PART III PROCESS SAFETY
3. Microbial Interactions – General Considerations
3.4. Microbial interactions – selected case studies
3.4.1. Microecology in humans
The largest and most important interface between a superior organism and its environment is accounted for by surfaces covered by epithelial cells. At birth the foetus is delivered from the essentially sterile uterine environment and interac- tions of the neonate with microorganisms occur from this point on. The main por- tals of entry of microorganisms are the skin, as well as mucosal surfaces of the
gastrointestinal, respiratory and urogenital tracts. In physiological terms, interac- tion with bacteria eventually leads to colonisation of such epithelial surfaces – this co-existence is usually harmonious and beneficial to the host (which is an example of commensalism). A complex, open ecosystem – formed by resident bacteria and other microorganisms that interact temporarily with the macro- organism - is thus established. However, the interaction with “endogenous”
microorganisms can, under specific conditions, be harmful to the host (i.e. para- sitism), so opportunistic infections may occur.
Interactions between the microflora and their host are characterised by active participation of both partners and the strategy of both of them seems similar: evo- lutionary co-existence has provided both the microorganisms and the immune sys- tem of the host with similar mechanisms of diversification and selection. Human individuals are thus complex ecosystems, formed by a normal microflora – that comprises mainly bacteria, as well as viruses, fungi and protozoa to a lesser extent.
Commensal bacteria exhibit an enormous diversity; not less than 1000 species are apparently involved. The commensal microflora is thus an integral part of the complex, natural mechanisms acting on mucosal surfaces and skin that safeguard resistance of the organism against pathogenic microorganisms. When the qualita- tive and quantitative profiles are at an optimum, attachment and multiplication of pathogenic microorganisms on these surfaces, subsequent invasion of epithelial cells and the circulatory system are prevented. This process is termed “colonisa- tion resistance”,. Intestinal microflora play an important role in anti-infectious resistance, both by direct interaction with pathogenic bacteria and by influence upon the immune system – during the early postnatal period, the intestinal microflora stimulates development of both local and systemic immunity; after- wards, these components evoke regulatory (inhibitory) mechanisms to keep both mucosal and systemic immunity in balance (Tlaskalov’a-Hogenov’a et al., 2004).
3.4.2. Microecology in dairy products
Although fermented milk products are regarded predominantly as a result of lac- tic acid fermentations, the frequent co-occurrence of yeasts and lactic acid bacte- ria has led to the suggestion of interactions that can influence product characteristics (and quality thereof). Presence of yeasts is obviously necessary for the desirable carbon dioxide and ethanol production in eastern European and Asian products, such as kefir, koumiss and airag. The mechanisms of these inter- actions may depend on stimulation or else inhibition of growth of one (or both) of those co-cultured species. Those organisms may in fact compete for nutrients that cause growth, or they may produce metabolites that inhibit each other’s growth; e.g. yeasts may produce vitamins that enhance growth of lactic acid bac- teria. Furthermore, mutual influence of microorganisms on each other’s metabo- lism may lead to different profiles of organoleptically important compounds in the final fermented milk (Narvhus and Gadaga, 2003).
The commensalistic interaction between L. acidophilus and a lactose fer- menting yeast called Kluyveromyces fragilis in acidophilus-yeast milk relies on
coexistence of both organisms for the formation of a product with good final quality. The co-culture of L. acidophilus with K. fragilis reduces the time of coagulation of milk due to acid production by the latter, whereas it raises the number of viable lactic acid bacteria, while inhibiting growth of Escherichia coli and Bacillus cereus.
Mutualistic synergism occurs between yeasts and lactic acid bacteria during fermentation of kefir. Yeasts provide such growth factors as free amino acids and vitamins for bacteria, which consequently entertain elevated acid production;
bacterial end-products are in turn used by yeasts as an energy source. This phe- nomenon creates a balanced stability of the final product. However, a decrease in alcohol production by yeasts may occur due to excessive lactic and acetic acid production by osmophilic lactic acid bacteria, coupled with competition for the carbon source – or even lysis of yeast cell walls by bacterial enzymes (Viljoen, 2001). A positive effect upon growth and kefiran production by Lactobacillus kefiranofaciens was observed (Cheirsilp et al., 2003) in a mixed culture with S. cerevisiae; physical contact with the latter is enhanced by capsular kefiran produced by L. kefiranofaciens.
3.4.3. Microecology in yoghurt
Yoghurt is produced via fermentation of pasteurised (full or skimmed) milk. The major agents in this process are Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. During incubation, in which the starter grows as mixed culture, a positive interaction between the two microor- ganisms is generally observed. Typically, S. thermophilus and L. bulgaricus are inoculated at a 1:1 ratio and then remain present throughout yoghurt production.
When both bacteria grow in association, the times required for milk coagulation are shorter than if either of them is grown separately. This process occurs because during growth, S. thermophilus produces formic acid which, in turn, stimulates the growth of L. bulgaricus. The activity of the latter on casein releases amino acids, which, in turn, stimulate growth of the former. However, Ginovart et al.
(2002) have reported that interaction between such bacterial cells is not only due to growth stimulation related to either formic acid or free amino acids but also to acidity of the medium.
3.4.4. Microecology in cheese
Microorganisms are an essential component of natural cheese varieties and play important roles during both cheese manufacture and ripening. They can be divided into two main groups; starter and secondary microflora. The former, typ- ically composed of Lactococcus lactis, S. thermophilus, Lactobacillus helveticus and L. delbrueckii – used either individually, or in various combinations depend- ing on variety in stake, are responsible for acid development during cheese mak- ing. Starters may be either blends of defined strains, or (as happens in the case of many cheeses manufactured by traditional methods) composed of more or less undefined mixtures of strains – which are adventitious and are present in cheese
milk. During cheese ripening, the starter culture coupled with the secondary microflora promotes a complex series of biochemical reactions that are vital for proper development of both flavour and texture. The secondary flora is normally composed of complex mixtures of bacteria, yeasts and moulds – which are dependent on the particular cheese variety, as they contribute significantly to its specific characteristics. The secondary microflora may also be added in the form of defined cultures; however, in many situations it is composed of adventitious microorganisms that gain access to cheese either from its ingredients or from the environment. During cheese manufacture and ripening, a number of interactions occur between individual constituents of the cheese microflora (Beresford et al., 2001).
Yeasts, which often originate in contamination during cheese making, con- tribute to ripening by metabolizing lactic acid, producing lipases and proteases, fermenting residual lactose and excreting growth factors, either as viable entities or following autolysis (Viljoen, 2001). All of these characteristics contribute to the sensory quality of the final cheese. The increase in pH arising from lactic acid utilization encourages growth of bacteria – which may not only affect flavour and textural quality but also pose a risk to public health. Studies on the interaction between yeasts and starter cultures in Cheddar and Gouda cheeses indicated that the former also play a significant role during ripening via supporting growth of starter cultures. The large number of viable yeasts present during the later stages of ripening is indicative of a possible mutualistic interaction within the microflora. During ripening, yeasts increase at a faster rate than starter cultures but no inhibition of either population is typically observed; therefore, said mutu- alistic interaction may contribute to the final product.
3.4.5. Microecology in probiotic foods
Additive and synergistic health-promoting effects, brought about by individual strains in multistrain probiotic foods, may be explained on the basis of possible relationships between strains in those mixed systems. Interrelationships may enhance certain probiotic characteristics, such as growth and metabolic activity.
Growth of probiotic microorganisms following inoculation is necessary to maintain sustainable numbers in the gastrointestinal tract. This growth can be stimulated by the presence of other species – as happens with certain starter cul- tures involved in manufacture of fermented dairy products. For such probiotic bacteria as L. acidophilus and Bifidobacterium spp., it is known that they grow slowly in milk because they lack proteolytic activity. Addition of typical yoghurt- bacteria – particularly L. delbrueckii subsp. bulgaricus, will enhance growth of such probiotic strains. This positive interaction is referred to as protocooperation – and is explained by the exchange of certain growth factors, such as amino acids, free peptides, formate and CO2(Timmerman et al., 2004). A progressive incre- ment of B. animalis growth was affected by presence of L. acidophilus – which hydrolyzes milk caseins using extracellular proteinases, thus yielding amino acids and peptides that stimulate growth of B. animalis. On the other hand,
growth of L. acidophilus is also enhanced by presence of B. animalis, possibly due to production of acetate (Gomes et al. 1998; Timmerman et al., 2004).
3.4.6. Microecology in sourdough
Sourdough is an intermediate food product, whose microflora is composed of sta- ble associations of lactobacilli and yeasts, based on metabolic interactions. As shown for certain industrial sourdough processes, such microbial associations may endure for years – even though the fermentation process is run under non- aseptic conditions. The importance of antagonistic and synergistic interactions between lactobacilli and yeasts is based on metabolism of carbohydrates and amino acids, coupled with production of carbon dioxide. Typical mutual associa- tions involve Lactobacillus sanfranciscensis and either Saccharomyces exiguus or Candida humilis. Maltose is the preferred energy source for L. sanfranciscensis but is not utilized at all by either of those latter species (such as maltose-negative yeasts, which use sucrose, glucose and fructose). Maltose is continuously released by flour amylases; when there is an excess of maltose coupled with envi- ronmental stress, several strains of L. sanfranciscensis hydrolyse maltose and accumulate glucose in the medium. This glucose affects the ecological system, as it may be metabolised by its producers, by other lactic acid bacilli (LAB) and by yeasts. It may however, initiate glucose repression in competitors for maltose and glucose may then be utilised by maltose-negative yeasts. Due to the faster con- sumption of maltose and especially glucose by S. cerevisiae, a decrease in metab- olism of L. sanfranciscensis is expected when the latter is associated with maltose-positive yeasts. However, disappearance of S. cerevisiae from the micro- bial population of sourdough during consecutive fermentations is related to repression of genes involved in maltose fermentation – so that maltose cannot be utilized - and to rapid depletion of sucrose. Sourdough yeasts do not affect cell yield of L. sanfranciscensis, because pH is the limiting factor for growth of lactobacilli – note that L. sanfranciscensis does not grow below pH 3.8 (de Vuyst and Neysens, 2004).
3.4.7. Microecology in wine
Alcoholic fermentation is dominated by growth of yeasts, because of their ability to develop at low pH (3.0–3.5) – as prevailing in grape juice; they produce ethanol, which inhibits growth of filamentous fungi and bacteria. The first 2-4 days of fermentation are characterized by growth of various species of Kloeckera/Hanseniaspora, Candida, Metschnikowia, Pichia and Kluyveromyces – which achieve populations of the order of 108cfu /ml, before progressively dying off according to their tolerance to increasing concentrations of ethanol. By this time, they have utilized sufficient amounts of sugars and amino acids in the juice and have generated sufficient end-products to provide the fingerprint of wine.
Saccharomyces cerevisiae also grows during these early stages; however, its unique tolerance of ethanol permits its continuing growth, until it eventually pre- dominates during the mid-to-final phases of fermentation. Further studies on wine