Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Economic losses in the U.S. dairy food supply (milk, cheeses, and other cultured milk products) may be caused by microbial contamination, by fermentation failures due to poor starter culture performance resulting from harsh processing conditions or virus infection. Microbial contamination of dairy foods may be reduced by the presence of food grade bacteria that produce simple or complex natural inhibitory substances. Marketability and nutritive value of dairy foods also may be improved by supplementation with other, nutritionally or pharmacologically active products, resulting from the activity of food grade microbes on key milk protein components. These objectives can be approached by developing dairy lactic fermentation bacteria to produce bioactive peptides or enzyme systems in amounts to achieve the desired effect. Dairy fermentation failures may be reduced by developing lactic starter
cultures with greater heat and acid resistance under harsh production conditions, and with increased resistance to virus attack. To achieve objectives, microbial biochemistry, molecular biology and biotechnology methods are used to optimize the level of gene expression in selected target microbes under a variety of conditions. Molecular biology research tools are used to identify stress proteins and virus (bacteriophage) resistance systems and transfer these properties to improve other starter cultures. 2. How serious is the problem? Why does it matter? Dairy foods are nutrient-rich and subject to contamination by undesirable microorganisms. Contaminated dairy foods may cause outbreaks of diseases, and may result in serious economic losses to dairy processors. Protection of dairy foods can be improved by developing dairy lactic fermentation bacteria to produce natural bioactive peptides for controlling undesirable microbes. New knowledge is also needed to maximize and stabilize
production levels in dairy fermentation cultures. Marketability and competitiveness of U.S. dairy foods can be expanded by enrichment with components arising from microbial activity on milk to yield products with health-promoting (probiotic, prebiotic) and pharmacological(antihypertensive, antitumor) properties. Strains with improved stress and bacteriophage defense systems survive better under stressful industrial conditions (high temperature and acid levels) and resist fermentation failures. Biotechnology research aids in the development of dairy cultures with improved performance to improve competitiveness and marketability of dairy products, protect the health of consumers against food borne diseases and to reduce economic losses to dairy food processors by preventing fermentation failures. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? The project plan is aligned with NP306 ('Quality and Utilization of
Agricultural Products') and addresses objectives under program components 'Quality Characterization, Preservation ad Enhancement' and 'New Processes, New Uses, and Value-Added Foods and Biobased Products'. The research leads to the development of dairy lactic fermentation bacteria as production systems of natural bioprotective peptides, proteins and enzymes for improving the health-promoting, nutritional and storage qualities of dairy foods. No other ARS project addresses the same objectives and interaction with peers is in-house and extramural. Collaboration involves researchers at the Rutgers University (antimicrobial peptides), the University of Wisconsin (bioactive peptides by lactobacilli), Universite Laval, Canada (bacteriophage research), Utah State University (probiotics, prebiotics, lactic acid bacteria genomics), University of Arkansas (bioactive peptides), NCAUR (bacterial culture screening and selection), and ERRC (DNA sequencing, bioactive peptide characterization and
experimental food systems). 4. What were the most significant accomplishments this past year? A. Single Most Signficant Accomplishment during FY 2003: Lactic fermentation bacteria selected from the NCAUR and ERRC culture collections were screened for the production of bioactive peptides with antimicrobial activity by several bioassay techniques. This research resulted in the identification of several cultures with antimicrobial activity AND the partial characterization of a new bacteriocin from a strain of the cheese and yogurt fermentation culture, Streptococcus thermophilus. The unique range of the antimicrobial activity of this bacteriocin included several pediococci that are major spoilage bacteria in beer and wine fermentations and listeria species that may be involved in the spread of food borne illness in dairy foods. The unusual range of activity implied that the bacteriocin may have applications as an adjunct controlling agent of Listeria contaminants in food systems and
spoilage bacteria such as pediococci that cause failures in industrial fermentations. B. Other Significant Accomplishment(s) if any: The resistance pattern of lactococci, lactobacilli, pediococci and streptococci selected from the ERRC and NCAUR culture collections to the milk-based antimicrobial peptides, casocidin, isracidin and lactoferricin was rechecked and confirmed. The majority of Over 275 cultures was verified to show resistance to these peptides validating further evaluation as peptide production systems. The availability of a compatible host culture would make it possible to produce milk-based bioactive peptides with applications in dairy food product development. Research was initiated on establishing the range of sensitivity of major groups of objectionable bacterial contaminants (coliforms, listeria, staphylococci) in dairy foods to the synthetic peptides casocidin, lactoferricin and isracidin which are present as peptide components of milk proteins. The research
revealed that effective concentrations of lactoferricin and casocidin required to suppress the growth of coliforms and staphylococci were in excess of 500 microgram per ml while isracidin was ineffective even at 1,000 microgram per ml concentration. These findings indicated a need for the reassessment of the feasibility of producing the three milk protein based antimicrobial peptides by biotechnologically modified lactic bacteria in sufficient quantities to attain the required inhibitory concentration levels in food systems. Research was continued on defining the optimum parameters of bacteriocin (pediocin) production by pediococci in dairy industrial effluents (whey). In nutritionally enhanced lactose hydrolyzed whey samples which included commercially available whey permeate preparations, high levels of pediocin production was achieved. This research implied the feasibility of large-scale production of bioactive peptides in media formulated from dairy industrial wastes. Research was
initiated on the coproduction of the lactic bacterial antimicrobial peptides nisin (a product of lactococci which is FDA- approved for food use) and pediocin (a product of pediococci, which is awaiting FDA approval for food use) by biotechnologically developed lactic fermentation bacteria. Nisin producing lactococci that possess natural resistance to pediocin served as production systems. The results demonstrated the feasibility of coproducing these two powerful bioprotective antilisterial agents in the same lactic fermentation bacterium and the potential for using biotechnologically modified food grade lactic fermentation bacteria in food systems. C. Significant Activities that Support Special Target Populations: None D. Progress Report: The Project Plan is an extension of the 'bridging' project 1935 41000 053 00D ('Genomic Potential of Lactic Acid Bacteria for Probiotic and Prebiotic Functions'), and Research Project 1935 41000 043 00D ('Genetic Engineering of Dairy Bacteria for
Food and Non-food Applications'), which resulted in characterization of new gene transport vectors, improved gene expression systems and new host strains capable of producing bioactive gene products. Research was initiated on the characterization of the mechanism of antibiotic (erythromycin) resistance in a strain of the cheese and yogurt starter culture, Streptococcus thermophilus, which was discovered in the course of screening lactic bacteria for antimicrobial substances. Since the occurrence of antibiotic resistance in food grade cultures is highly objectionable due to the potential for transferring the resistance character to human pathogens, a clear understanding of the resistance mechanism is imperative and requires further investigation. Research was initiated on the peptide inhibitors of the angiotensin converting enzyme (ACE) which is involved in the regulation of blood pressure. These peptides are generated from milk proteins by lactobacilli used in the preparation of
fermented milk products. Milk samples fermented by several strains of Lactobacillus helveticus and Lactobacillus bulgaricus yielded high levels of ACE-inhibitory activity. These encouraging results showed the need for further research on the genomic and proteomic evaluation of food grade lactobacilli to produce bioactive peptides with antihypertensive activity from different types of milk proteins and the characterization of the peptide products by sequence analysis. Interaction continued with Laval University on evaluating the genes for bacteriophage resistance systems and the potential for transferring these beneficial genes to other lactic cultures used in commercial dairy food production. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. In previous projects on the biotechnological development of dairy fermentation cultures (1935 41000 043 00D and 1935 4100 053 00D), high production levels of bioactive peptides and
proteins for food production were achieved. Research revealed the importance of maintaining the structural integrity of genetic elements needed to assure the production of bioactive peptides. Gene transport vehicles with greater stability were developed to allow for bioactive peptide production by modified dairy cultures. Microbial strains developed by biotechnology to produce different types of antimicrobials and other bioactive peptides are expected to have useful applications in the industrial production of dairy and other food products with improved nutraceutical, probiotic and biopreservative properties. Several types of DNA sequences were identified that stimulate peptide production in modified cultures. Transport molecules were designed for transferring the gene cluster for the antimicrobial peptide pediocin and the lysostaphin gene in dairy lactococci, streptococci and lactobacilli. DNA processing enzymes were characterized in dairy streptococci that may have impact on gene
stability in modified lactic cultures. Media combinations were evaluated for the efficient growth of bacteriocin producing dairy cultures. A special class of heat stress protein genes was discovered that aids dairy bacteria in surviving harsh industrial processing conditions. Genetic elements responsible for phage resistance was also discovered in a thermophilic lactic culture. Studies on the permeabilization on S. thermophilus cells were completed to maximize expression of cell-bound lactase enzyme which has application in the production of low-lactose milk products and the utilization of hydrolyzed-lactose whey media for bacteriocin production by genetically unmodified food-grade lactic cultures such as pediococci. In the current project, over 275 lactic fermentation cultures were selected to include major groups (lactococci, lactobacilli and streptococci) used in dairy food production. These strains were tested for sensitivity to the synthetic peptides casocidin, isracidin and
lactoferricin that correspond to antimicrobial sequences present in milk proteins. The majority of the cultures were resistant to these peptides, indicating a potential for application as production systems. Major types of food pathogens such as coliforms, listeria and staphylococci displayed relatively high levels of resistance to synthetic casocidin, lactoferricin and isracidin, indicating the need for reassessing the conditions of practical utilization of these antimicrobial peptides as bioprotective agents in food systems. A sensitive agarose/agar assay technique was also developed and improved for the detection of modified lactic fermentation bacteria with capacity to produce bioactive peptides. The concurrent evaluation of lactic fermentation cultures for antimicrobial activity resulted in the discovery and partial characterization of a bacteriocin from a Streptococcus thermophilus culture with unusual activity against Pediococcus species which may cause spoilage in beer and
wine fermentations and moderate activity against Listeria species which may be responsible for outbreaks of food borne disease. This bacteriocin may have potential for applications in the prevention of undesirable microbial contamination in food and beverage industries. The screen for antimicrobials also identified a yogurt starter culture with natural resistance to the macrolide antibiotics erythromycin and streptogramin B which indicated the need for further studies on the mechanism of resistance and the potential exchange of resistance between beneficial food fermentation bacteria and clinical pathogens. The gene cluster for the antilisterial pediocin of Pediococcus acidilactici was also successfully introduced into a nisin-producing Lactococcus lactis culture, achieving the coproduction of two important but unrelated bacteriocins. Lactic cultures with such dual capacity may have important applications as bioprotective agents in prepared foods. Conditions were also optimized for
the efficient production of antimicrobial peptides in whey-based media by genetically modified lactic fermentation cultures. Screening of lactic fermentation bacteria for capacity to produce bioactive peptides demonstrated that selected lactobacilli have the capacity to generate angiotensin converting enzyme inhibitors during milk fermentations. This implied the potential for enriching foods with dairy protein based antihypertensive peptides that result from action of selected strains of lactobacilli during milk fermentation. These accomplishments have cumulatively increased overall knowledge on the molecular biology properties of dairy lactic fermentation cultures and contributed to the development of cultures with the capacity to produce bioactive milk components and other natural biopolymers, leading to value-added dairy foods with improved health promoting, nutraceutical, functional and storage properties. 6. What do you expect to accomplish, year by year, over the next 3 years?
FY 2004 a) continue to improve assay techniques to detect bioactive peptide production in lactic fermentation bacteria, b) continue developmental research on molecular biology strategies to design functional genes for bioactive peptides occurring in milk proteins, c) continue to identify lactic fermentation cultures with capacity to produce bioactive products from milk proteins with potential as ingredients in value-added dairy foods with improved nutraceutical, functional and storage properties, d) continue evaluation of bioactive-peptide producing lactic bacteria in food systems to prevent or reduce microbial contamination, e) continue to improve whey-based media to optimize conditions for efficient production of bioactive peptides by selected dairy fermentation cultures, f) continue developmental research on the coproduction of natural antimicrobial peptides by biotechnologically improved lactic cultures, g) continue research on the mechanism of antibiotic resistance in lactic
cultures, h) initiate research on the biochemical and genetic requirements for developing lactic cultures with probiotic characteristics, i) initiate research on genomic and proteomic characteristics of lactic fermentation bacteria to discover applicability in discovering producers of bioactive peptides, and j)continue extramural interactions on antimicrobial peptide characterization and bacteriophage resistance mechanisms in lactic cultures. This project is coded to NP 306 which is currently involved in the OSQR Review Process. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Scientific data and research materials on bioactive-peptide producing lactic cultures, improved bioassay techniques, antibiotic and bacteriophage resistant cultures, and
bioactive peptide producing lactic bacteria were made available on request to other scientists and starter culture companies servicing dairy food processing industries.
Impacts
(N/A)
Publications
- Somkuti,G.A., Steinberg,D.H. Pediocin production by recombinant lactic acid bacteria. Biotechnology Letters. 2003. v.25.p.473-477.
- Somkuti,G.A., Steinberg,D.H. Thermophilin 110: a broad spectrum bacteriocin of Streptococcus thermophilus. 2003. American Dairy Science Association. Abstract No. 272.
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Progress 10/01/01 to 09/30/02
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Economic losses in the U.S. dairy food supply (milk, cheeses, and other cultured milk products) may be caused by microbial contamination, by fermentation failures caused by poor starter culture performance due to harsh processing conditions or virus infection. Microbial contamination of dairy foods may be reduced by the presence of food grade bacteria that produce natural inhibitory substances. Marketability and nutritive value of dairy foods also may be improved by supplementing with other, nutritionally or pharmacologically active products, resulting from the activity of food grade microbes on key milk protein components. These objectives can be approached by developing dairy lactic fermentation bacteria to produce bioactive peptides or enzyme systems in amounts to achieve the desired effect. Dairy fermentation failures may be reduced by developing lactic starter cultures with greater
heat and acid resistance under harsh production conditions, and with increased resistance to virus attack. To achieve objectives, microbial biochemistry, molecular biology and biotechnology methods are used to optimize the level of gene expression in selected target microbes under a variety of conditions. Molecular biology research tools are used to identify stress proteins and virus (bacteriophage) resistance systems and transfer these properties to improve other starter cultures. 2. How serious is the problem? Why does it matter? Dairy foods are nutrient-rich and subject to contamination by undesirable microorganisms. Contaminated dairy foods may cause outbreaks of diseases, and may result in serious economic losses to dairy processors. Protection of dairy foods can be improved by developing dairy lactic fermentation bacteria to produce natural bioactive peptides for controlling undesirable microbes. New knowledge is also needed to maximize and stabilize production levels in dairy
fermentation cultures. Marketability and competitiveness of U.S. dairy foods can be expanded by enrichment with components arising from microbial activity on milk to yield products with health-promoting(probiotic, prebiotic) and pharmacological(antihypertensive, antitumor) properties. Strains with improved stress and bacteriophage defense systems survive better under stressful industrial conditions (high temperature and acid levels) and resist fermentation failures. Biotechnology research aids in the development of dairy cultures with improved performance to improve competitiveness and marketability of dairy products, protect the health of consumers against foodborne diseases and to reduce economic losses to dairy food processors by preventing fermentation failures. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? The research project is aligned with NP306 ("New Uses, Quality and Marketability of Plant and Animal
Products") and leads to the development of dairy lactic fermentation bacteria as production systems of natural bioprotective peptides, proteins and enzymes for improving the health- promoting, nutritional and storage qualities of dairy foods. The research also contributes to NP108 ("Food Safety: Animal and Plant Products") as information generated on bioactive peptides may be useful in reducing the incidence of food-borne diseases and to NP301 ("Plant, Microbial, and Insect Genetic Resources, Genomics and Genetic Improvement"), as genetic/genomic research leads to dairy fermentation cultures with greater productivity and improved performance. No other ARS project addresses the same objectives and interaction with peers is in-house and extramural. Collaboration involves researchers at the University of Wyoming (antimicrobial peptides), the University of Alabama (lysostaphin gene), Universite Laval, Canada (bacteriophage resistance), University of Arkansas (bioactive peptides), Rutgers
University (bioactive peptides), NCAUR (culture screening), and ERRC (effect of prebiotics on lactic fermentation cultures). 4. What was your most significant accomplishment this past year? A. Single Most Significant Accomplishment during FY 2002: Screening of lactic fermentation bacteria is a prerequisite for selecting a compatible host system for the production of bioactive peptides. Lactic fermentation cultures received from the NCAUR and the ERRC Dairy Processing and Products culture collections were tested for resistance to three milk-based antimicrobial peptides, casocidin, isracidin and lactoferricin. The majority of over 275 cultures (lactococci, streptococci, lactobacilli, pediococci) tested showed resistance to these peptides indicating a need for further evaluation as peptide production systems. The availability of a compatible host culture would make it possible to produce milk-based bioactive peptides with applications in dairy food product development. B. Other
Significant Accomplishment(s) if any: The lactic fermentation cultures screened (275+) for resistance to milk- based antimicrobial peptides were also tested for capacity to produce their own peptides with antimicrobial activity. This resulted in the identification of several bacteriocin-producing lactococci, lactobacilli and one strain of Streptococcus that displayed activity against either Listeria or Pediococcus. Research was completed on producing bioprotective peptides (pediocin) by a lactose nonfermenting Pediococcus lactic culture in inexpensive dairy industrial wastes (whey) utilizing ARS-developed microbial technology for lactose hydrolysis to support high level production of bacteriocin. This approach has applications in the large-scale production of bioactive peptides in inexpensive dairy industrial effluents. Research also resulted in a more sensitive assay system to detect bacteriocin production by natural or biotechnologically developed lactic fermentation bacteria.
Research was completed on selected cultures with stress protein genes and genes for bacteriophage resistance systems, and interaction continued with Laval University on assessing the potential for transferring these beneficial genes to other lactic cultures used in commercial dairy food production. C. Significant Activities that Support Special Target Populations: None. D. Progress Report: Screening for compatible lactic fermentation bacteria: cultures of lactococci, lactobacilli, streptococci and pediococci from the DPPRU collection and cultures received from the NCAUR culture collection were systematically tested for sensitivity to the milk-based peptides casocidin, isracidin and lactoferricin used. Most cultures displayed resistance over a broad concentration range, which indicated potential for possible use as production systems of these peptides. Methodology was improved for measuring antimicrobial sensitivity by a double-layer agarose/agar assay. 5. Describe your major
accomplishments over the life of the project, including their predicted or actual impact? This Project Plan is a natural extension of the expiring "bridging" project 1935 41000 053 00D ("Genomic Potential of Lactic Acid Bacteria for Probiotic and Prebiotic Functions"), and Research Project 1935 41000 043 00D ("Genetic Engineering of Dairy Bacteria for Food and Non-food Applications"), which resulted in characterization of new gene transport vectors, improved gene expression systems and new host strains capable of producing bioactive gene products. In previous projects on the biotechnological development of dairy fermentation cultures (1935 41000 043 00D and 1935 4100 053 00D), high production levels of bioactive peptides and proteins for food production were achieved. Research revealed the importance of maintaining the structural integrity of genetic elements needed to assure the production of bioactive peptides. Gene transport vehicles with greater stability were developed to allow
for bioactive peptide production by modified dairy cultures. Microbial strains developed by biotechnology to produce different types of antimicrobials and other bioactive peptides are expected to have useful applications in the industrial production of dairy and other food products with improved nutraceutical, probiotic and biopreservative properties. Several types of DNA sequences were identified that stimulate peptide production in modified cultures. Transport molecules were designed for transferring the gene cluster for the antimicrobial peptide pediocin and the lysostaphin gene in dairy lactococci, streptococci and lactobacilli. DNA processing enzymes were characterized in dairy streptococci that may have impact on gene stability in modified lactic cultures. Media combinations were evaluated for the efficient growth of bacteriocin producing dairy cultures. A special class of heat stress protein genes was discovered that aids dairy bacteria in surviving harsh industrial processing
conditions. Genetic elements responsible for phage resistance was also discovered in a thermophilic lactic culture. Studies on the permeabilization on S. thermophilus cells were completed to maximize expression of cell-bound lactase enzyme which has application in the production of low-lactose milk products and the utilization of hydrolyzed-lactose whey media for bacteriocin production by genetically unmodified food-grade lactic cultures such as pediococci. In the current project, over 275 lactic fermentation cultures were selected to include major groups (lactococci, lactobacilli and streptococci) used in dairy food production. These strains were tested for sensitivity to 3 synthetic peptides, casocidin, isracidin and lactoferricin that corresponded to antimicrobial sequences present in milk proteins. The majority of the cultures were resistant to these peptides, indicating a potential for application as production systems. A sensitive agarose/agar assay technique was also developed
and improved for the detection of modified lactic fermentation bacteria with capacity to produce bioactive peptides. The concurrent evaluation of the targeted cultures themselves for antimicrobial activity yielded an in-house streptococcus culture with unusual activity against Pediococcus which may cause spoilage in beer and wine fermentations. The pediocin gene cluster was also successfully introduced into a nisin-producing Lactococcus culture, demonstrating the potential for dual production of unrelated bacteriocins. Research continued on the evaluation of whey-based media to optimize conditions for efficient production of bioactive peptides by genetically developed lactic fermentation cultures. Initiated research on molecular biology strategies to design functional genes corresponding to bioactive peptides. Also initiated research on the effect of selected biopolymers with prebiotic potential on lactic fermentation cultures. These accomplishments have cumulatively increased overall
knowledge on the molecular biology properties of dairy lactic fermentation cultures and contributed to the development of cultures with the capacity to produce bioactive milk components and other natural biopolymers, leading to value-added dairy foods with improved nutraceutical, functional and storage properties. 6. What do you expect to accomplish, year by year, over the next 3 years? Year 1: a) confirmation of the capacity of selected lactic cultures for the the production of bioactive antimicrobial peptides from milk proteins and other sources, b) improvement of bioassay techniques to detect bioactive peptide production, c) preparation of synthetic oligonucleotides based on the sequences of bioactive peptides, d) continuation of developmental research on molecular biology strategies to design functional genes for bioactive peptides, e) screening of lactic fermentation cultures with capacity to produce bioactive milk protein components and other biopolymers with potential to lead
to value-added dairy foods with improved nutraceutical, functional and storage properties, f) evaluation of the efficacy of bioactive-peptide producing lactic bacteria in dairy foods to prevent or reduce microbial contamination, g) improvement of whey-based media to optimize conditions for efficient production of bioactive peptides by selected dairy fermentation cultures, h) continue research on dual expression of selected antimicrobial peptides by lactic cultures, i) testing the effect of prebiotic polymers on lactic cultures, and j) continue extramural interaction on bacteriophage resistance mechanisms in lactic cultures. Year 2: a) continue to develop strategies for improving molecular cloning techniques for synthetic genes, b) construct cloning vectors of various classes for the transport of new genes encoding bioactive peptides, c) continue research on evaluating the structural integrity of vector constructs in lactic acid bacterial host systems, d) continue evaluation of
bioprotective effect of bioactive-peptide producing lactic bacteria in dairy foods, b) initiate research on developing lactic cultures into efficient production systems for bioactive gene products, c) initiate research on assessing production efficiency of cultures in model dairy food systems, and d) initiate research on defining control parameters of gene expression in selected dairy fermentation bacteria to impart added nutraceutical and functional qualities to dairy foods. Year 3: a) continue to optimize conditions for the microbial production of milk- and other protein based nutraceuticals and bioactive agents using modified lactic fermentation bacteria in dairy foods, and validate their impact on food quality, b) continue research on dairy fermentation cultures to increase knowledge on mechanisms of gene expression controlling the production of bioactive gene products with health- promoting or protecting effect, c) continue research on vector development for gene transport and
research on preserving the structural integrity of newly introduced genes, and c) continue research on the performance of the lactic fermentation cultures in prototype food systems. 7. What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the adoption durability of the technology? Scientific data on uses of bioactive-peptide producing lactic cultures, improved bioassay techniques, stress protein genes and bacteriophage resistance genes were made available on request to other scientists and starter culture companies servicing dairy food processing industries.
Impacts
(N/A)
Publications
- Somkuti, G.A., Steinberg, D.H. Agarose/agar assay system for the selection of bacteriocin-producing lactic fermentation bacteria. Biotechnology Letters. 2002. v. 24. p. 303-308.
- Somkuti, G.A., Steinberg, D.H. Coproduction of nisin and pediocin by lactic acid bacteria. 2002. Society for Industrial Microbiology. Abstract No. 48.
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