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  1. 1. Bio-preservation/ Natural PreservationP. Suresh KumarSenior Scientist (Fruit Science)National Institute of Abiotic Stress ManagementPune, Maharashtra,
  2. 2. Technological applications in food processing• Recent trends in food processing• Techniques & application of immobilized enzymes in food industry• Application of glucose oxidase, catalase and petinase in food processing• Single cell proteins for human food consumption• Biotechnology for natural & artificial flavour & fragrance production• Microbial biotechnology for food flavour production, oils & fats• Molecular high intensity low calorie sweeteners• Sources & production of vitamins under controlled conditions• Safety issues related to processed foods• Parealization• Nanotechnology• Hurdle technology• Bio-preservation/ Natural preservation• High electric light pulse technology• Asceptic pacakging/ vacuum packaging• Biodegradable plastics• Extrusion cooking
  3. 3. Bio-preservation• Biopreservation is the use of natural or controlled microbiota or antimicrobialsas a way of preserving food and extending its shelf life.• Beneficial bacteria or the fermentation products produced by these bacteria areused in biopreservation to control spoilage and render pathogens inactive infood. It is a benign ecological approach which is gaining increasing attention.• Of special interest are lactic acid bacteria (LAB). Lactic acid bacteria haveantagonistic properties which make them particularly useful as biopreservatives.When LABs compete for nutrients, their metabolites often include activeantimicrobials such as lactic and acetic acid, hydrogen peroxide, and peptidebacteriocins. Some LABs produce the antimicrobial nisin which is a particularlyeffective preservative.• A bacterium that is a suitable candidate for use as a biopreservative does notnecessarily have to ferment the food. But if conditions are suitable for microbialgrowth, then a biopreservative bacterium will compete well for nutrients withthe spoilage and pathogenic bacteria in the food. As a product of its metabolism,it should also produce acids and other antimicrobial agents, particularlybacteriocins. Biopreservative bacteria, such as lactic acid bacteria, must beharmless to humans.
  4. 4. • These days LAB bacteriocins are used as an integral part ofhurdle technology. Using them in combination with otherpreservative techniques can effectively control spoilagebacteria and other pathogens, and can inhibit theactivities of a wide spectrum of organisms, includinginherently resistant Gram-negative bacteria."[1]• In fish processing, biopreservation is achieved by addingantimicrobials or by increasing the acidity of the fishmuscle. Most bacteria stop multiplying when the pH isless than 4.5. Traditionally, acidity has been increased byfermentation, marination or by directly addingacetic, citric or lactic acid to food products. Otherpreservatives includenitrites, sulphites, sorbates, benzoates and essential oils
  5. 5. Sauerkraut• Sauerkraut directly translated: "sour cabbage", isfinely shredded cabbage that has been fermentedby various lactic acid bacteria, includingLeuconostoc, Lactobacillus, and Pediococcus.It hasa long shelf-life and a distinctive sour flavor, bothof which result from the lactic acid that formswhen the bacteria ferment the sugars in thecabbage.• It is not to be confused with coleslaw, whichconsists of fresh cabbage and may receive an acidictaste from vinegar.
  6. 6. Finalsauerkraut
  7. 7. Sauerkraut Fermentation• Cabbage contains enough lactic acid bacteria in order to ferment and producesauerkraut with salt alone. In order to obtain product of the highest quality allthose bacteria strains must ferment in a certain sequence. This happensnaturally as long as sauerkraut is fermented around 65° F (18° C).• Leuconostoc mesenteroides - they are the smallest and start thefermentation first producing around 0.25 to 0.3% lactic acid. They areheterofermenters, this means that they produce different compounds such aslactic acid, acetic acid (vinegar), ethyl alcohol, carbon dioxide (soda gas) andmannitol.• The last one is a bitter flavored compound which is metabolized later byLactobacillus plantarum. All those acids, in combination with alcohol fromaromatic esters, contribute to the characteristic flavor of the high qualitysauerkraut.• If the temperature is higher than 72° F (22° C) they might not grow and thatwould be detrimental to the flavor of sauerkraut. In about 2 days Leuconostocmesenteroides will produce 0.3% lactic acid and this increased acidity willrestrict its growth. Nevertheless, the enzymes it produced will continue todevelop flavor.
  8. 8. • Lactobacillus plantarum - this strain takes over the production of lacticacid from Leuconostoc mesenteroides and continues fermenting until anacidity level of 1.5 to 2% is achieved. L. plantarum will ferment attemperatures higher than 72° F (22° C) and it can grow at higher aciditylevels. It will ferment at lower temperatures as well, albeit at much slowerrate. Lactobacillus plantarum is the most popular lactic acid bacteria strainand it ferments sauerkraut, pickles, cheese and even meat.• This bacteria is a homofermenter what means that it produces onecompound only. It consumes sugar and produces lactic acid whichimparts acidic taste to fermented food.At the end of this stage sauerkraut has an acceptable quality and can beserved or canned. If there is enough sugar left, the fermentation willcontinue until all sugar supply is exhausted.• Lactobacillus pentoaceticus ( L.brevis) – continue fermenting until anacidity level of 2.5 – 3% is obtained. As there is no more sugar left in thecabbage the fermentation comes to the end.
  9. 9. Effect of Fermentation Temperature• The best quality sauerkraut is produced at 65-72° F (18-22° C)temperatures. Temperatures 45.5° F (7.5° C) to 65 F (18 C) favor thegrowth and metabolism of L.mesenteroides. Temperatures higherthan 72° F (22° C) favor the growth of Lactobacillus species.Generally, lower temperatures produce higher qualitysauerkraut, although at 45.5° F (7.5° C) bacteria are growing so slowthat the cabbage might need 6 months to complete fermentation.Higher temperatures produce sauerkraut in 7-10 days but of thelesser quality. This creates such a fast fermentation that some typesof lactic acid bacteria don’t grow at all and less reaction take placeinside what results in a less complex flavor.– Below 45.5° F (7.5° C) fermentation time is up to 6 months.– At 65° F (18° C) fermentation time is 20 days.– At 90-96° F (32-36° F) fermentation time is 10 days.
  10. 10. Moisture• Bacteria that love to spoil sauerkraut will havethe upper hand if you have an insufficientlevel of brine. Too low a water/brine level andyou’re giving the undesirable aerobic (oxygen-loving) bacteria and yeasts the food they needto grow on the surface. This can cause off-flavors and discoloration at minimum, or evenan allergic reaction to those with sensitivitiesto mold and yeast.
  11. 11. Oxygen Concentration• Lactobacillus plantarum, the primary bacteria responsible for StageTwo, works best without oxygen. Anaerobically (withoutoxygen), Lactobacillus plantarum does their job the way we wantthem to – they cause fermentation of cabbage via lactic acid.Aerobically (with oxygen), it will produce acetic acid (vinegar). Sincewe’re making sauerkraut, oxygen must be avoided.• Sauerkraut that is allowed oxygen will not contain any vitamin C inthe final product after just six days. It will also increase chances ofmold forming. If you are regularly getting mold on the top of yourcabbage, this is a visible sign you are allowing too much oxygen in.Oxygen also allows pink yeasts to grow and could result in soft‘kraut.• Finally, don’t mess with your brine. When brine is stirred, youintroduce air which make conditions more favorable for growth ofspoilage bacteria.
  12. 12. Nutrients• Nutrients also affect the outcome of sauerkraut, salt beingthe primary nutrient of concern.• Salt should be added at a ratio of about 2-3%. Much morethan this andthe Lactobacilli can’t thrive. A good rule ofthumb is one tablespoon of salt per two pounds ofcabbage.• Be sure to add salt as evenly as possible – if you createpockets of cabbage that aren’t salted, you are sending anopen invitation for spoilage bacteria to invade and turnyour cabbage brown, or for yeasts to turn it pink.• It is essential to use pure sea salt. Salts with added alkalimay neutralize the acid, resulting in a failed sauerkraut.
  13. 13. pH• pH is a measure of hydrogen ion concentration. Foodswith a pH above 4.6 are low acid and these foods won’tprevent bacterial spoilage.• However, since sauerkraut has a pH of 4.6 or lower, it istermed a high acid food. This acidic environment willnot permit the growth of bacterial spores and thus isresistant to spoilage.• Lactobacilli thrive in an acid environment, but so canmolds and yeasts. So it’s important to find out whatthe mold and yeast don’t like that Lactobacilli cantolerate in order to prevent mold and yeast fromgrowing at all. I discuss this next.
  14. 14. Kimchi• Kimchi, also spelled gimchi, kimchee, or kimchee, is a traditional fermented Korean dishmade of vegetables with a variety of seasonings.• It is Koreas national dish, and there arehundreds of varieties made with a mainvegetable ingredient such as napa cabbage,radish, scallion, or cucumber.• Kimchi is also a main ingredient for many Koreandishes such as kimchi stew, kimchi soup andkimchi fried rice.
  15. 15. Tips• Cabbage should contain up to 3.5% sugar. The sweeter rawcabbage is the better sauerkraut will be obtained.• Adding less than 2% salt might produce soft or even slimysauerkraut. Adding less than 1% will produce sauerkraut thatwould be soft and unacceptable commercially. Adding morethan 3.5% salt might inhibit growth of lactic acid bacteria.• The more lactic acid is produced the more acidic sauerkrautbecomes. There is a limit how much lactic acid can beproduced. Once the sugar supply is exhausted, lactic acidbacteria stop growing.• White scum on the surface of the sauerkraut is due to yeastsand should be removed daily. There is no reason to discardthe sauerkraut.
  16. 16. • It is possible to use the brine from the previous sauerkraut fermentationas a starter culture for a new production. This is a common method usedin production of bread or even salami (back slopping), where a part offermented product is saved for a new production. In theory at least, itshould produce a new batch with the same characteristics as the oldone.• If more sugar were added the fermentation will continue longer andmore lactic acid will be produced. That would result in increased acidityand very sour cabbage which is not desirable. Eventually the acidity levelwill be so high that lactic acid bacteria will not survive.• During fermentation glucose (sugar) is converted to about 50% lacticacid, 25% acetic acid and ethyl alcohol, and 25% carbon dioxide.• Keep the fermentation temperature below 80° F (27° C). For best qualitysauerktaut maintain fermentation temperature at around 65° F (18° C).
  17. 17. Bacteriocin• Bacteriocins are bacterial ribosomallysynthesized peptides or proteins withantimicrobial activity.• Nowadays, the term bacteriocin is mostlyused to describe the small, heat-stablecationic peptides synthesised by Grampositive bacteria, namely lactic acid bacteria(LAB), which display a wider spectrum ofinhibition
  18. 18. Compound Producer microorganisms Target microorganismsAcidsLactic acid All LAB All microorganismsAcetic acid Heterofermentative LAB All microorganisms, pH dependentAlcohols Yeasts heterofermentative LAB All microorganismsCarbon dioxide Heterofermentative LAB Most microorganismsDiacetyl Lactococcus ssp.Yeasts, gram-negative bacteria at⩾200ppm, gram-positive bacteria at⩾300ppm (butter flavour: 2–4 ppm)Hydroqen peroxide All LAB All microorganismsReuterin Lactobacillus reuteriBroad spectrum: gram-positivebacteria, gram-negative bacteria,fungiSiderophoresMost aerobic and facultatively anaerobicbacteria, including Pseudomonas ssp.Staphylocuccus ssp.Iron-dependent microorganismsBenzoic acid; mevalonic acid Lactobacillus plantarumPantoea aqqloberans (gram-negative bacteria),lactone; methylhydantoin Fusarum avenaceumMicrogard® Propionibacterium shermanii Most gram-negative bacteria,yeasts, fungiBioprofit® Lactobacilllus rhamnosus, Broad spectrum: fungi, yeasts,Propionibacterium freudenreichii heterofermentative LAB, Bacillusssp. shermanii ssp. (gram positive bacteria)Antimicrobial products of low molecular mass of microbial origin
  19. 19. Targets ApplicationsFermentation efficiencyLactose metabolism,proteolytic systemsNutritional properties Vitamin synthesisSensory properties Flavor, texture, appearanceShelf life, safety BiopreservativesPhage resistancePhenotype stabilisation Chromosomal integrationTherapeutic effects Cholesterol reductionCompetitive exclusionAnticarcinogenic activityColonization factorsNew traits Enzyme productionTable 6. Potential targets for the application of genetically modifiedLactobacilli
  20. 20. BacteriocinsEmergence of bacteriocin-resistant pathogens or spoilage bacteriaConditions that may destabilize biological activity of proteins (bacteriocins)Nonspecific proteolytic enzymesOxidationHeavy metalsExcessive agitation, foamingFreeze-thaw, shearingBinding to food componentsInactivation by other food additivesPartitioning into polar or nonpolar food components pH effectsSolubilityActivity dependent on narrow pH rangeBactriocinogenic lactic acid becteriaInadequate environment for growth or bacteriocin productionSpontaneous loss of bacteriocin producing abilityPhage infectionAntagonism by other floraDevelopment of bactericin resistant microfloraTable 7. Factors that may compromise the efficacy of bacteriocinsand bacteriocinogenic lactic acid bacteria in product applications