Part 2 covered natural food preservatives derived from animal and microorganism, legislatory aspects, future trends.

1. Part -2
Natural Preservatives in Food

2. Antimicrobials from animals
Lactoperoxidase
 Glycoprotein with high isoelectric point and one Fe group
 Occurs in milk, saliva and tears of many mammals.
 Function is to oxidize thiocynate and some halides to generate
products and affects the cytoplasmic membrane that kill or
inhibit growth microorganisms.
 Exert bacteriostatic effect on Listeria, Staphylococcus
 Addition of LP to starter culture for yogurt production inhibited
the acid production and new yogurt produced with extended
shelf life
 Used in oral care products toothpaste
 Preservative in ground beef products

3. Pleurocidin
 A 25 amino acid peptide isolated from the skin mucus
membrane of the winter flounder
 Active against Gram-positive and Gram-negative bacteria.
 Effective against foodborne organisms including Vibrioparahe
molyticus, L. monocytogenes, Saccharomyces cerevisiae, and
Penicillium expansum .
 It was inhibited by magnesium and calcium, which may limit
the use of this AMP

4. Pleurocidin…
 Antimicrobial activity was
reported at levels well
below the legal limit for
nisin (10,000 IU/g)
without significant effect
on human red blood cells

5. Research Going
 To evaluate the suitability of pleurocidin, originally
isolated from the winter flounder as a potential food
preservative
 Phase 1
 Phase 2
 Phase 3
 Pleurocidin, a novel antimicrobial peptide: Efficacy in
food safety and expression in genetically engineered
yeast Dissertation Abstracts International, Volume:
65-11, Section: B, page: 5474

6. Chitosan
 Natural biopolymer obtained from the exoskeletons of
crustaceans (crabs and shrimp) and arthropods
 Used as active material for its antifungal activity and
antibacterial activity.
 Liu et al. studied the efficacy of chitosan against E. coli and
concluded that low molecular weight chitosan is effective for
controlling growth.
 The strong antibacterial activity of chitosan was also observed
against S. aureus

7. Chitosan

8. Chitosan…
 Chitosan coating was formed by dipping the products in a
chitosan–lactic acid/Na-lactate solution and pH adjusted
 several bacteria and yeast were exposed to chitosan
concentrations varying from 40 to 750 mg/l. Gram-negative
bacteria seemed to be very sensitive for the applied
chitosan (MIC< 0.006% (w/v))
 The antimicrobial effect of chitosan were observed on
strawberries during 12 days.
 Chitosan: antimicrobial activity, interactions with food
componentsand applicability as a coating on fruit and
vegetables Food Microbiology 21 (2004) 703–714

9. Defensin
 Group of antimicrobial peptides widely found in nature
including mammalian epithelial cells and Immune cells.
Immune response is the first line of defense and occurs soon
after pathogen exposure
 Function by binding to the microbial cell membrane, and once
embedded, forming pore-like membrane defects that
allow efflux of essential ions and nutrients.
 Human defensins are active in vitro against Gram-positive and
Gram-negative bacteria, fungi, enveloped viruses, and other
pathogens

10. Antimicrobials from microorganisms
 Possible ways of application of bacteriocin in the preservation
of food
 Inoculation of food with LAB (starter or as preservative
culture)
 Addition of purified or semipurified bacteriocin as food
preservative
 Use of product previously fermented with the bacteriocin
producing strain as Single ingredient in food formulation

11. MIC and
MBC
 MIC - Minimum Inhibitory Concentration is the
lowest concentration of an antimicrobial agent needed to
stop the microbe from reproducing
 MBC - Minimum Bactericidal Concentration is the
lowest concentration of an antimicrobial agent needed to
kill the microbe

12. Reuterin
 Produced by some strains of Lactobacillus reuteri, during the
metabolism of glycerol to 1,3-propanediol activity against
Gram-negative and Gram-positive bacteria, yeasts, and
filamentous fungi
 Lactobacillus reuteri found in the gut of other mammals and
birds
 Resistant to the action of proteolytic and lipolytic enzymes
 Exhibit bacteriostatic activity against Listeria monocytogenes,
E. coli , S. choleraesuis , Y. enterocolitica,

13. Antibacterial spectrum of lactic acid
bacteria isolated
Lactococcus lactis S1 Listeria innocua, Enterococcu
s faecalis
Lactococcus lactis S2 Listeria innocua
Lactococcus lactis S7 Listeria innocua, Enterococcu
s faecalis Bacillus cereus
Lactococcus lactis S9 Listeria innocua, Bacillus cer
eus
Lactococcus diacetylactis V1 Enterococcus faecalis

14. Research
Going
 Nisin (N), Reuterin (R), and the Lactoperoxidase natural
biopreservatives at low concentrations, within the hurdle
concept of food preservation, useful tool to control the growth
of pathogenic microorganisms in nonacidified dairy products.
 J Dairy Sci 2008 Jan;91(1):70-5 , Antimicrobial activity of
nisin, reuterin, and the lactoperoxidase system on Listeria
monocytogenes and Staphylococcus aureus in cuajada, a
semisolid dairy product manufactured in Spain

15. Pediocin
 Produced by strains of Pediococcus acidilactici and P.
pentosaceus and is designated GRAS
 The bacteriocins produced by P. acidilactici are AcH, PA-1,
JD, and 5, and those produced from P. pentosaceus are A, N5p,
ST18, and PD1
 Pediocin AcH has proven efficacy against both spoilage and
pathogenic organisms, including L. monocytogenes, S. aureus,
and Cl.Perfringens
 Control growth of L. monocytogenes in fermented meat
product such as sausage and frankfurters.

16. Pediocin…
 Pediocin approved by USDA for use in reduced nitrite
bacon to aid in prevention of botulinum toxin production
 Recently, a pediocin by P.acidilactici containing
formulation is marketed under the commercial name Alta
2341

17. Sakacin
 Bacteriocin produced by certain strains of the lactobacillus
sakei
 43 amino acid peptide
 Inhibit the growth of gram positive bacteria including food
pathogen
 Sakacin types A, B, P, K
 Target on bacterial cytoplasmic membrane so leakage of
cellular component and depletion of proton motive force.

18. Sakacin…
 Sakacin A influence Listeria monocytogens growth, a
microorganism able to grow at 4 °C up to 1600 AU ml-1
 Sakacin-A one of the bacteriocin control growth of L.
monocytogenes in pasteurized ground beef

19. Nisin
 Only natural antimicrobial peptide approved by the FDA for
use as a food preservative
 Obtained from the culturing of L. lactis on natural substrates.
 Nisin is soluble in water and 100 & 300 IU/g decrease growth
of L.monocytogens, S.aureus
 Functions by interacting with the phospholipids in the
cytoplasmic membrane of bacteria, thus disrupting 450
membrane function and preventing outgrowth of spores

20. Nisin

21. Nisin…
 Highly active against many of the Gram-positive bacteria
 Completely degraded in alimentary tract so safe to use
 Used as a Food preservative in a number of Thermal
processed foods, particularly in Dairy products, Canned
foods, Plant protein Foods and Cured Meat products and
Marine Products.

22. Food Applications Nisin Level (mg/Kg or
mg/L)
Processed cheese 5-15/200-600
Ricotta cheese 2.5-5.0/100-200
Dipping sauces 1.25-6.25/50-250
Pasteurized milk products 0.25-10.0/10-400
Crumpets 4-6.25/150-250
Continental type cooked sausage 5-25/200-1000
Salad dressing 1.25-6.25/50-250
Beer 25-37/1000-1500
Canned foods (high acids) 2.5-5/100-200

23. Natamycin
 Also known as Pimaricin
 Produced by a pure culture of Streptomyces natalensis
bacteria through fermentation process.
 Centrifuged, filtered, and washed, to get pure end product.
 Targets ergosterol in the cell wall, a building block of
yeasts and molds, which is responsible for intracellular
nutrient transport

24. Natamycin…
 Effective at very low levels
 Natamycin is approved in different applications with
different level in the world
 In rats, the LD50 is ≥2300 mg/kg, and doses of
500 mg/kg/day over 2 years caused no detectable
differences in survival rate, growth, or incidence of tumors

25. DU POINT
DANISCO

26. CREAGRI INC
 Developed an antimicrobial from natural sources
 HIDROX is ingredient that contains high concentration of
olive polyphenols, especially Hydroxytyrosol, a potent
natural compound with the highest level of free radical
protection activity
 HIDROX® is certified GRAS
 It performs better against food pathogens such as
Salmonella, Listeria, E.coli
 Took 10 years R & D work

27. Legislatory Aspects
 Existing food legislation in most countries would not
favour the use of natural compound, unless this compond
have genuinely acquired as GRAS
 Lengthening procedure for marketing approval and
hampering economic implementation in practice
 As they are new, would require non-toxicity record
 Only Nisin and Pediocin were approved by U.S.FDA till
date

28. Issues
 Mechanism of action of natural preservatives is needed in
order to setup an effective preservation strategy for each
product
 Financially difficult in developing commercial bacteriocin
preparations, because of the costly production, low
production rates, unstable products
 Shown strong bactericidal effects against pathogens in
laboratory test systems and weak-moderate effects in real
foods so research reqquired

29. Future Trends
 Demand for safer food free from chemical additives will
increase in future
 Numerous bactericin inhibit spoilage but only nisin has been
licensed and received GRAS. But deficiency like instability at
neutral to alkaline pH
 Toxilogical data for natural antimicrobial are often lacking and
safe use is not guaranteed by well documented data so lot R &
D work required