinolytic activity

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Table 2. Technological characteristics of nisin producing L. lactis strains. Acidifying activitya ∆pH (6 h)

∆pH (24 h)

Extracellular proteolytic activity

+

0.33±0.01

1.72±0.02

-

+/+ + + + + +/+/+/+ + +

0.14±0.01 0.14±0.01 0.30±0.00 0.33±0.01 0.41±0.01 0.39±0.03 0.01±0.01 0.04±0.01 0.06±0.01 0.06±0.04 0.14±0.03 0.14±0.02

0.63±0.01 0.75±0.01 2.39±0.01 1.71±0.04 1.75±0.02 1.80±0.04 1.42±0.01 0.35±0.01 1.30±0.02 1.87±0.00 1.88±0.03 1.94±0.03

+ + + -

values presented are means of three replicates ± SD weak activity

2

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Table 3. Antibiotic susceptibility of nisin producing L. lactis strains.

L. lactis ATCC 11454

20

22

56

59

63

24R

25R

28R

31R

Minimum inhibitory concentrations (MIC) µg/mL Chloramphenicol 4 4 2 4 3 4 4 3 3 4 Clindamycin 0.016 0.032 0.047 0.125 0.016 0.023 0.032 0.064 0.047 0.047 Streptomycin 3 3 0.094 3 6 6 4 1.5 1.5 0.094 Gentamicin 0.125 0.19 0.19 0.094 0.19 0.25 0.25 0.064 0.023 0.023 a Tetracycline 0.125 0.19 6 0.19 0.19 0.125 0.25 128a 16a 8a Erythromycin 0.016 0.023 0.047 0.064 0.032 0.047 0.064 0.064 0.047 0.047 Ampicillin 0.047 0.094 0.125 0.094 0.064 0.125 0.25 0.094 0.023 0.023 a Values above the breakpoint provided by EFSA (2012).

3 0.094 6 0.19 0.19 0.094 0.19

2 0.094 6 0.19 0.19 0.047 0.125

3 0.094 3 0.125 0.25 0.094 0.125

10R

23R

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Nisin producing L. lactis strains

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Table 4. Enzymatic activities of nisin producing L. lactis strains evaluated by the API-ZYM test.

Cystine arylamidase

3 0 0 5

15

20

22

56

59

63

10R

23R

24R

25R

28R

31R

0 0 0 0 4 1

1 0 1 0 3 1

0 1 1 0 3 1

1 1 1 0 3 1

2 2 2 0 5 2

1 1 1 0 4 0

1 1 1 0 3 1

1 3 2 0 2 1

1 3 2 0 3 1

1 1 1 0 3 1

1 1 0 0 2 0

1 0 0 0 4 1

1 0 1 2

2 0 1 1

1 0 0 3

1 0 0 4

2 0 1 5

0 0 0 5

1 0 0 4

1 0 0 2

1 0 1 1

1 0 1 4

0 0 0 4

1 0 0 4

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Alkaline phosphatase Esterase (C4) Esterase lipase (C8) Lipase (C14) Leucine arylamidase Valine arylamidase

L. lactis ATCC 11454 1 1 1 1 5 3

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Nisin producing L. lactis strainsa

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Trypsin α-chymotrypsin Acid phosphatase Naphthol-AS-BI3 1 2 2 2 4 2 2 3 2 1 2 phosphohydrolase α-galactosidase 0 1 0 0 0 0 0 0 0 0 0 0 β- galactosidase 0 0 0 1 0 1 1 0 2 3 1 1 β- glucuronidase 0 0 0 0 0 0 0 0 0 0 0 0 α-glucosidase 0 0 0 1 1 1 1 0 2 3 1 2 β-glucosidase 1 0 0 0 0 0 0 0 5 5 1 3 N-acetyl-β0 0 0 0 0 0 0 0 0 0 0 0 glucosaminidase α-mannosidase 0 0 0 0 0 0 0 0 0 0 0 0 α-fucosidase 0 0 0 0 0 0 0 0 0 0 0 0 a Color reaction grade 0 was interpreted to correspond to a negative reaction, grades 1 and 2 corresponded to a weak reaction (5 to <20 nmol) and grades 3, 4, and 5 corresponded to a strong reaction (>20 nmol).

4

2 0 1 0 1 3 0 0 0

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Table 5. Properties of nisin producing L. lactis strains that influenced the elimination of certain strains from cheese experiments

Nisin GLc03

15 20 22* 56 59 63 10R 23R 24R 25R 28R 31R

+ + + + -

-

negative result positive result

+ + + -

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Production of harmful enzymes + + + + +

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Nisin Z

Antibiotic resistance

Lacking technological characteristics

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Nisin A

Safety evaluation

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*- was excluded from cheese experiment because of the specific unpleasant odour during

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acidifying activity test.

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Fig. 1. Amino acid sequences of nisin variants showing amino acid changes deducted by the sequencing of nisin region from 12 Lactococcus

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lactis strains. Strains 15 and 20 were isolated from raw cow milk and encode nisin A gene, strains 22, isolated from raw cow milk and 56, 59, 63,

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isolated from raw goat milk, encode nisin Z gene, while strains 10R, 23R, 24R, 25R, 28R, 31R isolated from fermented wheat and buckwheat samples encode novel nisin variant GLc03 gene.

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Fig. 2. Antibacterial activity of nisin producing Lactococcus lactis strains. Non-pathogenic

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Lactobacillus delbruecki ATCC 12315 strain was used to test the antimicrobial effect of bacteriocins and combination of other antimicrobial compounds produced by L. lactis like organic acid, hydrogen peroxide and others. The origin of strains is indicated by the colour code: light grey = isolated from raw cow milk, dark grey = isolated from raw goat milk, black = isolated from wheat or buckwheat. Values are means ± standard deviations of three replicates.

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Fig. 3. Effect of nisin producing L. lactis strains on the growth of L. monocytogenes in fresh model cheeses during 7 days of storage at 4°C. Values are means ± standard deviations of three

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replicates. Significant differences noted with respect to control cheese: *P<0.001.

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Highlights

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12 Lactococcus lactis strains were producers of bacteriocin nisin A, Z or novel variant GLc03 Evaluation of technological properties of L. lactis strains with novel nisin variant GLc03 Selected nis+ L. lactis strains significantly reduced listeria numbers in fresh cheese

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