Bacteriocin based strategy for enhaced food safety

Bacteriocin based strategy for enhaced food safety

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  Bacteriocins based strategyfor enhanced food safety Diwas Pradhan Dairy Microbiology Division Oct., 2015 National Dairy Research Institute Karnal-132001
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   Introduction toFood Safety  Bacteriocins of Lactic Acid Bacteria  Classification of bacteriocins  Detection of bacteriocin activity  Applications of bacteriocin in foods Outline of the presentation
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  Food Safety FoodQuality Making food desirable to eat- Good taste Color Texture Making food safe to eat and free from disease causing agents- Infectious agents Toxic chemicals Foreign objects Major Concerns of the Food Industry
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  Product Diversification Upgraded Quality EnhancedShelf Life Ready to Eat Fresh- Tasting Nutritious and Vitamin Rich Minimally- Processed Consumers’ Preference
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  Lactic acid Propionic acid Aceticacid Antimicrobial substances LAB produces Organic acids Low molecular weight compounds Bacteriocins Reuterin Diacetyl & Acetaldehyde H2O2 Fatty acids Phenyl lactic acids Nisin Lacticin Pediocin Gassericin Antimicrobial compounds produced by LAB Today’s area of discussion
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  “Bacteriocins are ribosomally-synthesizedpeptides or proteins with antimicrobial activity, produced by different groups of bacteria” BACTERIOCINS  Bacteriocins of lactic acid bacteria play a defining role in the preservation & microbial safety of foods  Bacteriocins may have broad or narrow spectrum of activity  Bacteriocins with broad spectrum of activity are of great importance in food safety while Bacteriocins with narrow spectrum of activity may be used for specified use
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  Bacteriocins of lacticacid bacteria Safe and efficacious use of nisin for >40 years in several countries (GRAS status) Effective under wide pH & temperature range Activity is not lost in the presence of food additives and effective in dairy foods during storage Effective in low concentrations Consumer resistance to traditional chemical preservatives and concern over the safety of existing food preservatives such as sulfites and nitrites Do not alter acceptance quality of food and are safe for human consumption What makes LAB bacteriocins as promising agent for their use in biopreservation
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  Lantibiotics Unmodified peptides Large proteinsCircular peptides Class I Class II Class III Class IV Heng et al., 2006 Classification of bacteriocins So far many researchers have classified the LAB bacteriocins into many groups and hence led to some controversies. An appropriate classification of bacteriocins has been given by Hegg and Tagg in 2006 According to them, LAB bacteriocins can be divided into four classes
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  Circular peptides characterized bya peptide bond between the C- and N-terminus Large heat labile proteins with modest prospects as food biopreservatives Post-translationally modified peptides that contain lanthionine amino acid Heat stable non-modified peptides and is the largest class among Gram positive bacteriocins Class I Class II Class III Class IV Different classes of bacteriocins Bacteriocins
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  Classification of bacteriocins IngolfF. Nes et al., 2015
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  Detection of BacteriocinActivity Agar well assay method Indicator / sensitive strain or in soft agar is overlaid on TGE hard agar plate and 6 mm well are cut with sterile borer. 100 µl of cell free culture supernatant (CFCS) is poured into the wells and incubated at 37 ̊C for 24 h after diffusion of CFCS Spot-on-lawn method Indicator / sensitive strain or in soft agar is overlaid on TGE hard agar plate 5µl of cell free culture supernatant (CFCS) is spotted onto the overlaid surface and incubated at 37 ̊C for 24 h after diffusion of CFCS
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  Measurement of Lowconcentrations of potassium ions Bacteriocins usually act by permeabilizing the cell membrane of indicator strain and release of K+ ions Low concentrations of potassium ions were measured, so that the released potassium ions from a bacteriocin-sensitive indicator strain directly correlated to concentrations of crude bacteriocin present in fermentation broth injected into the cell. Bactericidal action of bacteriocin by potassium ion efflux and increased ATP demand from K+ ATPase (adapted from Garneau et al., 2002)
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  PCR methods canalso be used to detect genes responsible for bacteriocin production and regulation in bacterial cultures. The DNA from bacteriocin positive strains can be subjected to a bacteriocin-specific PCR array with primers representing known structural genes of bacteriocins from LAB. PCR based methods
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  Contd……. Optical density measurement Opticaldensity measurement in a microplate system is also a convenient approach to check the inhibition of target bacteria and determine the inhibitory effects of bacteriocins. ELISA technique has also been tested for the detection of bacteriocins that mainly uses affinity-purified anti-nisin immunoglobulin for the binding of nisin and anti-nisin peroxidise linked with the substrate. ELISA technique
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  1 How to addbacteriocins in foods…………… Using a purified/semi-purified bacteriocin preparation as an additive in food Incorporation of an ingredient previously fermented with a bacteriocin-producing strain Use of a bacteriocin-producing culture to replace the starter culture in fermented foods to produce the bacteriocin in situ Traditional methods for incorporation of bacteriocin preparations in foods
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  Prototypical lantibiotic, havingbeen first marketed in England in 1953 and has since been approved for use in over 50 countries. Nisin has been assessed to be safe for food use by the Joint Food and Agriculture Organization/World Heath Organization Expert Committee on Food Additives in 1969. In 1983, this bacteriocin was added to the European food additive list as number E234 (indeed it is the only natural antibacterial to have been approved for as a food preservative by the EU) In 1988, it was approved by the US Food and Drug Agency (FDA) for use in pasteurized, processed cheese spreads and is currently used in a wide variety of foods across the world 1. Use of purified/ semi purified bacteriocins Commercially available purified / semipurified Bacteriocins Nisin Lactoccocus lactis ssp. lactis Pediocin PA-1 Pediococcus acidilactici Delves-Broughton et al., 1996
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  Use of nisinin the safety & biopreservation of dairy products In pasteurized, processed cheese products to prevent outgrowth of spores of Clostridium tyrobutyricum 100-400 IU/g of nisin – for good quality cheese Extends the shelf life of dairy desserts which cannot be fully sterilized Vegetables Target Organisms Bacteriocin Fermented Vegetables L. monocytogenes Nisin Non-fermented vegetables E. faecium Nisin-EDTA Treatment Canned Vegetables Clostridium spp Nisin Soy Milk Bacillus cereus Nisin EDTA-Na Acetate- Citric Acid-K. sorbate
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  Use of Pedicinin the safety & biopreservation of dairy products Pediocin AcH: Active against both spoilage and pathogenic organisms L. monocytogenes Enterococcus faecalis Staphylococcus aureus Clostridium perfringens Pediocin PA-1: Inhibits Listeria in dairy products such as cottage cheese, ice cream, and reconstituted dry milk
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  2) Use ofan ingredient previously fermented with a bacteriocin-producing strain Pediocin 34 produced by Pediococcus pentosaceus 34 Spectrum of activity of pediocin 34 Gram positive Gram negative * Staphylococcus aureus Escherichia coli Listeria monocytogenes Pseudomonas spp. Bacillus spp Salmonella spp. Enterococcus spp. * In the presence of 20mM EDTA Micrococcus spp. Combination of Pediocin and Nisin has Synergistic Effect Bacterial cells resistant to one bacteriocin can be sensitive to another bacteriocin and the antibacterial spectrum of bacteriocins is effective by using a combination of bacteriocins
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  Contd….. MicroGARDTM A product fromDANISCO, Denmark Produced from skim milk fermented by a strain of Propionibacterium freudenreichii ssp. shermanii Used as biopreservative & flavour enhancer Approved by FDA (1990) and granted GRAS status (1996 ALTA™ 2341 Quest International, US Produced from Pediococcus acidilactici fermentation and has to rely on the inhibitory effects of pediocin PA- 1/AcH Added to Mexican soft cheese to prevent Listeria contamination LACTICIN 3147 fermentate Lacticin 3147: Lc. lactis DPC3147 fermentate De-mineralized whey spray dried to produce a bioactive lacticin 3147 powder Effective in inhibiting L. monocytogenes Scott A and Bacillus cereus in natural yoghurt, cottage cheese and soups
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  3)Use of abacteriocin-producing culture to replace the starter culture in fermented foods to produce the bacteriocin in situ The use of cultures to produce bacteriocins in situ A more natural method of shelf-life extension and improving the safety of foods BS-10® Nisin producing L. lactis spp. Lactis, Chr. Hansen BIOPROFIT™ L. rhamnosus LC705, BioGaia BOVAMINE Meat CulturesTM Texas Tech University HOLDBAC™ L. plantarum, L. rhamnosus, L. sakei, L. paracasei and Propionibacterium freundenreichii ssp. shermanii, DANISCO
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  Development of resistanceagainst antimicrobial Natural degradation over time Complex interaction with food matrix Consumption of whole of the antimicrobial in killing of target microbes (Balasubramanian et al., 2011) Drawbacks of instant addition of antimicrobial Antimicrobial becomes ineffective due to:-
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  Antimicrobial packaging (AMP) Microencapsulation Nanoencapsulation Hurdle technology 1 2 3 4 Contd……. Newlyemerged techniques for incorporation of bacteriocin preparations in foods
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  1) ANTIMICROBIAL PACKAGING(AMP) “Anti Microbial Packaging (AMP) is the packaging system that is able to kill or inhibit spoilage and pathogenic organism that are contaminating foods” AMP Bacteriocins of LAB Pre-existing Packaging concept Antimicrobial packaging film prevents microbial growth on food surface by direct contact of the package with the surface of foods The classical protective function of packaging is supported by the antimicrobial action of relevant bacteriocins
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  AMP Nisin is ahighly surface-active molecule that can bind to different compounds, such as fatty acids of phospholipids It a suitable feature for adsorption to solid surfaces and killing bacterial cells that subsequently adhere silica gel, gelatine, collagen casings, cast protein films; polymer film coatings, calcium alginate, methyl cellulose films, corn zein, wheat gluten films Delivery systems used for immobilization of bacteriocins Duraisamy et al., 2015
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  2) Microencapsulation “It isa technology whereby the target molecule is packaged in miniature, sealed capsules to protect it from external factors and deliver in the targeted site under specific conditions. ” Minimizes bacteriocin resistance development and helps to achieve controlled release of bacteriocins
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  Food-grade polymers likealginate, chitosan, carboxy methyl cellulose (CMC), carrageenan, gelatin and pectin are mostly applied, using various microencapsulation technologies 2) Microencapsulation
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  3) Nanoencapsulation Bacteriocins arevery small molecules to be efficiently retained in porous microcapsules which may lead to leakage or inefficient delivery at the target site. Hence many times primarily encapsulation of bacteriocins in nanosized liposomes is popularly followed. Nanoliposomes provide more surface area Improve the distribution in the food system along with the bioavailability of the encapsulated bacteriocins Added directly to the food items Malheiros et al. 2012
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  4) Bacteriocins aspart of Hurdle Technology “ Use of hurdles of differing levels of intensity to bring microbiological growth under control”
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  Bacteriocins as partof Hurdle Technology
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  Bioengineered Nisin Deferred antagonismagar diffusion assay highlighting the enhanced bioactivity of a Nisin K22T producer, relative to a Nisin A producer, against S. agalactiae AT CC13813 (colony size = 10 mm) Cotter et al., 2012 The enhanced strain was found to produce a nisin that possessed a lysine to threonine change at position 22 (K22T) of the nisin peptide,
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  Bioengineered Nisin Cotter etal., 2012 Nisin T was enhanced against S. agalactiae, Streptococus mutans, Clostridium difficile, several S. aureus strains, L. lactis and a variety of mycobacteria Nisin V was enhanced against this same selection but differed by virtue of also exhibiting enhanced activity against Listeria monocytogenes, Enterococcus faecium and Bacillus cereus Some other examples of bioengineered nisin prepared with food grade approach
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  MOVING TOWARDS BETTERTOMORROW Cotter et al., 2012 Identify new bacteriocins for application in foods Altering the specificity of existing bacteriocins Increasing the level of bacteriocin production Development of bacteriocin producing lactic starters through gene transfer system Continued study of physical and chemical properties
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  CONCLUSIONS Bacteriocins of LABand bacteriocin-producing cultures are attractive options in food safety & biopreservation Further research into the synergistic reactions of these compounds and other natural preservatives, in combination with advanced technologies could result in replacement of chemical preservatives. Application of bacteriocins could allow less severe processing treatments, while still maintaining adequate microbiological safety and quality in foods
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  PROVIDING SAFE FOODIS NOW NOT A MATTER OF CHOICE, IT IS A NECESSITY !!
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  Winter School, NDRI,2015 THANK Y U