MIC 303 INDUSTRIAL AND ENVIRONMENTAL MICROBIOLOGY INDUSTRIAL PRODUCTS FROM MICROBIAL PROCESSES

Advantages of using micro-organisms very rapid growth rates. utilise waste products as substrates e.g. agricultural wastes. can be grown continuously and on a large scale so there are less shut-downs and less re-sterilisations → economic advantages. high protein content. high yields from small factories. can be genetically manipulated. usually produce less toxic or non-toxic waste products. living organisms are used → temperatures used are lower than in chemical production which is therefore reduced cost

FOOD PRODUCTION Soya Sauce Miso (Tempe) Citric acid Lactic acid

It probably arrived in Japan from China with the introduction of Buddhism. Five types of soya sauce Typical compositions of five varieties of soy sauce Koikuchi (Deep brown) – 90% of total market. Usukuchi (Light brown) Tamari/ Tanari (Dark brown) Saishikomi (Dark brown) Shiro (Yellow) All soy sauces comprise 17-19% salts, seasoning and flavour enhancer. Classification of Soy Sauce

Classification of Soya Sauce by Japan Classification Koikuchi Most abundant in Japan 90 % Japanese production Reddish chocolate in colour Used in all food for cooking Aromatic and strong flavour Usukuchi 10 % Japanese production Colour not very dark, flavor and strong Used for cooking purpose

Classification of Soya Sauce by Japan Classification Tamari/ Tanari Produced and originated in China Strong flavor and dark chocolate in colour Sweet and liquid tanari Saishikomi Process alcohol Preferred in Japan Shiro Possess reducing sugar Often very sweet High amount of reducing sugar

Soya beans Soaking and cooking of soya beans Cooked in water (10-12 hours) Cooked until soft cooled 50 % Wheat Roasting and cracking of wheat Roasted Crushed roasted 50 % Rice steamed / wheat bran + soya beans (soaked in water) + Aspergillus oryzae spore Mix 0.1 – 0.2 % Seed koji Incubate (3-5 days) at 30ºC Koji Production PRODUCTION OF SOYA SAUCES

Market Brine Fermentation and Mash (Moromi) stage Bottling Pasteurization (5-10 min) Spread on trays (5 cm thick) Filter Pressed Mix with NaCl (17-19% + water) Incubate ( ºC) for 3 days Lactic acid fermentation (Pediococcus or P. soyae, followed by yeast fermentation by S. rouxii for 2 months) Refining stage

Production of soya sauces Two different processes: Soaking and cooking of soybeans Roasting and cracking of wheat Soaking and cooking of soybeans The soybeans (starting material) are soaked at room temperature (30°C) for h → doubling of their weight. The water either flows continuously over the beans or is added batch wise with changes every 2-3 h → prevent heat accumulation and the development of spore- forming bacteria. The swollen material is drained, recovered with water and steamed → induced softening and afford pasteurization. Followed by rapid cooling to less than 14°C on 30cm trays over which air is forced to avoid spoilage.

Production of soya sauces Roasting and cracking of wheat Wheat (or wheat flour or bran) is roasted to generate the desired flavouur characteristics. Products include vanillin and 4-ethylguaiacol from the degradation of lignin and glycosides. The degree of roasting will also impact the colour.

Production of soya sauces Koji production “Koji’ means ‘bloom of mould’. Involves the cultures of mixed strains of Aspergillus oryzae or Aspergillus sojae on either steamed polish rice or a mix of wheat bran and soybean flour – % to produce koji. Important characteristics of selected strains, different ability to generate high levels of several enzymes (protease, amylase, lipase, cellulase and peptidase) A 1:1 soybean: wheat mixture is spread in 5cm layers on bamboo (or steel) trays with the koji starter for 2-5 days. Moisture and temperature (25-35 °C) control is important to allow mycelial growth and reduce formation of spores.

Production of soya sauces Brine Fermentation and Mash (moromi) stage Mature Koji, mixed with an equal volume of saline (15-19% sodium chloride). Less → allows the development of putrefactive organisms. If [salts] is too high → inhibition of desirable osmophilic and halophilic organisms. Function of salt: to destroys the koji mycellium.

Production of soya sauces Refining The final process in soy sauce fermentation is refining, includes pressing, filtration, pasteurization and packaging. The aged moromi is pressed in a vertical automatic press to separate the soy sauce from the residue. After pressing, the filtered raw soy sauce is pasteurized in a heat-exchanger at ~ for a few minutes to ensure clarity, to inactivate residual enzymes, and to inactivate any undesirable microorganisms. It may be necessary to clarify the soy sauce additionally by centrifugation or sedimentation. The sauce is treated with caramel as a coloring agent, and then packaged either in clean glass bottles, enameled gallon cans or in plastic containers.

Soya beans + barley soaked in water Drain Cooked by steamed Spread on trays + cooled (30 days) at ºC Inoculate ascospores 70 % 30 % Soya beans (soaked in water) + steamed Mix Seed koji Cooled to get 30 – 60 % moisture content PRODUCTION OF MISO Drain Incubate days at 30ºC

Seed Koji Mixture Addition of NaCl (14-15%) Fermentation (2-3 months at ºC) by lactic acid bacteria Followed by yeast fermentation Mix blending Market Set aside for 2-3 weeks (aging) Package + Pasteurization

INDUSTRIAL ORGANIC ACID PRODUCTION

CITRIC ACID widely used in the food industry as an acidulant and flavouring agent in beverages, confectionary and other foods and in leavening systems for baked foods. Until 1920s, citric acid mainly prepared from lemon juice. In 1923, Pfizer began operate a fermentation-based process in USA, using A. niger grown in surface culture on a medium of sucrose and mineral salts. In 1940s, submerged fermentations have become the principle mode. Many bacteria, filamentous fungi, yeasts (Candida lypolytica) were used, but A. niger remains predominant industrial producer.

Citric acid Biosynthesis Primary metabolites. Metabolic pathway invlved Embden- Meyerhof-Parnas (EMP) pathway and TCA cycle. – Removal of iron, an activator of aconitase → Inhibition of continuation of TCA cycle and to accumulate citrate. Ex: addition of copper to diminishes aconitase activity.

CITRIC ACID PRODUCTION Substrate preparation (Aspergillus niger) Inoculums fermented Production fermented fermented Harvest tank Broth filter (Mould mycelium throw up) Filtrate receiver Fermented liquor Lime addition (to produce precipitate Ca citrate)

Precipitation of calcium citrate + H 2 SO 4 Regeneration of citric acid Active carbon (Decolorization treatment) Mother liquor recycle Crystalization Centrifugation Sieving Drying Packaging Filtration (CaSO 4 ) Gypsum disposal Filtration and washing Filtrate to effluent treatment Filtration Concentration (Convert Ca Citrate to respective organic acid)

LACTIC ACID Primarily used in the food industries as a preservative, an acidulant or in the preparation of dough conditioners. Lactic acid is produced in 20,000 – 100,000L fermentations using Lactobacillus delbruckii or other homolactic bacteria (L. bulgaricus). The media contain a complex nitrogen source and vitamin supplements, with up to 12% (w/v) sucrose or glucose as carbon source. Thse carbohydrates are metabolized to pyruvate via the EMP pathway, which is then converted to L- lactate dehydrogenase.

LACTIC ACID PRODUCTION 15 % maize sugar 10% CaCO 3 0.5% malt sprout 74.4 % H 2 O O.25 (NH 4 ) 2 PO 4 Filter Press Shake flask culture (Lactobacillus) Production fermenter Batch fermentation (1 week) Slurry + Lime Evaporator

Calcium lactate Filter Press Acid conversion tank Filter (remove CaSO 4 ) Cake slurry tank Storage Evaporator + H 2 SO 4 Lactic acid [50%]