PART III PROCESS SAFETY
3. Bacteria
discovered and elucidated. Microbial hygiene was understood and pasteurization of perishable foods was introduced to combat infectious diseases. Initially, starter cultures were isolates from earlier fermentations that were maintained and prop- agated at the site of production. Christian Hansen was the first to isolate and propagate special yeasts to be used in breweries. The first starter culture was born. During the same period microbial enzymes were also developed such as proteases, amylases and invertases (Mongensen et al., 2002).
Fermentation or supplementation with selected intestinal flora started around 1935, by the Shirota Institute for Research on Protective Bacteria, and fermentation at a large scale with these probiotic microorganisms started after1990 (Bosschaert et al., 2001).
TABLE1. Contribution of lactic acid bacteria as natural ingredients, health/ nutrition addi- tives and specialty fermented foods (Lee, 1996a)
Items Applications
Ingredients Preservatives
(nisin, bacteriocins, Lactoperoxide- Preservation of various foods thyocyanate)
Enzymes
Proteinases, exopeptidases, esterases Cheese ripening, enzymes-modified cheese flavours, new protein hydrolysates, functional and bioactive peptides.
Lactase, Superoxide dismutases Lactose-hydrolyzed whey syrups Antioxidants for lipids.
Polysaccharides (dextran). Gums and thickeners, culture viscosity stabilizers.
Flavors (diacetyl, acetoin) Butter and yoghurt flavours.
Health/Nutrition
Probiotic cultures Lactose digestion, control of intestinal pathogen systems, hypercholesterolemia reduction tumour inhibition, reduction of protein allergenicity, reduction of osteoporosis, increase in vitamins K, B complex, (yoghurt, Bifidus and kefir).
Lac. acidophilus, Lac. casei, Bif. bifidum Special foods
Reduction of toxic compounds Removal of pesticides in wine/ penicillin in milk Foods for elderly and babies Immunostimulating easily digestible,less constipating
pasted foods, cereal-carrying cultures
Pet foods Bioprotection ingredient against pathogens,upgrade by-products dried foods containing live lactic acid bacteria for disaster scene and unsanitary conditions.
Emergency foods
Ice cream Bif. spp. and Lac. acidophilus added.
The beneficial effect of pre-digestion of proteins is relatively small for healthy people, with normal gastro-intestinal function. Nevertheless, it is known that the clots of curd that form in the stomach after digestion of yoghurt are notably smaller than after the ingestion of milk and this will increase proteolysis in the stomach.
Predigestion of carbohydrates may also greatly improve the tolerance for cer- tain foods or the possibility to produce alcoholic beverages. For example, it is a well known fact that due to the low lactose content of yogurt in combination with the lactase activity of the microbial culture, yogurt is better tolerated than milk by lactose deficient individuals. The lactase activity of Lactobacillus bulgaricus and Streptococcus thermophilus resists passage through the stomach and assists in the digestion of lactose in the intestine. Several lactobacilli, such as strains of Lectobacillus buchneri, L, brevis, L. cellobiosis, L. fermentum and L. salivarius, posses alpha-galactosidase activity and can be used to eliminate the gas- producing carbohydrate fraction in legumes and Soya milks. The acid formed during fermentation of carbohydrates may help lowering the pH of the gastric
contents and this is certainly beneficial for elderly people, who often have a reduced gastric acid secretion.
In what concerns the fats many bacteria, yeasts and moulds used in fermen- tation display lipolytic activity, the nutritional significance of this is probably small.
Fermentation may reduce the content of non-digestible material in plant foods, like cellulose, hemicellulose and polygalacturonic and glucuronic acids, Breakdown of these compounds may lead to an improved bioavailability of min- eral and trace elements.
Fermented milks are claimed to contain a number of biologically active com- pounds, which contribute to human health. These compounds include bacteria used for fermentation, their metabolic products and components derived from milk. Certain strains of lactic acid bacteria are probiotic in vivo, nevertheless fer- mented milks peptides derived from hydrolyzed milk proteins may also con- tribute to the probiotic growth and subsequent probiotic properties. In fact, peptides with different activities have been detected in various milk proteins upon proteolysis with digestive enzymes or starter bacteria.
These peptides include opiod, antimicrobial, anti-cancer, anti-hypertensive, immunomodulatory and mineral carrier activity. Both casein fractions and whey proteins are known to act as precursors for abovementioned bioactive peptides.
The potential to repress pathogens and spoilage organisms is another health benefit mainly of Lactic acid bacilli (LAB), which possess a number of antago- nistic properties, which can contribute to the increased of the shelf safe life of fer- mented foods. These properties are:
– Decreasing of the pH by the production of organic acids, lactic acid, acetic acid, etc,
– Consumption of the available nutrients, – Decreasing the redox potential,
– Production of hydrogen peroxide, under aerobic conditions,
– Production of special inhibitory components like antimicrobials and bacteriocins.
3.2. Starter cultures
From a modern point of view starter cultures are defined as: preparations which contain living microorganisms, which are applied with the intention of making use of their microbial metabolism.
Generally, the preparations used as starters today can be classified within three categories:
– Undefined cultures, which in the dairy industry are called mixed-strain cul- tures. These starters are based on the use of fermenting substrate, taken from selected process that resulted in good quality end products.
– Single strains cultures which contain one defined strain
– Multiple strain cultures, whi ch contain one or more defined strains, respec- tively. However, bacteriophage attack, mutations and seasonal variations in composition of the inoculation material (milk, vegetables, grapes etc), may often constrain stability of such multi-strain, wild types cultures. As conse- quence, development of starters based on single strains or defined multi-strain starter cultures were initiated.
– Back slopping cultures, These are inoculation processes based on the contin- uous inoculation of foods from a previous batch culture and is still currently in industrial use. This method is used in Italy for the preparation of Sieroinnesto.
In general, all types of microorganisms have been used for food fermentations, but Lactic Acid Bacteria (LAB) and yeasts are more common than moulds (see Table 2 ). A combination of LAB and yeasts are used for some products and in a few cases lactic fermentation combined with moulds make up the flora.
In order to explore the potential development of cultures of single strains, it is important that sufficient numbers of acceptable strains are available. These are strains that raise no question as to their use in a food context, meaning that they should be safe.
The GRAS status (Generally Recognized As Safe) in granted to species or cul- tures that have a record of safe use in foods.
Lactic acid bacteria are consumed in a large extent, primary through consump- tion of fermented foods. According to the latest statistics as published in Bulletin No 355 of the I.D.F. the average annual consumption of fermented milk products TABLE2. Fermented foods which are produced by a process involving LAB, Yeasts and moulds (Buckenhuskes ,1993)
Foods Raw material LAB +
Sauekraut White cabbage Back slopping
Various vegetables Various vegetable Back slopping
Olives Green and Black Back slopping
Soy products. (Similar to
dairy products) Soy protein
Soy sauce Soybeans Yeast and mould
Sour dough Wheaten flour or rye flour Yeasts
Kwass Malt, bread Yeasts
Cacao Yeasts
Coffee
Wine Grapes Yeasts
Beer Malt Yeasts
Fermented sausages Meat from various animals
Fishes Fishes
Cheese Milk
Yogurt Milk
Cream Milk
Butter Milk
Kefir Milk Yeasts
Wine Malolactic fermentation Wine
Yogurt +probiotic Milk Probiotic bacteria
is 22 kg per capita in Europe. In total, this amount is about 8.5 billion kg fer- mented milk per year. With an average microbial content in these fermented prod- ucts of 108bacteria/g or ml, this represents ca. 8.5×1020of LAB. Assuming one bacteria cell weighs 4×10−12g, this means that 3400 tones of pure lactic acid bac- teria cells are consumed every year in Europe, and this is only for fermented milk.
During the last decade we have witnessed a dramatic progress in the develop- ment of methodologies for the genetic modification of bacteria. Obviously the modifications one would like to take place are dictated by the applications whereby bacteria are used. Without any further specification of the sort of appli- cation only very general features for strain improvement can be given, like:
– Improvement of product yield,
– Combination of properties into a single strain, – Elimination of undesirable properties, – Introduction of novel properties,
– Improvement of existing properties (see Table 3).
3.3. Genetic engineering of species
In order to make the ideal culture for any particular food application, it is neces- sary to understand the function that is required of the culture, and to have tools to improve the function of the culture. Both aspects have been advanced consider- ably through scientific achievements during the last few years. The search for a starter culture has until recently relied on the screening of a large number of iso- lates in small-scale food fermentations. The starter culture finally selected would be the one giving a satisfactory performance in the process and also giving an acceptable organoleptic evaluation of the food product. Excellent cultures have been isolated this way, and the method will certainly also in the future be used to expand the pool of microorganisms to be used as starter cultures. The last two decades of research have, however, generated tools allowing us to specifically tar- get the individual genes and metabolic pathways responsible for desired per- formance parameters of a starter culture. Specific targeting makes screening by high throughput methods possible, and it opens the possibility to use mutant selection and genetic engineering to construct starters that are superior to the ones found in nature (Hansen, 2002).
Recombinant DNA technology, developed in the 1970s, together with tradi- tional mutagenesis and selection have impacted industrial microbiology and have been major influences in constructing bacterial strains with enhanced fermenta- tive qualities. Conjugation involves the transfer of plasmids from one microor- ganism to another and necessarily implicates that a close relationship exists between the donor and recipient microorganism. Chromosomal genes may also be transferred, and in this case pure DNA, modified or unmodified, is introduced into recipient cells, without the need for closely related species (Dass, 1999)
Genetic engineering may be a powerful tool but it should not at all affect the GRAS status of those microorganisms.
TABLE3. Genetic transfer systems in Lactic Acid Bacteria (Lee B, 1996b)
Process Major use Limitation
IN VIVO Conjugation
Donor/recipient cell- to Transfer of transmissible Cell surface recognition
cell contact plamids required
The host DNA must be homologous for recombination to occur
Conjugal plasmid transfer Genetic transfer of at high frequencies in chromosomal DNA lactococci and leuconostocs with homologous DNA
Mobiliation of non transmissible plasmids Transduction
Bacteriophage-mediated Useful technique for making Only small fragment of transfer of DNA fine structural changes in DNA transfer
genetic material.
Transformation
Uptake of naked DNA by host General DNA transfer Plasmid DNA maintained, but
procedure chromosomal DNA must be
homologous for recombination to occur
These systems (protoplast, Electroporation technique is whole cell, electroporation) current choice for plasmid
DNA transfer into lactic acid bacteria
IN VITRO Recombinant DNA
Insertion of foreign DNA Transfer of single gene or No theoretical limitation, into a plasmid or phage groups of linked genes but must overcome stability cloning vector, from any source without and expression of introduced transformation into the barrier to a desirable gene; must meet safety
host; Screening of the host strain. requirements
target gene.
Among the microorganisms that have been genetically altered through proto- plast fusion, one may find Aspergillus oryzae, Penicillium caseicolum, P. nal- giovense and Rhizopus niveus. Penicillium roqueforti, Mucor circinelloides and A. sojae are starter organisms that have been manipulated by transfection.
Hybrids produced form the mesophilic Saccharomyces cerevisiae and the cryophilic S. bayanus have promising wine-making qualities at 10˚C, releasing more flavour compounds than either of the parent species.
The undesirable properties of microorganisms may also be removed by using gene disruption. In yeasts and moulds, homologous DNA fragments tend to inte- grate at the homologous site on the fungal genome, resulting in activation of the gene. This process, called homologous recombination is being introduced for the development of starter strains without particular undesirable qualities (Dass, 1999).
Important aspects to be taken into account when applying genetic engineering include the need to carefully compare the activity of genetically manipulated microorganisms with that of parent strains, as well as ensure that no changes occur in the food products wherein they are included that may be harmful to con- sumer’s health or to the organoleptic quality of the product.