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Part -2 Natural Preservatives in Food

Antimicrobials from animals

Lactoperoxidase

Glycoprotein with high isoelectric point and one Fe groupOccurs 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, StaphylococcusAddition of LP to starter culture for yogurt production inhibited the acid production and new yogurt produced with extended shelf lifeUsed in oral care products toothpastePreservative in ground beef products

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

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

Research GoingTo evaluate the suitability of pleurocidin, originally isolated from the winter flounder as a potential food preservativePhase 1Phase 2Phase 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

ChitosanNatural 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

Chitosan

Chitosan Chitosan coating was formed by dipping the products in a chitosanlactic acid/Na-lactate solution and pH adjustedseveral 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) 703714

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 themicrobialcell membrane, and once embedded, forming pore-like membrane defects that alloweffluxof essential ions and nutrients.

Human defensins are activein vitroagainst Gram-positive and Gram-negative bacteria, fungi, enveloped viruses, and other pathogens

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

MIC and MBCMIC - Minimum Inhibitory Concentrationis the lowest concentration of an antimicrobial agent needed to stop the microbe from reproducing

MBC - Minimum Bactericidal Concentrationis the lowest concentration of an antimicrobial agent needed to kill the microbe

Reuterin

Produced by some strains of Lactobacillus reuteri, during the metabolism ofglycerolto1,3-propanediol activity against Gram-negative and Gram-positive bacteria, yeasts, and filamentous fungi

Lactobacillus reuteri found in the gut of other mammals and birdsResistant to the action of proteolytic and lipolytic enzymes

Exhibit bacteriostatic activity against Listeria monocytogenes, E. coli , S. choleraesuis , Y. enterocolitica,

Antibacterial spectrum of lactic acid bacteria isolatedLactococcuslactisS1 Listeriainnocua,EnterococcusfaecalisLactococcuslactisS2ListeriainnocuaLactococcuslactisS7Listeriainnocua,EnterococcusfaecalisBacilluscereusLactococcuslactisS9Listeriainnocua,BacilluscereusLactococcusdiacetylactisV1 Enterococcusfaecalis

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 Sci2008 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

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.

PediocinPediocin approved by USDA for use in reduced nitrite bacon to aid in prevention of botulinum toxin production

Recently, a pediocin byP.acidilacticicontaining formulation is marketed under the commercial name Alta 2341

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.

SakacinSakacin 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

Nisin

Only natural antimicrobial peptide approved by the FDA for use as a food preservative

Obtained from the culturing ofL. lactison 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

Nisin

NisinHighly 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.

Food Applications Nisin Level (mg/Kg or mg/L)Processed cheese5-15/200-600Ricotta cheese 2.5-5.0/100-200Dipping sauces1.25-6.25/50-250Pasteurized milk products0.25-10.0/10-400Crumpets4-6.25/150-250Continental type cooked sausage5-25/200-1000Salad dressing1.25-6.25/50-250Beer25-37/1000-1500

Canned foods (high acids)2.5-5/100-200

Natamycin

Also known asPimaricin

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

NatamycinEffective at very low levels

Natamycin is approved in different applications with different level in the world

In rats, the LD50is 2300mg/kg, and doses of 500mg/kg/day over 2 years caused no detectable differences in survival rate, growth, or incidence oftumors

DU POINTDANISCO

CREAGRI INCDeveloped 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

HIDROXis certified GRASIt performs better against food pathogens such as Salmonella, Listeria, E.coliTook 10 years R & D work

Legislatory AspectsExisting 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

IssuesMechanism 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

Future TrendsDemand 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

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