The following points highlight the three main foods prepared by mould fermentation process produced by soya bean. The food are:- 1. Tempeh 2. Soy Sauce and Rice Wine 3. Mycoprotein.
Tempeh is a traditional mould-fermented food in Indonesia, though it has also attracted interest in the Netherlands and United States. The most popular type of tempeh is produced from soya beans and is also known as tempeh kedele. The process of tempeh production is outlined in Figure 9.17. Whole clean soya beans are soaked overnight in water to hydrate the beans.
A bacterial fermentation occurs during this stage decreasing the pH to 4.5-5.3. The hydrated beans are de-hulled and the moist cotyledons cooked; a process which pasteurizes the substrate, destroys the trypsin inhibitor and lectins contained in the bean and releases some of the nutrients required for fungal growth.
After cooking, the beans are drained and may be pressed lightly to remove excess moisture before spreading into shallow bamboo trays and allowing to cool. Starter culture is added either by mixing some tempeh in with the cooked soya beans prior to packing in the trays or by sprinkling a spore inoculum, prepared by extended incubation of a piece of tempeh, on to the beans.
The fermentation is invariably a mixed culture of moulds, yeasts and bacteria but the most important component appears to be Rhizopus oligosporus, although other Rhizopus and Mucor species are often isolated. Over two days incubation at ambient temperature (30-35 °C), the mycelium develops throughout the mass of beans knitting it together.
During fermentation the pH rises to around 7, fungal proteases increase the free amino acid content of the product and lipases hydrolyse over a third of the neutral fat present to free fatty acids. Unlike many fermented foods, tempeh production is not a means of improving the shelf-life of its raw material which is in any case inherently quite stable.
Tempeh contains antioxidants which retard the development of rancidity but will keep for only one to two days as sporulation of the mould dis-colours the product and a rich ammoniacal odour develops as proteolysis proceeds.
Tempeh production does however improve the acceptability of an otherwise rather unappealing food. Fresh tempeh has a pleasant nutty odour and flavour and can be consumed in a variety of ways, usually after frying in oil.
In addition to improving acceptability, fermentation also improves the nutritional quality of soya beans. In part this stems from the reduction or removal of various anti-nutritional factors at different stages in the processing.
Destruction of the trypsin inhibitor and lectins during cooking of the beans has already been mentioned and levels of phytic acid, which can interfere with mineral nutrition, are also reduced by about a third in the course of processing.
The notorious ability of beans to produce flatulence is also regarded as an anti-nutritional property and flatulence-inducing oligosaccharides such as stachyose and raffinose are partially leached out of the beans during the soaking stage.
Despite the extensive proteolytic changes which occur during fermentation, studies have failed to show that the protein in tempeh is more easily digested. With the exception of thiamine which decreases, other vitamins increase to varying degrees during fermentation.
Vitamin B12, the anti-pernicious anaemia factor, shows the most marked increase and this is associated with the growth of the bacterium Klebsiella pneumoniae during fermentation. The usual source of this vitamin in the diet is animal products and it has been suggested that tempeh could be an important source of B12 for people subsisting on a largely vegetarian diet.
Tempeh can be made from a number of different plant materials including other legumes, cereals and agricultural by-products. One variety that has achieved some notoriety is tempeh bongkrek which is made in central Java using the presscake remaining after extraction of coconut oil.
Tempeh bongkrek has been associated with occasional serious outbreaks of food poisoning due to the bacterium Pseudomonas cocovenenans growing in the product and elaborating the toxins bongkrekic acid and toxoflavin (Figure 9.18). Since 1951, at least 1000 people are known to have died as a result of this intoxication and in 1988 the Indonesian Government prohibited the production of tempeh bongkrek.
Two factors are thought to give rise to this problem. Reduction or omission of an initial soaking of the presscake may fail to give a lactic fermentation sufficient to reduce the pH below 6, a level at which the bacterium cannot grow.
Also, the fungal inoculum may be too small since it has been shown that P. cocovenenans cannot grow if Rhizopus oligosporus has more than a tenfold numerical superiority (estimated by plate counts).
Ontjom is a tempeh-like product produced in Indonesia from peanut presscake which normally has a fruity/mincemeat character. It can be produced using the tempeh mould but Neurospora intermedia is also often used. This mould has strong α- galactosidase activity which can further contribute to the reduction of flatulence- inducing oligosaccharides.
2. Soy Sauce and Rice Wine:
Though they are markedly different in character, rice wine and soy sauce share sufficient common features in their production to warrant discussing them together. Both are representatives of products which involve mould activity in a two-stage fermentation process. The mould starter used is often known as koji, a Japanese term derived from the Chinese character for mouldy grains.
In the koji stage, aerobic conditions allow moulds to grow on the substrate producing a range of hydrolytic enzymes necessary for utilizing the macromolecular material present. In the case of soy sauce (Figure 9.19), soaked and cooked soya beans are mixed with roasted cracked wheat in about equal proportions and inoculated with tane koji or seed koji.
This has been previously grown-up on a similar mixture of substrates and contains a mixture of strains of Aspergillus oryzae. The moulds are then allowed to grow throughout the mass of material spread as layers about 5 cm deep for 2-3 days at 25-30 °C.
In the second, mash or moromi stage, conditions are made anaerobic so no further mould growth can occur. In soy sauce production, this is achieved by mixing the koji with an approximately equal volume of brine to give a final salt concentration of 17-20%.
Although the moulds can no longer grow, the activity of a whole battery of hydrolytic enzymes continues breaking down proteins, polysaccharides and nucleic acids to produce a liquid rich in soluble nutrients.
Yeasts and lactic acid bacteria dominate the microflora producing a number of flavour components and converting roughly half of the soluble sugars to lactic acid and ethanol so that the final soy sauce normally has a pH of 4.5-4.9 and ethanol and lactic acid contents of 2-3% and 1% respectively.
The halophilic lactic acid bacterium Tetragenococcus halophilus (formerly Pediococcus halophilus) and the yeasts Zygosaccharomyces rouxii and Torulopsis have been identified as being important in this stage.
The moromi stage can be quite pro acted, lasting up to a year or more, at the end of which the mash is pressed to remove the solid residues which may then be mixed with brine to undergo a second fermentation and produce a lower grade product. The liquid is pasteurized and filtered, possibly after a period of maturation, and then bottled.
Rather similar steps are involved in the production of soya bean pastes known as miso in Japan and chiang in China. These include up to 40% of a grain such as rice or barley, use dry salt rather than brine, and employ a shorter fermentation so the product has the consistency of a paste rather than a liquid.
In the brewing of the Japanese rice wine, sake, a koji prepared on steamed rice is used. Although the mould used is the same species as in soy sauce production, Aspergillus oryzae, the strains used in sake production are particularly noted for their ability to produce amylolytic enzymes.
In the moromi stage, water is added along with strains of the yeast Saccharomyces cerevisiae specially adapted to the sake fermentation. During this stage amylolytic enzymes from the mould continue to break down the starch in the rice to produce fermentable sugars which are then converted to ethanol by the yeast.
The high alcohol content of around 20% v/v achieved in such fermentations is thought to be due to a combination of factors. Particularly important is the slow rate of fermentation which results from the relatively low fermentation temperature (13-18 °C) and the slow release of fermentable sugars.
The high solids content in the moromi is also thought to help in keeping the yeast in suspension and active at such high alcohol concentrations. At the end of fermentation which typically lasts for three weeks, the product is settled, filtered and blended before being pasteurized and bottled.
A number of other rice-based mould starters are used in the countries of East and Southeast Asia to fulfill a similar role to koji (Table 9.11). They are used to produce sweetened rice products which can be consumed fresh or added to other products or can be used as a base for the production of rice wine and rice vinegar.
The microbiological composition of these generally differs from that of koji and comprises primarily Rhizopus and Mucor species and amylolytic yeasts.
Products such as tempeh and koji contain a significant amount of mould biomass and a reasonable extension of this type of approach would be to grow up mycelium itself as a source of food. Of the many investigations into the growth of moulds on readily available substrates one has successfully emerged as a commercial product.
Mycoprotein, marketed as Quorn, is essentially the mycelium of Fusarium graminearum grown in continuous culture in a medium containing glucose, ammonium salts and a few growth factors. Advantages associated with the use of a filamentous organism are that it can be harvested by filtration and washing and can be readily textured to give the product an acceptable mouth-feel.
To be acceptable as a food for human consumption it is necessary to reduce the level of RNA, which is nearly 10% based on mycelial dry weight, to below the levels likely to lead to kidney-stone formation or gout.
This is achieved by a mild heat treatment prior to filtration which activates the mould’s RNAases and leads to a dramatic reduction of RNA to about 1% which is acceptable. The product has a useful protein content of 44% and is high in ‘fibre’ because of the cell walls of the filamentous fungal structure.