Biopreservation of Foods

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1 (e-text and Learn More) Component-I (A) - Personal Details:

2 Role Name Affiliation National Coordinator Professor R.C. Kuhad University of Delhi South Campus New Delhi Paper Coordinator Professor A. K. Puniya National Dairy Research Institute (NDRI), Karnal Content Writer/Author 1. Dr. Tejpal Dhewa Bhaskarcharya College of Applied Sciences (University of Delhi), Sector-2, Phase-1, Dwarka, New Delhi Dr. Monica Puniya Dairy Division, National Dairy Research Institute (NDRI), Karnal Content Reviewer Language Editor (LE) Technical Conversion

3 Component-I (A) - Module Structure: Structure of Module/Syllabus of a module (Define Topic of module and its subtopic) Introduction, Biological methods for food preservation (Lactic Acid Bacteria, Bacteriocins from Lactic Acid Bacteria), Application of bacteriocins in selected foods, Limitations of Biopreservation process. Component-II - Description of Module Description of Module Subject Name Technology Paper Name Module Name Module Id Pre-requisites Objectives Keywords FT/FM/13 Types of bacteriocins, application of bacteriocins To study about types and food applications of bacteriocins for preservation of various types of foods. Bacteriocins, GRAS status, Lactic Acid Bacteria, Nisin

4 TABLE OF CONTENTS 1. Introduction 2. Biological methods for food preservation 2.1 Lactic Acid Bacteria Bacteriocins from Lactic Acid Bacteria 3. Application of bacteriocins in selected foods 4. Limitations of Biopreservation process 5. Summary 6. Glossary 7. Did you know 8. Web links / references

5 1. Introduction The main purpose of the use of preservatives is to restrict the number of microorganisms present in the food. At ancient time, human being used to preserve the food by the addition of several types of artificial chemicals but increasing consumer awareness of potential health risks associated with some of these substances, for example: the toxicity of the preservatives (nitrites), the alteration of the nutritional and organoleptic properties of foods. Recently, modern consumer trends in purchasing and consumption, with demands for safe but minimally processed products without any additives. Therefore, researchers exploring the possibility of using natural additives as a preservative to reduce the risk of health hazards. Some natural preservative are present in several types of food: a) Egg white contains lysozyme. b) Lactoperoxidase system in raw milk shown to be useful to extend the keeping quality of milk in developing countries where pasteurization is not possible immediately after milking and refrigerated transport systems are poorly developed. c) Plant origin antimicrobials such as the extracts of herbs are being frequently used in preservation of foods. d) Microorganisms also producing some secondary metabolites of antimicrobial potential such as antibiotics and bacteriocins. Therefore, such natural compounds may be added as preservatives in food to control the microbial growth without any adverse effect on health. Technically, this process is called biopreservation and the natural compounds used in this process are called biopreservatives. Biopreservation may be defined as the extension of shelf life and enhanced safety of foods by the use of natural or antimicrobial compounds or controlled microbiota. Keeping consumer demands in mind alongwith the necessary safety standards, traditional methods of controlling microbial spoilage and safety hazards in foods are being replaced by combinations of various modern innovative technologies that include biological antimicrobial systems such as lactic acid bacteria (LAB) and their metabolites i.e. bacteriocins. It has been scientifically proved the use of lactic cultures or their metabolites bacteriocins, either single or in combination with gentle treatments and low concentrations of natural chemical preservatives, may be an efficient way of increasing keeping quality (shelf life) of food and safety of food may also achieved by the inhibition of the growth of foodborne pathogens and subsequent spoilage without changing the nutritional quality the food and food products. Therefore, the addition of an antimicrobial compounds (bacteriocins) of microbial origin recognised as a potential natural preservative, as a result of scientific researches on LAB and their antimicrobial products have been made to available which can be used in food preservation.

6 2. Biological methods for food preservation As earlier mentioned, biopreservation is practiced to extend the shelf life and food safety through the use of natural or controlled microbiota and or their antimicrobial compounds. In general, the easiest forms of food biopreservation are fermentation, a process based on the growth of microorganisms in foods, whether natural or added. Conventionally, a several number of foods have been protected against spoiling by natural processes of fermentation. For example, lactic acid bacteria (starter/protective cultures), which produce organic acids and other compounds that, in addition to antimicrobial properties, also confer unique flavours and textures to food products. Nowadays, in the dairy industry and other industries involved in the production of fermented foods such as meat, spirits, vegetable products, and juices using starter culture to initiate the process of fermentation during food production. The microorganism used is selected depending on food type with the aim (providing attractive flavour properties for the consumer) and it must fulfil the standards of GRAS status. It is expected that a starter culture should have both the metabolic and antimicrobial qualities. 2.1 Lactic Acid Bacteria A natural group of Gram-positive, non-motile, non-spore forming, rod- and coccus-shaped organisms that can ferment carbohydrates to form chiefly lactic acid; they also have low proportions of G+C in their DNA is more than 55%. The genera of LAB include: Lactococcus, Streptococcus, Lactobacillus, Pediococcus, Leuconostoc, Enterococcus, Carnobacterium, Aerococcus, Oenococcus, Tetragenococcus, Vagococcus, and Weisella. Lactic acid bacteria have several following important physiological properties and technological applications: a) Ability of adhesion and colonization of the intestinal tract b) Fermentation of lactose and citrate c) High resistance to freezing and lyophilization d) Production of antimicrobial metabolites. e) Production of exo-polysaccharides f) Proteolytic activity and g) Resistance to bacteriophages Lactic acid bacteria have play very important role in food fermentation as starter cultures or protective cultures in the manufacture of dairy, meat and vegetable products because of Generally Regarded As Safe status. The main contribution of such microbes is in the preservation of the nutritional qualities of the raw material by extended shelf life and the inhibition of spoilage and foodborne pathogenic bacteria due to competition for nutrients and the presence of inhibitor agents produced i.e. organic acids (lactic acid),

7 bacteriocins and H 2 O 2. Several scientific reports demonstrate that bacteriocin-producing lactic acid bacteria able kill or inhibit the spoilage and pathogenic bacterial population. Beside these above mentioned applications of lactic acid bacteria, various strains are recognized to be probiotics (health beneficial microbes). Probiotics may be defined as a preparation of or a product containing living, defined microbial cells in sufficient numbers to alter the intestinal microbiota of the host and thus, exert beneficial health effects. Lactic acid bacteria have qualified following selection criterion suggested by food regulatory authorities for probiotics: a) Ability of adherence to cells. b) Exclusion or reduction of pathogenic adherence, Persistence and multiplication. c) Production of antimicrobial compounds i. e. organic acids, H 2 O 2 and bacteriocins d) Be safe, non-invasive, non-carcinogenic, and non-pathogenic (GRAS status); and e) Collective to form a normal balanced flora. Most common probiotics strains that are used for human being have been isolated from the human gastrointestinal tract (GIT) and typically belong to species of the genera Lactobacillus and Bifidobacterium. In addition, lactic acid bacteria other strains i. e. E. faecium, E. faecalis, S.thermophilus, and P. acidilactici are also used in as potential probiotics Bacteriocins from Lactic Acid Bacteria Bacteriocins are the antimicrobial ribosomally synthesized peptides produced by LAB and othe bacteria. Such peptides are effective to kill only closely related microorganisms and very sensitive in nature, they are inactivated by proteases in the GIT. The bacteriocins can effectively be used to inhibit some gram-positive bacteria, spore-forming bacteria, and food-borne pathogens. The major classes of bacteriocins produced by LAB include in Table The bacteriocins derived from lactic acid bacteria have following attractive qualities that make them suitable biopreservative agent in food preservation: a) Broad spectrum of bactericidal activity most Gram-positive bacteria and some damaged Gramnegative bacteria and pathogenic bacteria (L. monocytogenes, Bacillus cereus, S. aureus, and Salmonella). b) Good thermal resistance capacity, can maintain antimicrobial activity after pasteurization and even sterilization. c) Made up of proteins, inactivation by proteolytic enzymes of GIT. d) Non- toxic nature (non-toxic to laboratory animals tested) and generally non-immunogenic also (inactive against eukaryotic cells) e) Regulatory genes of bacteriocins production are mainly located in plasmid. Hence, there is wide scope of genetic manipulation.

8 Table 13.1: Major classes of bacteriocins produced by Microorganisms. Class Class I Class II Class III Class IV Description with examples Comprises the lantibiotics (lanthionine-containing peptides with antibiotic activity); Small peptides that are differentiated from other bacteriocins by their content in dehydroamino acids and thio-ether amino acids. Examples: Nisin, discovered in 1928, most studied. Lacticin 481 from L. lactis Citolysin from E. faecalis and Lacticin 3147 from L. Lactis. Small heat stable and non-lantibiotic linear peptides comprises the (<10 kda) They are grouped into three subclasses on the basis of either a distinctive N-terminal sequence: ClassII.1: the pediocin-like bacteriocins. Example: Pediocin PA-1/AcH produced by Pediococcus. Class II.2: the lack of leader peptide. Example: Enterocin EJ97 produced by E. faecalis Class II.3 neither of the above traits. Example: Enterocin L50A produced by E. faecalis Large heat labile proteins of > 30 KDa, include many bacteriolytic extracellular enzymes i. e. hemolysins and muramidases that may resembles the physiological activities of bacteriocins. Examples: Helveticin J produced by L. helveticus and Bacteriocin Bc-48 produced by E. faecalis Complex proteins whose activity requires the association of carbohydrates or lipid moieties; circular antibacterial peptide, an intriguing and novel type of antimicrobial substance produced by bacteria, plants and mammalian cells. The unique characteristic is the existence of head-to-tail peptide chain ligation. Example: Enterocin AS-48 ( the first circular peptide)

9 Above motioned qualities of bacteriocins make them more interesting for use as biopreservatives in food and also explores the potential of these peptides. Out of four classes, first two classes have received increased attention as biopreservatives. The most extensively studied bacteriocin is nisin, which is widely in the food industry, produced by Lactococcus lactis (FDA-approved bacteriocins, GRAS microorganism) and is used as a food additive in more than 50 countries. In Spain, nisin is listed as E-234, and may also be cited as nisin preservative or natural preservative. The main foods are: processed cheese, dairy products and canned foods, where nisin is effective against foodborne pathogens (L. monocytogenes and several other Gram-positive spoilage causing microbes. 3. Application of bacteriocins in selected foods Nowadays, modern consumers have been consistently concerned with the nutritive values of their diet as well as about possible adverse health effects from the presence of chemical additives in their foods. Resulted, consumers are focused on the natural and fresh foods with no chemical preservatives added. This insight has stimulated research interest in finding natural effective preservatives such as bacteriocins, which can be used to inhibit or destroy undesired microorganisms in foods to increase food safety and extend shelf-life. In general, there are three approaches, commonly used in the application of bacteriocins for preservation of several kinds of food: a) Inoculation lactic acid bacteria in the food where bacteriocins are produced in situ. Here, the ability of the starter culture to grow and conditions to produce bacteriocins in the products is very critical for its successful application. b) Addition intact bacteriocins, which may be purified or semi-purified as food preservatives. c) Use of a food product earlier fermented with a bacteriocins producing strain as an ingredient in food processing. A number of research group has been evaluated the potential of lactic acid bacteria (pediococci, lactobacilli, and enterococci) as bacteriocins producer and their successful application in the control of undesirable and pathogenic microorganisms. Although, several types bacteriocins have been isolated from food-associated LAB, but only few of them are necessarily found effective in all food systems. Undoubtedly, many bacteriocins definitely do have potential in food applications under the appropriate conditions. The following table 13.2 summarized few examples where bacteriocins producing cultures or their bacteriocins in a variety of food system, which show potential for their future applications as biopreservative, to inhibit or kill foodborne microorganisms.

10 Table 13.2: Application of bacteriocins in variety of food system as biopreservatives. Vegetables products Fish Pathogen & Spoilage organism (if any) Bactericins or bacteriocins producer Tinned - Nisin vegetables & fruit juices Salad & fruit - pediocin PA-1/AcH juice Rice, vegetables & fruit juices B. cereus, E. coli O157:H7, S. aureus, Alicyclobacillus acidoterrestris enterocin AS-48 Fresh and stored C. botulinum, L. Combination of nisin and Microgard; fish monocytogenes Carnobacterium divergens Rainbow trout - synergistic effect (lactic acid, NaCl or fish nisin) Meat Meat products L. monocytogenes nisin, enterocin AS-48, enterocins A and B, sakacin, leucocin A, and especially pediocin PA-l/AcH, alone or in combination with several physicochemical treatments. Dairy Cheese Clostridium butulinum nisin and/or nisin-producing strains products (Camembert, Ricotta & Manchego) Milk L. monocytogenes, S. aureus, & E. coli O157:H7 pediocin AcH; lacticin 3147 (against undesirable LAB) ; B. cereus Cheddar, Cottage B. cereus, S. aureus, L. enterocin AS-48 cheese & yogurt monocytogenes Sea food vacuum-packed L. monocytogenes Nisin

11 4. Limitations of Biopreservation process The main functional limitations for the application of bacteriocins in food preservation: its relatively narrow inhibiting activity spectrum and usually active against only one target microorganism. They are generally not active against gram-negative bacteria. Moreover, the activity of bacteriocin is not stable and loss occurs when it interacts with food components by binding with some other food components such as lipids and proteins or being degraded by proteolytic enzymes. To conquer above limitations, several researchers use the concept of hurdle technology to improve shelf-life and enhance food safety: a) It has been scientifically proved that gram-negative bacteria become sensitive to bacteriocins if the permeability barrier properties of their outer membrane are impaired by chelating agents, which can bind magnesium irons from the lipopolysaccharide (LPS) layer and disrupt the outer membrane of these bacteria. Disruption of outer memberane hence, permitting nisin to gain access to the cytoplasmic membrane. b) Interestingly, nisin also enhances thermal inactivation of bacteria and reducing the treatment time and resulting in better food qualities. In a study, Boziaris and others (1998) found that addition of nisin in media, liquid whole egg, or egg white caused a reduction of required pasteurization time of up to 35%. c) In another study, Budu-Amoako and others (1999) showed that nisin reduced the heat resistance of L. monocytogenes in lobster meat and appreciably reduced the treatment time compared with thermal treatment alone. Such finding shows that the significant reduction in drained weight loss would allow substantial cost savings. The combination (synergistic) effect between bacteriocins and other available processing technologies on the inactivation of microorganisms has also been commonly reported in the literature. For example, synergistic effects between sodium diacetate and pediocin; nisin and the lactoperoxidase system; combinations of various bacteriocins nisin and leucocin F10 provides greater activity against L. Monocytogenes and concurrent application of bacteriocins and non-thermal processing technologies (HP : High Hydrostatic Pressure; PEF: Pulsed Electric Field, enhance bacterial inactivation and also improve shelf-life of foods. Non-thermal technologies typically have better nutritional and sensory and qualities as compared to conventional thermal processing methods. Hence, non-thermal methods are considered more useful and attractive in food production. 5. Summary The purpose of Biopreservation is to extend the shelf life and enhanced safety of foods. It is acheieved by the use of natural or controlled microbes or their antimicrobial compounds, for example, use of certain lactic acid bacteria (LAB). Such organisms revealed antimicrobial properties commonly associated with foods, are

12 being assayed to increase the safety and lengthen the shelf life of foods. In general, the antagonistic attrubutes of LAB originate from competition for nutrients and the production of numerous antimicrobial active metabolites (like organic acids: lactic and acetic; H 2 O 2 and certain antimicrobial peptides:bacteriocins etc.). Nowadays, the application of bacteriocins or bacteriocin-producing strains of LAB are of great interest for food manufacturers because they are generally recognized as safe (GRAS) organisms and their antimicrobial products considered as effective biopreservatives. Although, it is required to continuousely extend our understanding concerning the influences of environmental parameters, survival of bacteriocin producing microbes for future food applications. a. References Adams M.R. and Moss M.O., 4th edition, New Age International (P) Limited Publishers, New Delhi, India, Banwart J.M. Basic, 1stedition. CBS Publishers and Distributors, Delhi, India, Boziaris IS, Humpheson L, Adams MR. Effect of nisin on heat injury and inactivation of Salmonella Enteritidis PT4. International Journal of 43 (1998):7-13. Budu-Amoako E, Ablett RF, Harris J, Delves-Broughton J. Combined effect of nisin andmoderate heat on destruction of Listeria monocytogenes in cold-pack lobster meat. Journal of Protection 62 (1999): Cutter, C.N. Microbial control by packaging: A Review. Critical Reviews in Science and Nutrition 42 (2002): Frazier W.C. and Westhoff D.C., 3rd edition. Tata McGraw-Hill Publishing Company Ltd, New Delhi, India, Jay J.M., Loessner M.J. and Golden D.A. Modern food microbiology, 7th edition, CBS Publishers and Distributors, Delhi, India, Mahapatra A.J, Muthukuarappan, K and Julson, J.L. Applications of ozone, bacteriocins and irradiation in food processing: A Review. Critical Reviews in Science and Nutrition, 45 (2005): Manas P. and Pagan R. Microbial inactivation by new technologies of food preservation. Journal of Applied 98 (2005): Raso Javier, Barbosa-Canovas G.V. Non-thermal preservation of foods using combined processing techniques. Critical Reviews in Science and Nutrition 43, no.3 (2003):