FOOD AND INDUSTRIAL MICROBIOLOGY

Module 4. Microbiology of food preservation

Lesson 15
NATURAL ANTIMICROBIAL COMPOUNDS

15.1 Introduction

Natural foods preservatives have been used and recognized to mankind since long time. Apart from the preservatives, in all-natural preservation, freezing, pickling, deep frying, salting and smoking also come. Organic substances such as salt, sugar, vinegar and alcohol. These are utilised in both raw and cooked food stuff to increase the shelf value of food so that aroma, taste as well as the foods itself may be stored for a longer period of time. Some preservatives targets enzymes in fruits and vegetables that continue to metabolize after they are cut. For instance, citric and ascorbic acids from lemon or other citrus juice can inhibit the action with the enzyme phenolase which turns surfaces of cut apples and potatoes brown.

15.2 Natural Food Preservatives have Many Advantages

They do not alter the color of the food and gives the required flavour. Artificial preservatives are responsible for causing a lot of health trouble pertaining to respiratory tract, heart, blood and other. There is no main health concern associated with the use of natural food preservatives. These compounds naturally do the process of osmosis and are completely safe for consumption.

15.2.1 Activated lactoferrin (ALF, Activin)

Lactoferrin is an antimicrobial protein present as normal component of fresh milk. It has antibacterial, antifungal and antiviral activity.It also occurs in saliva, tears, and some other body fluids. Activin is activated lactoferrin and is a more potent antimicrobial than plain lactoferrin. It has been accorded GRAS status by the U.S. Food and Drug Administration. It has antimicrobial activity due to its capacity to chelate Fe²⁺ along with HCO⁻³ . It binds to cell surfaces and has a high affinity for the outer membrane proteins (OMP) of Gram-negative bacteria. It also inhibits growth and neutralizes endotoxins. It has been approved at a level of 65.2 ppm for beef carcasses, and may be applied either as a mist or by spraying. It is not acidal agent but acts primarily by preventing pathogens from establishing a niche on meat surfaces.

15.2.2 Ozone (O₃ )

This gaseous compound possesses antimicrobial activity. Like chlorine, it is the most powerful oxidant available for conventional water treatment and is highly reactive. It is 1.5 times more potent than chlorine. It is effective in solution and in its gaseous form as it is unstable it must be generated on site and used. Because it is more effective in killing Cryptosporidium parvum than chlorine, its use in water treatment systems is increasing. It is normally supplied from ozone generators. The cell target for O₃ is the membrane where it disrupts permeability functions. Ozone is GRAS for bottled water use, and for use on a variety of fresh foods, but its strong oxidizing power does not recommend its use for red meats. A typical concentration used is 0.1-0.5 ppm, which is effective against Gram-positive and Gram-negative bacteria as well as viruses and protozoa. Ozone treatment can be used in vegetables, fruits, beef etc. to destroy pathogens like E.coli O157:H7, S. typhimurium, Giardia lamblia, etc.

15.2.3 Hydrogen peroxide (H₂O₂ )

Hydrogen peroxide is a strong oxidizing agent and it is formed to some extent by all aerobic organisms, and it is enzymatically degraded by the enzyme catalase :

2H₂O₂ → 2H₂O + O₂

It is used as a sterilant for food-contact surfaces of olefin polymers and polyethylene in aseptic packaging systems. Hydrogen peroxide vapors have microbiocidal properties. The antimicrobial effect of hydrogen peroxide attributes to a strong oxidizing effect on the bacterial cells and to the destruction of basic molecular structure of cellular proteins. H₂O₂ also prevent spores of Bacillus cereus from swelling properly during the germination process. It is used in the treatment of vegetables, fruits and fruit juice in the form of vapors.

15.2.4 Sodium chloride and sugars

These compounds are grouped together because of the similarity in their modes of action in preserving foods. NaCl has been employed as a food preservative since ancient times. The early food uses of salt were for the purpose of preserving meats. This use is based on the fact that at high concentrations, salt exerts a drying effect on both food and microorganisms. Salt (saline) in water at concentrations of 0.85–0.90% produces an isotonic condition for non-marine microorganisms. Because the amounts of NaCl and water are equal on both sides of the cell membrane, water moves across the cell membranes equally in both directions. When microbial cells are suspended in high salt concentration such as 5% saline solution, the concentration of water is greater inside the cells than outside. In diffusion, water moves from its area of high concentration to its area of low concentration. In this case, water passes out of the cells at a greater rate than it enters. The result to the cell is plasmolysis, which results in growth inhibition and possibly death. This is essentially what is achieved when high concentrations of salt are added to fresh meats for the purpose of preservation. Both the microbial cells and those of the meat undergo plasmolysis (shrinkage), resulting in the drying of the meat, as well as inhibition or death of microbial cells. Enough salt must be used to effect hypertonic conditions. The higher the concentration, the greater are the preservative and drying effects. In the absence of refrigeration, fish and other meats may be effectively preserved by salting. The inhibitory effects of salt are not dependent on pH, as are some other chemical preservatives. Most nonmarine bacteria can be inhibited by 20% or less NaCl, whereas some molds generally tolerate higher levels. Organisms that can grow in the presence of and require high concentrations of salt are referred to as halophiles; those that can withstand but not grow in high concentrations are referred to as halodurics. Sugars are involved in the preservation of food. Sugars, such as sucrose, exert their preserving effect in essentially the same manner as salt. One of the main differences lies in relative concentrations. It generally requires about six times more sucrose than NaCl to affect the same degree of inhibition. The most common uses of sugars as preserving agents are in the making of fruit preserves, candies, jams, jellies, fruit juices, condensed milk etc. The shelf stability of certain pies, cakes, and other such products is due in large part to the preserving effect of high concentrations of sugar, which, like salt, makes water unavailable to microorganisms. Microorganisms differ in their response to hypertonic concentrations of sugars, with yeasts and molds being less susceptible than bacteria. Some yeasts and molds can grow in the presence of as much as 60% sucrose, whereas most bacteria are inhibited by much lower levels. Organisms that are able to grow in high concentrations of sugars are designated osmophiles; osmoduric microorganisms are those that are unable to grow but are able to withstand high levels of sugars. Some osmophilic yeasts such as Zygosaccharomyces rouxii can grow the presence of extremely high concentrations of sugars.

15.2.5 Flavoring agents

Many flavoring agents possessing definite antimicrobial effects are used in foods. Flavor compounds generally have more antifungal activity than antibacterial. The non-lactic, Gram-positive bacteria are the most sensitive, and the lactic acid bacteria are rather resistant. The minimal inhibitory concentrations (MIC) of many flavoring compounds are 1,000 ppm or less against either bacteria or fungi. All were pH sensitive, with inhibition increasing as pH and temperature of incubation decreased. Diacetyl is one of the most effective flavoring agents, which is produced by lactobacillus leuconostoc and strectococus. It is somewhat unique in being more effective against Gram-negative bacteria and fungi than against Gram positive bacteria diacetyl reacts with the arginine bingding proteins of gram negative bacteria. 2, 3-pentanedione is inhibitory to a limited number of Gram-positive bacteria and fungi at 500 ppm or less. Menthol, which imparts a peppermint like aroma inhibits S. aureus at 32 ppm, and E. coli and C. albicans at 500 ppm. Vanillin and ethyl vanillin are inhibitory, especially to fungi at levels <1,000 ppm.

15.2.6 Spices and essential oils

Many spices possess significant antimicrobial activity. The antimicrobial activity is due to specific chemicals or essential oils, Such as eugenol and Allicin. Antimicrobial substances vary in content from the allicin of garlic (with a range of 0.3–0.5%) to eugenol in cloves (16-18%). Cinnamon and clove oils are also highly effective against Aspergillus parasiticus aflatoxin production. Plant EOs such as cumin, caraway and coriander have inhibitory effects on organisms such as Aeromonas hydrophila, Pseudomonas fluorescens and Staphylococcus aureus. Basil has high activity against B. cereus, Enterobacter aerogenes, Escherichia coli, and Salmonella. Lemon balm and sage EOs have adequate activity against L.monocytogenes and S. aureus. Oregano and thyme EOs had comparatively high activity against enterobacteria. Minimum inhibitory concentration (MIC) of oregano and thyme at a range of 190 ppm and 440 ppm, respectively for E. cloacae and for lactic acid bacteria MIC of oregano and thyme at a range of 5 ppm and 440 ppm, respectively,

15.2.7 Phosphates

These salts are commonly added to certain processed meats to increase their water holding capacity. They also contribute to flavor and antioxidative activity. Food-grade phosphates range from one phosphate (e.g. trisodium phosphate) to at least 13 (sodium polyphosphate). They possess antibotulinal activity, especially when combined with nitrites. Combination of 140 ppm NaNO 2 , 0.26% potassium sorbate, and 0.14% sodium acid pyrophosphate (SAPP) delay C. botulinum neurotoxin production .

15.2.8 Fatty acids and esters

Acetic, propionic, and sorbic acids are short-chain fatty acids used as preservatives have antimicrobial activity. The fatty acids and esters have a narrow range of effectiveness and GRAS substances such as EDTA, citrate, and phenolic antioxidants also have limitations as antimicrobial agents when used alone. Although EDTA possesses little antimicrobial activity by itself, it renders Gram-negative bacteria more susceptible by rupturing the outer membrane and thus potentiating the effect of fatty acids or fatty acid esters.

15.2.9 Acetic and lactic acids

The organic acids are commonly used as preservatives. These acids are present in the fermented foods such as pickles, sauerkraut, and fermented milks due to their production within the food by lactic acid bacteria. lactic acid bacteria, produce acetic, lactic, and propeonic acids during fermentation. These acids posses’ antimicrobial effect which is due to both the depression of pH below the growth range and specific toxicity by the undissociated acid molecules. Lactic acid function as a permeabilizer of the outer membrane of Gram-negative bacteria and thus possibly acts as a potentiator of other antimicrobials. Organic acids are employed to wash and sanitize animal carcasses after slaughter to reduce their carriage of pathogens and to increase product shelf life.

15.2.10 Antibiotics

Antibiotics are secondary metabolites produced by microorganisms that inhibit or kill a wide spectrum of other microorganisms. These antibiotics are produced by molds and bacteria of the genus Streptomyces, penicillium and a few by Bacillus . S ubtilin and tylosin have been used as heat adjuncts for canned foods, Chlortetracycline and oxytetracycline at 7ppm concentration has been applied to poultry and natamycin is used as a food fungistat. Tetracycline has been permitted for fresh and other see foods. The antibiotics may be applied as a dip or an ice.

Antimicrobial peptides

Antimicrobial peptides were first isolated from natural sources in the 1950s when nisin was isolated from lactic acid bacteria for potential application as a food preservative. Subsequently, antimicrobial peptides were isolated from other natural sources, such as plants, insects, amphibians, crustaceans, and marine organisms. Antimicrobial peptides (AMPs) are widely distributed in nature and are used by many if not all life forms as essential components of nonspecific host defence systems. Antimicrobial peptides present a promising solution to the problem of antibiotic resistance because, unlike traditional antimicrobial agents, specific molecular sites are not targeted, and their characteristic rapid destruction of membranes does not allow sufficient time for even fast-growing bacteria to mutate. Lactoferrin bovine and activated lactoferrin (ALF), an iron binding glycoprotein present in milk, has antimicrobial activity against a wide range of Gram-positive and negative bacteria, fungi, and parasites. Lactoferrin has been applied in meat products and approved for their application in preservation of beef in USA.

Biopreservation is the use of natural or controlled microbiota or antimicrobials as a way of preserving food and extending it's shelf life . Beneficial bacteria or the fermentation products produced by these bacteria are used in biopreservation to control spoilage and render pathogenic inactive in food. It is a benign ecological approach which is gaining increasing attention and lactic acid bacteria (LAB) are the important among them. 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 bacteriocin. Some LABs produce the antimicrobial nisin which is a particularly effective preservative. 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 inhibiting the activities of a wide spectrum of organisms, including inherently resistant Gram-negative bacteria.