21st Century Guidebook to Fungi, SECOND EDITION, by David Moore, Geoffrey D. Robson and Anthony P. J. Trinci

Table of Contents

1. Chapter 16: Fungi as pathogens of animals, including man
2. Chapter 17: Whole organism biotechnology
   1. Fungal fermentations in submerged liquid cultures
   2. Culturing fungi
   3. Oxygen demand and supply
   4. Fermenter engineering
   5. Fungal growth in liquid cultures
   6. Fermenter growth kinetics
   7. Growth yield
   8. Stationary phase
   9. Growth as pellets
   10. Beyond the batch culture
   11. Chemostats and turbidostats
   12. Uses of submerged fermentations
   13. Alcoholic fermentations
   14. Citric acid biotechnology
   15. Penicillin and other pharmaceuticals
   16. Enzymes for fabric conditioning and processing, and food processing
   17. Steroids and use of fungi to make chemical transformations
   18. The Quorn fermentation and evolution in fermenters
   19. Production of spores and other inocula
   20. Natural digestive fermentations in herbivores
   21. Solid state fermentations
   22. Digestion of lignocellulosic residues
   23. Bread; the other side of the alcoholic fermentation equation
   24. Cheese and salami manufacture
   25. Soy sauce, tempeh and other food products
   26. Chapter 17 References and further reading
   27. Buy a PDF of Chapter 17
3. Chapter 18: Molecular biotechnology

17.25 Soy sauce, tempeh and other food products

Fungi are used in the processing of several food products that enjoy large markets in Asia. In these the fungus is mainly responsible for the characteristic odour, flavour, or texture and may or may not become part of the final edible product, though many microorganisms may be involved in these microbial ecosystems. Growing fungi on water-soaked seeds of plants is a popular way to produce soy sauce and various other fermented foods (Moore & Chiu, 2001; Hutkins, 2006; Montet & Ray, 2015; Wolfe & Dutton, 2015; Joshi, 2016).

Soy sauce has been used in China for more than 2,500 years so it is one of the world’s oldest condiments. It is a seasoning agent with a salty taste and a distinctly meaty aroma, although it is made by a complex fermentation process of a combination of soy beans and wheat in water and salt, in which carbohydrates are fermented to alcohol and lactic acid and proteins broken down to peptides and amino acids. This traditional brewing, or fermentation, method can take 6 months or more to produce the finest soy sauce, which should be a dark brown, transparent, salty liquid with balanced flavour and aroma. The brown colour is a result of sugar caramelisation during the 6 to 8 month maturation process. There is an alternative non-brewed method: an acid hydrolysis that often produces opaque liquor with a harsher flavour but takes only two days to make. We note the alternative but will not discuss it further.

The raw materials are:

• soy beans (seeds of Glycine max; also known as soya or soja beans) are mashed prior to mixing them with other ingredients;
• pulverised wheat grains are mixed with the crushed soy beans (soy sauce produced by chemical hydrolysis generally does not use wheat);
• salt (specifically NaCl) is added at the start of second fermentation to 12-18%; this obviously contributes to flavour but it also establishes a selective environment for the lactic acid bacteria and yeasts to complete the fermentation; high salt concentration also serves as a preservative.

Manufacturing through the traditional brewed method occurs in three steps (Luh, 1995):

• Koji-making: in which the crushed soy beans and wheat are blended together in water that is boiled until the grains are thoroughly cooked and softened. The mash, as it is known, is allowed to cool to about 27°C and fermented with the filamentous ascomycetes Aspergillus oryzae and A. sojae. Depending on the size of the factory, the soy beans may be fermented in fist-sized balls (the traditional method) or on trays. The culture of soy, wheat, and mould is known as koji. The main function of these moulds is to break down the proteins in the mash; this process takes 3 days and the fungi (in what is known as the seed starter) are often closely-guarded proprietary strains because this step has a vital role in determining the flavour of the final soy sauce, and methods are now being developed to use genomics for strain improvement (Zhong et al., 2018).
• Brine fermentation: when the substrate has become overgrown with the mould fungus the koji is transferred to fermentation tanks and mixed with water and salt to produce a mash called moromi. Lactic acid bacteria (Pediococcus halophilus), and 30 days later yeast (Saccharomyces rouxii), are then added to complete the fermentation. The moromi ferments for several months, during which the soy and wheat paste is digested into a semi-liquid, reddish-brown ‘mature mash’ that contains over 200 flavour compounds produced as the bacteria and yeast enzymatically digest the protein and other residues to produce amino acids and derivatives.
• Refinement: the raw soy sauce is separated from the wheat and soy residue after 6 to 9 months of moromi fermentation by pressure-filtration through cloth. The filtrate is pasteurised (which forms additional flavour compounds) and bottled ready for sale. Soy sauce press cake is a valuable by-product as an animal feed.

Clearly, three major groups of microorganisms are involved in soy sauce fermentation: Aspergillus fungi involved in the koji production, and communities of halotolerant bacteria and yeasts are responsible for the moromi fermentation. Enzymes produced by all these various microbes hydrolyse the raw materials during the complex soy sauce fermentation process. Present-day manufacture is done in large factories using carefully controlled production conditions. Soy beans are now steamed instead of being boiled and temperature and humidity controls are applied during koji production. But in the traditional method in China, moromi is fermented in the open air; and there are significant differences between the products produced in different seasons of the year (Cui et al., 2014).

Soy sauce is the liquid produced as soy beans are fermented, several other traditional soyfoods are made mainly from whole soy beans (Moore & Chiu, 2001; Hutkins, 2006). Soy beans are highly nutritious, containing large amounts of protein and other nutrients, and the traditional soyfoods offer real health benefits. Traditional soyfoods are classified into nonfermented and fermented products (Liu, 2008). Non-fermented soyfoods include soymilk, tofu, soy sprouts, yuba (soymilk film), okara (soy pulp), vegetable soybeans, soynuts and toasted soy flour. Fermented soyfoods include the bacterial products natto (fermented with the rice straw bacterium Bacillus natto), soy yoghurt (soymilk fermented by bacteria), and the fungus-dependent products:

• sufu, which is pickled tofu made by fermenting with the zygomycete fungi Actinomucor elegans, Mucor racemosus, or Rhizopus spp.
• Soy nuggets; large whole soybeans soaked and steamed and mixed with roasted wheat- or glutinous-rice flour before fermentation with the koji mould Aspergillus oryzae. After several days of incubation, the resultant soybean koji is packed in kegs with salt water and various spices, seeds, and/or root ginger slivers (and occasionally rice wine), then aged for several months. The soy nuggets are then sun dried.
• Tempeh is a fermented food made by the controlled fermentation of cooked germinated soybeans with the filamentous zygomycete Rhizopus oligosporus. Fermentation by Rhizopus binds the soybeans into a protein-rich compact white cake that can be used as a meat substitute. Tempeh has been a favourite food and staple source of protein in Indonesia for several hundred years (http://www.tempeh.info/)
• Miso is possibly the most important fermented food in Japanese cuisine. It is basically fermented soy bean paste but rice and several cereal grains and even other seeds can be combined into an extremely wide variety of miso that differ according to the combination of ingredients. The basic production process is essentially the same for all recipes: rice, barley or soy beans are steamed, cooled, and inoculated with the koji mould Aspergillus oryzae. When the koji has become established it is added, as a seed culture, to a mixture of washed, cooked, cooled and crushed soy beans, water and salt. This is placed in vats and allowed to ferment for 12 to 15 months to allow the proteins in the mixture to be broken down slowly and naturally, forming a paste that is flavoursome and nutritious.

Another fermented product of this sort, ang-kak is a bright reddish purple fermented rice product popular in China and the Philippines which is fermented using Monascus species (Ascomycota, order Eurotiales). In Petri dish cultures, the mycelium is white in early growth stages, however, it rapidly changes to a rich pink and subsequently to a distinctive yellow-orange colour (illustrated in Pattanagul et al., 2007). Monascus purpureus produces the characteristic pigments and ethanol which are used for red rice wine and food colouring. The pigments are a mixture of red, yellow and purple polyketides and about ten times more pigment is obtained from solid state fermentation than from submerged liquid fermentation.

The rice is first soaked in water until the grains are fully saturated and may then be directly inoculated, or steamed prior to inoculation. Inoculation is done by mixing M. purpureus spores or powdered red yeast rice together with the processed rice. The mix is then incubated at room temperature for 3 to 6 days. At the end of this time, the rice grains should have turned bright red in the centre and reddish purple on the outside. Red rice is sold as a pasteurised wet aggregate in jars, as whole dried grains, or as a ground powder (to be used as a red food colourant)( Lin et al., 2008; Pattanagul et al., 2007; Panda et al., 2010).

Updated July, 2019