1. Bio-preservation/ Natural Preservation
P. Suresh Kumar
Senior Scientist (Fruit Science)
National Institute of Abiotic Stress Management
Pune, Maharashtra, India
psureshars@gmail.com

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

4. Bio-preservation
• Biopreservation is the use of natural or controlled microbiota or antimicrobials
as a way of preserving food and extending its shelf life.
• Beneficial bacteria or the fermentation products produced by these bacteria are
used in biopreservation to control spoilage and render pathogens inactive in
food. It is a benign ecological approach which is gaining increasing attention.
• Of special interest are lactic acid bacteria (LAB). Lactic acid bacteria have
antagonistic properties which make them particularly useful as biopreservatives.
When LABs compete for nutrients, their metabolites often include active
antimicrobials such as lactic and acetic acid, hydrogen peroxide, and peptide
bacteriocins. Some LABs produce the antimicrobial nisin which is a particularly
effective preservative.
• A bacterium that is a suitable candidate for use as a biopreservative does not
necessarily have to ferment the food. But if conditions are suitable for microbial
growth, then a biopreservative bacterium will compete well for nutrients with
the spoilage and pathogenic bacteria in the food. As a product of its metabolism,
it should also produce acids and other antimicrobial agents, particularly
bacteriocins. Biopreservative bacteria, such as lactic acid bacteria, must be
harmless to humans.

5. • These days LAB bacteriocins are used as an integral part of
hurdle technology. Using them in combination with other
preservative techniques can effectively control spoilage
bacteria and other pathogens, and can inhibit the
activities of a wide spectrum of organisms, including
inherently resistant Gram-negative bacteria."[1]
• In fish processing, biopreservation is achieved by adding
antimicrobials or by increasing the acidity of the fish
muscle. Most bacteria stop multiplying when the pH is
less than 4.5. Traditionally, acidity has been increased by
fermentation, marination or by directly adding acetic,
citric or lactic acid to food products. Other preservatives
include nitrites, sulphites, sorbates, benzoates and
essential oils

6. Sauerkraut
• Sauerkraut directly translated: "sour cabbage", is
finely shredded cabbage that has been fermented
by various lactic acid bacteria, including
Leuconostoc, Lactobacillus, and Pediococcus.It has
a long shelf-life and a distinctive sour flavor, both
of which result from the lactic acid that forms
when the bacteria ferment the sugars in the
cabbage.
• It is not to be confused with coleslaw, which
consists of fresh cabbage and may receive an acidic
taste from vinegar.

7. Final
sauerkraut

8. Sauerkraut Fermentation
• Cabbage contains enough lactic acid bacteria in order to ferment and produce
sauerkraut with salt alone. In order to obtain product of the highest quality all
those bacteria strains must ferment in a certain sequence. This happens
naturally as long as sauerkraut is fermented around 65° F (18° C).
• Leuconostoc mesenteroides - they are the smallest and start the
fermentation first producing around 0.25 to 0.3% lactic acid. They are
heterofermenters, this means that they produce different compounds such as
lactic acid, acetic acid (vinegar), ethyl alcohol, carbon dioxide (soda gas) and
mannitol.
• The last one is a bitter flavored compound which is metabolized later by
Lactobacillus plantarum. All those acids, in combination with alcohol from
aromatic esters, contribute to the characteristic flavor of the high quality
sauerkraut.
• If the temperature is higher than 72° F (22° C) they might not grow and that
would be detrimental to the flavor of sauerkraut. In about 2 days Leuconostoc
mesenteroides will produce 0.3% lactic acid and this increased acidity will
restrict its growth. Nevertheless, the enzymes it produced will continue to
develop flavor.

9. • Lactobacillus plantarum - this strain takes over the production of lactic
acid from Leuconostoc mesenteroides and continues fermenting until an
acidity level of 1.5 to 2% is achieved. L. plantarum will ferment at
temperatures higher than 72° F (22° C) and it can grow at higher acidity
levels. It will ferment at lower temperatures as well, albeit at much slower
rate. Lactobacillus plantarum is the most popular lactic acid bacteria strain
and it ferments sauerkraut, pickles, cheese and even meat.
• This bacteria is a homofermenter what means that it produces one
compound only. It consumes sugar and produces lactic acid which
imparts acidic taste to fermented food.
At the end of this stage sauerkraut has an acceptable quality and can be
served or canned. If there is enough sugar left, the fermentation will
continue until all sugar supply is exhausted.
• Lactobacillus pentoaceticus ( L.brevis) – continue fermenting until an
acidity level of 2.5 – 3% is obtained. As there is no more sugar left in the
cabbage the fermentation comes to the end.

11. 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 the
growth and metabolism of L.mesenteroides. Temperatures higher
than 72° F (22° C) favor the growth of Lactobacillus species.
Generally, lower temperatures produce higher quality sauerkraut,
although at 45.5° F (7.5° C) bacteria are growing so slow that the
cabbage might need 6 months to complete fermentation. Higher
temperatures produce sauerkraut in 7-10 days but of the lesser
quality. This creates such a fast fermentation that some types of
lactic acid bacteria don’t grow at all and less reaction take place
inside 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.

12. Moisture
• Bacteria that love to spoil sauerkraut will have
the upper hand if you have an insufficient
level of brine. Too low a water/brine level and
you’re giving the undesirable aerobic (oxygen-
loving) bacteria and yeasts the food they need
to grow on the surface. This can cause off-
flavors and discoloration at minimum, or even
an allergic reaction to those with sensitivities
to mold and yeast.

13. Oxygen Concentration
• Lactobacillus plantarum, the primary bacteria responsible for Stage
Two, works best without oxygen. Anaerobically (without oxygen),
Lactobacillus plantarum does their job the way we want them to –
they cause fermentation of cabbage via lactic acid. Aerobically
(with oxygen), it will produce acetic acid (vinegar). Since we’re
making sauerkraut, oxygen must be avoided.
• Sauerkraut that is allowed oxygen will not contain any vitamin C in
the final product after just six days. It will also increase chances of
mold forming. If you are regularly getting mold on the top of your
cabbage, 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, you
introduce air which make conditions more favorable for growth of
spoilage bacteria.

14. Nutrients
• Nutrients also affect the outcome of sauerkraut, salt being
the primary nutrient of concern.
• Salt should be added at a ratio of about 2-3%. Much more
than this andthe Lactobacilli can’t thrive. A good rule of
thumb is one tablespoon of salt per two pounds of
cabbage.
• Be sure to add salt as evenly as possible – if you create
pockets of cabbage that aren’t salted, you are sending an
open invitation for spoilage bacteria to invade and turn
your cabbage brown, or for yeasts to turn it pink.
• It is essential to use pure sea salt. Salts with added alkali
may neutralize the acid, resulting in a failed sauerkraut.

15. pH
• pH is a measure of hydrogen ion concentration. Foods
with a pH above 4.6 are low acid and these foods won’t
prevent bacterial spoilage.
• However, since sauerkraut has a pH of 4.6 or lower, it is
termed a high acid food. This acidic environment will
not permit the growth of bacterial spores and thus is
resistant to spoilage.
• Lactobacilli thrive in an acid environment, but so can
molds and yeasts. So it’s important to find out what
the mold and yeast don’t like that Lactobacilli can
tolerate in order to prevent mold and yeast from
growing at all. I discuss this next.

18. Kimchi
• Kimchi, also spelled gimchi, kimchee, or kim
chee, is a traditional fermented Korean dish
made of vegetables with a variety of seasonings.
• It is Korea's national dish, and there are
hundreds of varieties made with a main
vegetable ingredient such as napa cabbage,
radish, scallion, or cucumber.
• Kimchi is also a main ingredient for many Korean
dishes such as kimchi stew, kimchi soup and
kimchi fried rice.

19. Tips
• Cabbage should contain up to 3.5% sugar. The sweeter raw
cabbage is the better sauerkraut will be obtained.
• Adding less than 2% salt might produce soft or even slimy
sauerkraut. Adding less than 1% will produce sauerkraut that
would be soft and unacceptable commercially. Adding more
than 3.5% salt might inhibit growth of lactic acid bacteria.
• The more lactic acid is produced the more acidic sauerkraut
becomes. There is a limit how much lactic acid can be
produced. Once the sugar supply is exhausted, lactic acid
bacteria stop growing.
• White scum on the surface of the sauerkraut is due to yeasts
and should be removed daily. There is no reason to discard
the sauerkraut.

20. • It is possible to use the brine from the previous sauerkraut fermentation
as a starter culture for a new production. This is a common method used
in production of bread or even salami (back slopping), where a part of
fermented product is saved for a new production. In theory at least, it
should produce a new batch with the same characteristics as the old
one.
• If more sugar were added the fermentation will continue longer and
more lactic acid will be produced. That would result in increased acidity
and very sour cabbage which is not desirable. Eventually the acidity level
will be so high that lactic acid bacteria will not survive.
• During fermentation glucose (sugar) is converted to about 50% lactic
acid, 25% acetic acid and ethyl alcohol, and 25% carbon dioxide.
• Keep the fermentation temperature below 80° F (27° C). For best quality
sauerktaut maintain fermentation temperature at around 65° F (18° C).

21. Bacteriocin
• Bacteriocins are bacterial ribosomally
synthesized peptides or proteins with
antimicrobial activity.
• Nowadays, the term bacteriocin is mostly
used to describe the small, heat-stable
cationic peptides synthesised by Gram
positive bacteria, namely lactic acid bacteria
(LAB), which display a wider spectrum of
inhibition

23. Compound Producer microorganisms Target microorganisms
Acids
Lactic acid All LAB All microorganisms
Acetic acid Heterofermentative LAB All microorganisms, pH dependent
Alcohols Yeasts heterofermentative LAB All microorganisms
Carbon dioxide Heterofermentative LAB Most microorganisms
Diacetyl Lactococcus ssp.
Yeasts, gram-negative bacteria at
⩾200 ppm, gram-positive bacteria
at ⩾300 ppm (butter flavour: 2–
4 ppm)
Hydroqen peroxide All LAB All microorganisms
Reuterin Lactobacillus reuteri
Broad spectrum: gram-positive
bacteria, gram-negative bacteria,
fungi
Siderophores
Most aerobic and facultatively anaerobic
bacteria, including Pseudomonas ssp.
Staphylocuccus ssp.
Iron-dependent microorganisms
Benzoic acid; mevalonic acid Lactobacillus plantarum
Pantoea aqqloberans (gram-
negative bacteria),
lactone; methylhydantoin Fusarum avenaceum
Microgard® Propionibacterium shermanii Most gram-negative bacteria,
yeasts, fungi
Bioprofit® Lactobacilllus rhamnosus, Broad spectrum: fungi, yeasts,
Propionibacterium freudenreichii heterofermentative LAB, Bacillus
ssp. shermanii ssp. (gram positive bacteria)
Antimicrobial products of low molecular mass of microbial origin

24. Targets Applications
Fermentation efficiency
Lactose metabolism,
proteolytic systems
Nutritional properties Vitamin synthesis
Sensory properties Flavor, texture, appearance
Shelf life, safety Biopreservatives
Phage resistance
Phenotype stabilisation Chromosomal integration
Therapeutic effects Cholesterol reduction
Competitive exclusion
Anticarcinogenic activity
Colonization factors
New traits Enzyme production
Table 6. Potential targets for the application of genetically modified
Lactobacilli

25. Bacteriocins
Emergence of bacteriocin-resistant pathogens or spoilage bacteria
Conditions that may destabilize biological activity of proteins (bacteriocins)
Nonspecific proteolytic enzymes
Oxidation
Heavy metals
Excessive agitation, foaming
Freeze-thaw, shearing
Binding to food components
Inactivation by other food additives
Partitioning into polar or nonpolar food components pH effects
Solubility
Activity dependent on narrow pH range
Bactriocinogenic lactic acid becteria
Inadequate environment for growth or bacteriocin production
Spontaneous loss of bacteriocin producing ability
Phage infection
Antagonism by other flora
Development of bactericin resistant microflora
Table 7. Factors that may compromise the efficacy of bacteriocins
and bacteriocinogenic lactic acid bacteria in product applications