Food Biotechnology Dr. Tarek Elbashiti

1. Food Biotechnology Dr. Tarek Elbashiti 5. Process Developments in Solid-State Fermentation for Food Applications

2. Solid-state (substrate) fermentation (SSF) has been defined as the fermentation process occurring in the absence or near absence of free water. • SSF processes generally employ a natural raw material as the carbon and energy source. • SSF can also employ an inert material as the solid matrix, which requires supplementing a nutrient solution to contain necessary nutrients as well as a carbon source. • The solid substrate (matrix), however, must contain enough moisture.

3. Solid substrates should generally have a large surface area per unit volume (in the range of 103–106 m2/cm3), to allow for ready growth on the solid–gas interface. • Several agro crops such as cassava and barley, and agro-industrial residues such as wheat bran, rice bran, sugarcane bagasse, cassava bagasse, various oil cakes (e.g., coconut oil cake, palm kernel cake, soybean cake, ground nut oil cake),

4. fruit pulps (e.g., apple pomace), corn cobs, sawdust, seeds (e.g., tamarind, jack fruit), coffee husk and coffee pulp, tea waste, and spent brewing grains are the most often, most commonly used substrates for SSF processes. • There are several other important factors which must be considered for development of SSF processes. • These include physico-chemical and biological factors such as pH of the medium; temperature and period of incubation; age, size and type of the inoculum; nature of the substrate; and type of microorganism employed.

5. SIGNIFICANCE OF SSF • It is cost effective due to the use of simple growth and production media comprising agro-industrial residues, and it uses small amounts of water and therefore releases considerably less effluent, thus reducing pollution concerns. • SSF processes are simple, use low volume equipment (lower cost), and are effective in that they provide high product titres (concentrated products).

6. Further, the aeration process (availability of atmospheric oxygen to the substrate) is easier. • There is increased diffusion rate of oxygen into moistened solid substrate, supporting the growth of aerial mycelium. • SSF can be effectively used at smaller volumes, which makes it suitable for rural areas.

7. SSF PROCESSES FOR FOOD APPLICATIONS • Many kinds of fermented foods, such as single-cell protein (SCP) probiotics, flavoring products, beverages, pigments, and peptide sweeteners, are produced largely by SSF. • In addition to these, several enzymes, organic acids, and exopolysaccharides have been produced using SSF.

8. Fermented foods can be stored for longer periods and used for food supply during the off season. • Fermentation also contributes to the digestibility and enhances the nutritional value of the product. • It can also increase fibre digestibility. • However, there are chances of accidental contamination of SSF food products by mycotoxins, which are a class of unwanted compounds and could be accidentally present or produced in SSF products.

9. Historical Developments • Historically, SSF processes have been used since ancient time for food applications. • SSF dates back to 6000 BC when Babylonians made beer from natural yeast. • Egyptians used this technique for bread making in 2600 BC, using brewer’s yeast. • Cheese making with Penicillium roquefortii was recorded in Asia before the birth of Christ. Koji processing reported to be migrated from China to Japan in the seventh century.

10. Miso, tempeh, tamari sauce, soy sauce, ang-kak, natto, tou-fu-ru, and minchin are some of the other ancient fermented foods known for centuries, which are prepared through SSF. • Tempeh and tamari sauce are soybean products, the former is an Indonesian food fermented by Rhizopus species and the latter is a Japanese food produced by using Aspergillus tamari. • Soy sauce, a brown, salty, tangy sauce, is obtained from a sterile mixture of wheat bran and soybean flour, fermented initially by lactic acid bacteria, followed by alcoholic fermentation and ripening.

11. In the eighteenth century, SSF was used to make vinegar from apple pomace. • The beginning of the twentieth century marked the use of SSF for the production of enzymes and organic acids using molds. • The period of the 1970s saw a major focus on production of SCP.

12. New Developments • It has been used particularly for the production of enzymes, organic acids, pigments, SCP, exopolysaccharides, and aroma compounds.

13. SSF PROCESSES FOR FOOD ENZYMES • Almost all the microbial enzymes can be produced using SSF systems. • Industrially important food enzymes, which include alpha amylase, glucoamylase, lipase, protease, pectinase, inulinase, glutaminase, and tannase, have been widely studied. • Enzyme production in SSF has often resulted in higher yields in comparison to SmF.

14. Studies conducted to examine the reasons why SSF produced higher enzyme yields than SmF have been unable to explain it fully, although some of the characteristics of SSF provide conditions for the microbes more like the habitat from which they were isolated, and this may be the major reason for higher enzyme production. • Most of the SSF processes for the production of food enzymes involve agro-industrial residues as the substrate, although inert materials such as polyurethane foam are also used Table 4.1.

17. 1. Amylases • α–Amylase (endo-α-1,4-D-glucan glucohydrolase, EC- 3.2.1.1) is an extracellular enzyme, which randomly cleaves the 1,4-α-D-glucosidic linkages in the interior of the amylose chain. • Amylases which produce free sugars are termed as saccharogenic amylases, and those which liquefy starch without producing free sugars are known as starch-liquefying amylases. • β-Amylase (α-1,4-glucan maltohydrolase, EC- 3.2.1.2) is an exoacting enzyme, which cleaves the nonreducing chain ends of amylose, amylopectin, and glycogen molecules, yielding maltose.

18. Glucoamylase [also known as amyloglucosidase, glucogenic enzyme, starch glucogenase, and gamma amylase (exo-α-1,4-D-glucan glucanohydrolase, EC- 3.2.1.3)] produces single glucose units from the nonreducing ends of amylose and amylopectin in a stepwise manner. • Unlike α- and β-amylase, most glucoamylases (GA) are able to hydrolyse the α-1,6 linkages at the branching points of amylopectin, although at a slower rate than 1,4-linkages. • Thus, glucose, maltose, and limit dextrins are the end products of GA starch hydrolysis.

19. Extensive studies have been done on the production of amylases, in particular for α-amylase and GA in SSF, employing several microorganisms and various kinds of agroindustrial residues, among which starchy substrates have been preferred (Table 4.1) • Reports on microbial production of β-amylase are scanty and very few in SSF. • A mutant strain of Bacillus megaterium, B6 mutant UN12, was described for the comparative production of beta amylase in SmF and SSF. • The starchy wastes used as substrates were from maize, potato, rice, rice husk, tamarind kernel, cassava, water chestnut, wheat, and wheat bran. wheat bran gave the highest yields.

20. Although the sources of α-amylases are fairly extensive, the principal commercial preparations are derived from some bacterial and fungal species. • The Bacillus species is considered the most prolific producer of α-amylase by SSF. • B. amyloliquefaciens and B. licheniformis are considered potent species for thermophilic α-amylase. • Irrespective of enzyme properties (such as temperature, pH optima, and range), SSFs are typically performed at mesophilic temperatures such as 30°C using agro-industrial residues for 24–96 h.

21. GA production in SSF was first demonstrated in 1914. • An extensive study was carried out on the production of GA in solid cultures by a strain of A. niger. • The study included screening of various agro-industrial residues, including wheat bran, rice bran, rice husk, gram flour, wheat flour, corn flour, tea waste, and copra waste, individually and in various combinations.

22. Apart from the substrate particle size, which showed profound impact on fungal growth and activity, substrate moisture and water activity also significantly influenced the enzyme yield. • Enzyme production in trays occurred in optimum quantities in 36 h in comparison to the 96 h typically required in flasks. • Supplementation of wheat bran medium with yeast extract increased glucoamylase synthesis by the fungal culture.

23. 2. Lipases: • Microbial lipases (glycerol ester hydrolases, EC 3.1.1.3)catalyse a wide range of reactions, specifically hydrolysis and interesterification. • They also catalyse alcoholysis, acidolysis, esterification, and aminolysis. • Microbial lipases are used for the production of desirable flavor in cheese and other foods, and for the interesterification of fats and oils to produce modified acyl glycerols, which cannot be obtained by conventional esterification.

24. Aspergillus lipases are highly selective for short chain acids and alcohols. • Candida rugosa lipase is more selective for propionic acid, butyric acid, butanol, pentanol, and hexanol. • The production of flavor esters by the lipases of Staphylococcus warneri and S. xylosus has been reported. • Mucor miehei and Rhizopus arrhizus lipases are more selective for long chain acid and acetates.

26. Several agro-industrial residues and inert supports have been used to produce lipases in SSF. • These include peanut press cake, coconut oil cake, wheat bran, rice bran, babassu oil cake, olive oil cake, sugarcane bagasse, and amberlite. • Enzyme production in SSF has been reported to be superior in comparison to SmF

27. 3. Proteases • Proteolytic enzymes find wide applications in food and other industries. • They account for nearly 60% of the industrial market in the enzyme technology. • Proteases are produced extracellularly by fungi and bacteria such as A. oryzae, A. flavus, R. oligosporus, Penicillium citrinum, P. chrysosporium, Bacillus subtilis, B. amyloliquefaciens, and Pseudomonas species. • In recent years, different types of proteases such as acid, neutral, and alkaline proteases are being produced by SSF.

28. Proteases production is generally inhibited by carbon sources, indicating the presence of catabolic repression of the biosynthesis. • It is interesting to note that although a number of substrates such as wheat bran, rice bran, and oil cakes have been employed for cultivating different microorganisms, wheat bran has been the preferred choice. • Acid protease production was increased when the gas had 4% CO2 (v/v) and it was directly related with the fungus metabolic activity as represented by the total CO2 evolved.

29. 4. Pectinases • Pectinases are a group of hydrolytic enzymes, usually referred as pectolytic enzymes, which find important applications in the food and beverages industries, in addition to various other industries. • Pectolytic enzymes degrade pectic material and reduce the viscosity of a solution, making it easier to handle. • They are used industrially in fruit processing to facilitate pressing and to help in the separation of the flocculent precipitate by sedimentation, filtration, or centrifugation in the extraction of the clarified juice. • Pectinases are used for the elimination of pectin in coffee and tea processing plants, and maceration of vegetable tissue.

30. Pectinases can be obtained from several fungi, bacteria, actinomycetes, and yeast, • Among these, A. niger is considered as the most important for production in SSF. • Various agro-industrial residues such as apple pomace, citrus waste, orange and lemon peels, soy bran, sugar cane bagasse, wheat bran, coffee pulp, cocoa pulp, and cranberry and strawberry pomace are used as the substrates. • Citrus pulps or peels could be used as inducers for enzyme synthesis. • In the case of pectinases, generally enzyme yields are higher and the enzyme is said to be more stable (over a wider range of pH and temperature) in SSF in comparison to SmF.

31. SSF PROCESSES FOR ORGANIC ACIDS • Production of organic acids such as citric acid for food application by SSF has been employed since olden times. • For example, citric acid production by SSF (the Koji process) was first developed in Japan, and is the simplest production method. • SSF can be carried out using several raw materials. • Generally, the substrate is moistened to about 70% depending on the water-holding capacity of the substrate. • The initial pH is normally adjusted to 4.5–6.0 and the temperature of incubation can vary from 28 to 30°C.

32. One of the important advantages of the SSF process is that the presence of trace elements does not affect citric acid production negatively as it does in SmF. • Commercially, citric acid is produced mainly using the filamentous fungus A. niger, although Candida sp. has also been used, employing both molasses- and starch-based media. • In SSF, production has been obtained using crops and crop residues such as apple pomace, grape pomace, coffee husk, cassava bagasse, beet molasses pineapple waste, and carrot waste as substrates by A. niger. • Citric acid production is largely dependent on the microorganisms, production techniques, and substrates employed.

33. Generally the addition of methanol increased citric acid production in SSF. • It has been proposed that overproduction of citric acid was related to an increased glucose flux through glycolysis. • At low glucose fluxes, oxalic acid could accumulate. • Table 4.2 shows SSF for citric acid production using agro-industrial residues by different strains of A. niger.

35. Another important organic acid required for food applications is lactic acid, which has been produced in SSF using fungal as well as bacterial cultures. • The commonly employed cultures belong to Rhizopus sp. and Lactobacillus sp., e.g., R. oryzae, L. paracasei, L. helveticus, and L. casei. • Different crops such as cassava and sweet sorghum, and crop residues such as sugarcane bagasse, and carrot-processing waste served as the substrate.

36. A comparative study involving fungal strains of R. oryzae to evaluate L-(+) lactic acid production in SmF and SSF showed that SSF was superior in production level and productivity. • Fermentation yields were 77% (irrespective of media) and yields were 93.8 and 137.0 g/l in SmF and SSF, respectively. • The productivity was 1.38 g/l per hour in SmF and 1.43 g/l per hour in SSF.

37. PRODUCTION OF AMINO ACIDS • The production of L-glutamic acid in SSF with sugarcane bagasse was used as solid inert substrate and a bacterial strain of Brevibacterium sp. was used for the fermentation. • The study not only showed good L-glutamic acid synthesis in solid culturing but also demonstrated that with media and process parameters manipulation, bacterial strains can also be successfully cultivated in SSF. • Yield as high as 80 mg glutamic acid per g dry fermented matter was obtained.

38. MUSHROOM PRODUCTION • Mushrooms have entered the new era of food technology as a common universally accepted nutritive food. • Their commercial cultivation involving SSF has rapidly spread globally, due to their innumerable applications. • They are a rich source of protein, carbohydrates, vitamins, and minerals. • Folic acid content in mushrooms has been found to be higher than in liver and spinach. • In addition to their nutritive value, mushrooms also possess medicinal properties.

39. They demonstrate antibacterial, antifungal, and antiprotozoal activities, due to the presence of polyacetylene compounds. • Today more than 2000 species of mushrooms are known, although only about 20 are cultivated commercially. • Button mushrooms, Japanese mark forest mushrooms, Chinese mushrooms, oyster mushrooms, and winter mushrooms are some of the various types of popular mushrooms being cultivated world wide. • However, button mushrooms alone account for about 60% of total world production.

40. Button mushrooms (Agaricus bisporus)

41. Mushroom cultivation involves SSF at three different stages, namely composting, spawn manufacture, and the growth of mushroom on the moist substrate. • A temperature range of 22–27°C, substrate moisture content of 55–70%, and a pH of 6.0–7.5 are generally considered as the most suitable conditions. • Animal manure from horses and chickens, and agro-industrial residues such as wheat straw, paddy straw, barley straw, rice bran, saw dust, banana, maize stover, tannery waste, wool waste, and sugarcane bagasse are used as substrates.

42. Spawn or inoculum production involves the growth of mycelia of mushroom on cereal grains such as rye, wheat, sorghum, and millet. • A mixture of sawdust and coffee husk was found quite suitable for spawn preparation for Agaricus bisporus, Pleurotus sp., Lentinus edodus, Flammulina velutipes, and Volvariella volvacea. • Depending upon the nature of the substrate, optimum conditions were moisture content at 40–60%, a pH of 6.5–7.0, and an incubation temperature of 25°C. • The final product spawn should be stored at 2–5°C.

43. Pleurotus sp.

44. The development of the fruiting body requires a lower temperature than the optimum for mycelial growth; it also requires proper ventilation, which helps in releasing the accumulated carbon dioxide, which retards the formation of fruiting bodies. • At this stage, substrate moisture should be generally higher than previous stages, (i.e., compost formation and spawn preparation). • High relative humidity (80–95%) is also a desirable condition in order to control the heat, mass, and gaseous exchange. • After harvesting of the fruiting body, the leftover solid residue can be used as manure or as animal feed, depending upon the raw material used as substrate.

45. PRODUCTION OF PIGMENTS • Pigments are the normal constituents of the cells or tissues that give color. • Pigments play a vital role in the food industry, to make food decorative and appealing. • Natural pigments contain provitamin A, and have anticancer activity, and other desirable properties such as stability to heat, light, and pH. • There are pigments which are chemo-synthetic counterparts of regular food components, that are referred as natural-identical. • Microbial production of pigments has usually been carried out in SmF, though SSF processes have also been applied.

46. Monascus pigments have good properties as food colorants possessing reasonable light and chemical stability, tinctorial strength, and water solubility when complexed with appropriate agents. • Monascus pigments may be used as substitutes for traditional food additives, such as nitrites for the preservation of meats, and as a potential replacement for synthetic food dyes.

47. Monoscus anka and M. pupureus are cultivated in SSF for red pigment production (Table 4.3). • Steamed rice is used as the substrate, although oats, wheat, and barley have also been used. • The culturing period is approximately three weeks. • Certain sugars, amino acids, and metals have been found important for the production, and yields are typically 10-fold higher in both SSF and SmF.

48. Pigment formation could be inhibited by the presence of glucose in the fermentation medium, but could be increased by limited aeration in SmF. • It was also observed that an increase in the partial pressure of CO2 increases the pigment production. • For isolating the pigment from fermented matter, simple extract with solvents such as ethanol is effective.

50. PRODUCTION OF AROMA COMPOUNDS • One of the significant applications of SSF in food applications involves production of food aroma compounds. • There are two main advantages of SSF as an alternative technology for the production of aroma compounds: • release of the products from the microbial membranes is facilitated by the higher concentration in liquid phase, and • sometimes the solid substrates or byproducts can be used directly in SSF without any pretreatment of the starting substrates.