Food and Industrial Microbiology

Module 4. Microbiology of food preservation

Lesson 18
BIOTECHNOLOGY IN FOOD PRESERVATION

18.1 Introduction

In response to consumer demand for more natural food preservation methods, biotechnology has been used to find ways of replacing synthetic preservatives in food. Some of the examples of food preservation methods that employ biotechnology include: recombinant antifreeze proteins that extend the shelf life of frozen dairy products and fruits, fermented whey that is high in acids to preserve cheese and rosmarinic acid produced from rosemary plant cell cultures as an oxidant. Moreover the microbial cultures and their metabolites have been used for the preservation of foods.

Since the role of micro-organisms in spontaneous food fermentation processes became clear, man has tried to apply ‘controlled’ fermentations in order to preserve food products. An increasing number of consumers prefer minimally processed food products, prepared with less or without chemical preservatives. The consumer wants food products to be ‘fresh’, ‘natural’, ‘healthy’ and ‘convenient’. Many of the new ready-to-eat and novel food types bring along new health hazards and new spoilage associations. Against this background and relying on improved understanding and knowledge of microbial interactions, milder preservation approaches such as bio preservation have been

18.2 Biopreservation

Biopreservation or biological preservation can be defined as a preservation method to improve safety and stability of food products in a natural way by using ‘desired’ microorganisms (cultures) and/or their metabolites without changing the sensory quality

Cultures can be defined as protective or antagonistic micro-organisms that are added to a food product only to inhibit pathogens and/or to extend the shelf-life, while changing the sensory properties of the product as little as possible. These cultures differ from starter cultures in their functional objectives. Starter cultures are, by definition, used in food fermentations in order to modify the raw material to give it new sensory properties and this relying on the metabolic activity (acid production) of the culture, while the preservation effect (antimicrobial action) is of secondary importance. For a protective culture, the functional objectives are the inverse.

Biopreservation can be applied in food products by two basic methods:

• Adding crude, semi-purified or purified microbial metabolites.

• Adding pure and viable micro-organisms.

The use of micro-organisms or their metabolites as food preservatives is not meant as a primary means of preservation but as a way to contribute to the hurdle approach in food preservation.

18.3 Antimicrobial Metabolites of Lactic Acid Bacteria

18.3.1 Organic acids

The most important and best characterized antimicrobials produced by LAB are lactic and acetic acid. The amount and type of acids produced during fermentation influence the subsequent microbial activity in the fermented material. Acetic acid, for example, is more antagonistic against yeasts compared to lactic acid. Some oxidative yeasts are able to utilize organic acids as a carbon and energy source and consequently cause spoilage through deacidification in fermented, especially plant material where they are naturall y present . The inhibitory effect of organic acids is mainly caused by undissociated form of the molecule, which diffuses across the cell membrane towards the more alkaline cytosol and interferes with essential metabolic functions. The toxic effects of lactic and acetic acid include the reduction of intracellular pH and dissipation of the me mbrane potential .

18.3.2 Hydrogen peroxide

Antimicrobial activity of hydrogen peroxide is attributed to its strong oxidizing effect on the bacterial cell and to the destruction of basic molecular structures of cell proteins . In raw milk, hydrogen peroxide produced by lactic acid bacteria can, after being catalyzed by lactoperoxidase, oxidise endogenous thiocyanate. The oxidized intermediary products are toxic to different bacteria . Hydrogen peroxide production has been considered as the main metabolite of LAB that could protect against urogenital infections, especially in the case of bacterial vaginosis .

18.3.3 Carbon dioxide

The influence of carbon dioxide on product preservation is twofold. Namely, except for its own antimicrobial activity, it creates an anaerobic environment by replacing the existent molecular oxygen. The antifungal activity of CO₂ is due to the inhibition of enzymatic decarboxylations and to its accumulation in the membrane lipid bilayer resulting in dysfunction in perm eability .

18.3.4 Reuterin

Reuterin is a pH neutral, water soluble, low molecular weight substance, which is non-bacteriocin and resistant to nuclease, protease and lipolytic enzymes. It is active over a wide range of pH values and capable of inhibiting growth of a wide spectrum of microorganisms, but it is labile to heat (100⁰C for 10 minutes). The unique and most attractive feature of reuterin is its strong antimicrobial activity. It has been found that concentrations of reuterin in the range of 15-30 µg/ml effectively inhibit growth of Gram-positive and Gram-negative bacteria, and lower eukaryotic organisms including yeast, fungi and protozoa.

18.3.5 Bacteriocins

The bacteriocins most studied for their biopreservative effect in food products, and more specific in meat and meat products, include nisin, pediocins and sakacins Nisin, produced by Lc. lactis subsp. lactis, is the only bacteriocin that has found practical application in food products. It is mainly applied in the prevention of late-blowing of cheese by inhibiting the outgrowth of Clostridium spores and in selected pasteurised cheese spreads to inhibit Clostridium and Listeria. Typical levels that are used in food products range from 2.5 to 100 ppm. Pediocins, in particular pediocin PA-1 (also AcH) from P. acidilactici, have been used successfully to control growth of L. monocytogenes in cottage cheese, half-and-half cream and cheese sauce, raw or fresh meat, cooked meat products and fermented meat products. Pediocin PA-1 was also found to be active towards L. curvatus in a meat product model. In general, the pediocins seem to be more effective in meat products than nisin but they are not approved for use.

18.4 Fermentates of Selected Organisms

The Microgard^TM products marketed by Danisco are fermentates of Propionibacterium freundenreichii subsp. shermanii that are commonly used commercially as biopreservatives in cottage cheese. Their inhibitory activity is attributed to propionic acid, acetic acid and a heatstable peptide (Guinane et al., 2005). Alta^TM and Perlac^TM are fermented whey-based products used as shelf-life extenders.

18.5 Biopreservation by Means of Protective Lactic Cultures

Since lactic acid bacteria are commonly used as starter cultures in food fermentations, investigators have explored the use of bacteriocin producers as protective cultures. Bioprotective culture may act as starter cultures in the food fermentation process or they may protect foods without any detrimental organoleptic changes. Natural bacteriocin producers, such as Lactobacillus plantarum, Pediococcus acidilactici, Enterococcus faecalis and Enterococcus faecium have been used as protective cultures in various products e.g. the outgrowth of clostridia spores in cheese milk was completely prevented when a nisin A producing strain was mixed at 10% rate with the starter culture. Similarly the application of bacteriocin- producing LAB in the meat industry also offers a promising way of natural food preservation.

Table 18.1 Bacteriocin based bioprotective cultures

Table 18.2 Desired properties of a protective LAB- culture

18.6 Applications of Protective Lab in Different Food Products

The effectiveness of Protective culture has been studied in different food products (Table 18.3.). The majority of these inoculation experiments were performed with the intention of demonstrating the effectiveness of bacteriocinogenic strains in controlling L. monocytogenes.

Table 18.3 Studies on the effect of non-bacteriocinogenic protective cultures in different types of food products

18.6.1 Milk and dairy products

Cheese suffers from spoilage through Clostridium spp. (late blowing) and is, furthermore, susceptible to contamination with L. monocytogenes. This latter problem arises mainly in cheeses in which the pH increases during ripening, such as the Italian cheeses Taleggio, Gorgonzola and Mozarella. The addition of this paired nisin producing starter system to make cheddar cheese provided enough nisin to increase the shelf life of pasteurised processed cheese, made from this cheddar, from 14 to 87 days at 22°C and to control L. monocytogenes, Cl. sporogenes and S. aureus. Antilisterial effects were also observed for a bacteriocinogenic Enterococcus faecium strain during Taleggio production.

18.6.2 Vegetable products

Bacteriocinogenic LAB are reported to have potential for the biopreservation of foods of plant origin, especially minimally processed vegetables and fermented vegetables. In minimally processed vegetables such as pre-packaged mixed salads and different types of sprouts, bacteriocinogenic LAB have been found to act on coliforms and enterococci and on L. monocytogenes). Moreover, bacteriocinogenic starter cultures may be useful for the fermentation of sauerkraut or olives to prevent spoilage. Biocompetitive control or the use of biocompetitive micro-organisms to inhibit mycotoxin forming moulds can be obtained by (1) the use of biocompetitive non-aflatoxinogenic moulds or (2) the use of antagonistic yeasts or bacteria.

18.6.3 Fish, fish products and seafood

Spoilage of fresh fish is generally caused by Gram-negative bacteria. However, when vacuum packaged the spoilage of fresh fish, smoked fish and seafood is dominated by mainly Gram-positive bacteria, in particular LAB, and also L. monocytogenes can cause problems. The potential of Carnobacterium spp. to control L. monocytogenes in cold-smoked salmon, the divercin V41 producing Carnobacterium divergens V41 the bacteriocinogenic Carnobacterium maltaromaticum (previous piscicola) A9b and the non-bacteriocinogenic C. maltaromaticum A10a have been used.

18.6.4 Other food products

Bacteriocin-producing and acid producing LAB have applications of in refrigerated ready-to-eat food products, e.g. soups, meals and salads, to prevent them from growth of food born pathogens, in particular Cl. botulinum and/or L. monocytogenes. A mixture of a nisin-producing Lc. lactis and a pediocin A-producing P. pentosaceus are effective to prevent growth of Cl. botulinum and botulinal toxin formation after 10 days at 10°C .

18.7 Food Preservation Through Fermentation

Fermentation is the process of bioconversion of organic substances by microorganisms and/or enzymes (complex proteins) of microbial, plant or animal origin. It is one of the oldest forms of food preservation which is applied globally. Indigenous fermented foods such as bread, cheese and wine, have been prepared and consumed for thousands of years and are strongly linked to culture and tradition, especially in rural households and village communities. It is estimated that fermented foods contribute to about one-third of the diet worldwide. During fermentation processes, microbial growth and metabolism i.e. the biochemical processes whereby complex substances and food are broken down into simple substances, result in the production of a diversity of metabolites. D uring fermentation breakdown of carbohydrates under limited supply of oxygen or under anaerobic conditions take place e.g. yeasts converts sugar to alcohol and CO₂ . Some aerobic, specific conversions may a lso be referred as fermentation such as Acetobacter convert ethylalcohol to acetic acid in the presence of oxygen . In nature, natural fermentations occur continuously. In technically advanced societies, fermented foods are produced to add special tastes to human diet, in less developed areas fermentation is still one of the major preservation methods. In contrast to most preservation methods, fermentation encourages growth and multiplication of selected microorganisms in foods. The application of microorganisms to food preservation practices must be such that a positive protection is available to control contamination. Lactic acid fermentation are of great importance in food preservation. The sugar in foodstuff may be converted to lactic acid and other end products and in such amounts that the environment is controlling over other organisms. Lactic acid fermentation is efficient and the fermenting organisms grow rapid ly . Natural inoculations are such that in a suitable environment the lactic acid bacteria will dominate, as in souring of milk. There is another fermentation which involves much gas production. It is useful in food preservation. In gassy fermentations sugar molecules are altered to form acids, alcohols and carbon dioxide. It is usually necessary to include some other controlling influence, such as adding sodium chloride to a substrate, with this form of fermentation. Sodium chloride is useful in a fermentation process of foods by limiting the growth of putrefactive organisms and by inhibiting the growth of large numbers of other organisms. Sodium chloride is one of the most important food adjuncts in food preservation. In fermentations salt can exert a role in sorting the organisms permitted to grow. Fermentation is globally applied in the preservation of a range of raw agricultural materials (cereals, roots, tubers, fruit and vegetables, milk, meat, fish etc.). Commercially produced fermented foods which are marketed globally include dairy products (cheese, yogurt, fermented milks), sausages and soy sauce. Foods fermented by Lactic acid bacteria are cucumbers, olives, cabbage, coffee cherries, vanilla beans, meat, dairy, by Yeasts are malt, grapes, wines, wines, bread doughs and by mould are soybeans.

18.8 Genetically Modified Organisms in Preservation

Microorganisms are an integral part of the processing system during the production of fermented foods. Microbial cultures can be genetically improved using both traditional and molecular approaches and improvement of bacteria, yeasts and moulds is done. One of the traits which have been considered for commercial food applications in both developed and developing countries include the ability to produce antimicrobial compounds (e.g. bacteriocins, hydrogen peroxide) for the inhibition of undesirable microorganisms. In many developing countries, the focus is also on the degradation or inactivation of natural toxins (e.g. cyanogenic glucosides in cassava), mycotoxins (in cereal fermentations) and anti-nutritional factors (e.g. phytates).

18.9 Recombinant Antifreeze Protein

Antifreeze proteins are potent cryogenic protection agents for the cryopreservation of food and pharmaceutical materials. A food-grade expression and fermentation system (BSE- and antibiotic-free) for the production and secretion of high levels of rAFP was developed. A novel recombinant type I antifreeze protein analogue (rAFP) was produced and secreted by Lactococcus lactis, a food-grade microorganism of major commercial importance. Lyophilized, crude rAFP produced by L. lactis was tested in a frozen meat and frozen dough processing model. The frozen meat treated with the antifreeze protein showed less drip loss, less protein loss, and a high score on juiciness by sensory evaluation. Frozen dough treated with the rAFP showed better fermentation capacity than untreated frozen dough. Breads baked from frozen dough treated with rAFP acquired the same consumer acceptance as fresh bread.