MIC 303 INDUSTRIAL AND ENVIRONMENTAL MICROBIOLOGY

1. MIC 303INDUSTRIAL AND ENVIRONMENTAL MICROBIOLOGY INDUSTRIAL PRODUCTS FROM MICROBIAL PROCESSES

2. 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

3. FOOD PRODUCTION • Soya Sauce • Miso (Tempe) • Citric acid • Lactic acid

4. Classification of Soy Sauce • 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.

5. 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

6. 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

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

8. Spread on trays (5 cm thick) Incubate (25 - 30ºC) for 3 days Brine Fermentation and Mash (Moromi) stage Mix with NaCl (17-19% + water) Lactic acid fermentation (Pediococcus or P. soyae, followed by yeast fermentation by S. rouxii for 2 months) Pressed Filter Refining stage Bottling Pasteurization (5-10 min) Market

9. 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 12-15 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.

10. 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.

11. 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 – 0.1-0.2% 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.

12. 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.

13. 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 70-80 ~ 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.

14. PRODUCTION OF MISO Soya beans + barley soaked in water Drain Soya beans (soaked in water) + steamed Cooked by steamed Drain Spread on trays + cooled (30 days) at 20 -30ºC Cooled to get 30 – 60 % moisture content Inoculate ascospores 70 % Incubate 40-50 days at 30ºC Seed koji 30 % Mix

15. Mix Seed Koji Mixture Addition of NaCl (14-15%) Fermentation (2-3 months at 25-30 ºC) by lactic acid bacteria Followed by yeast fermentation Set aside for 2-3 weeks (aging) blending Package + Pasteurization Market

16. INDUSTRIAL ORGANIC ACID PRODUCTION

17. 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.

18. 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.

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

20. Filtration and washing Precipitation of calcium citrate (Convert Ca Citrate to respective organic acid) + H2SO4 Filtrate to effluent treatment Filtration Regeneration of citric acid (CaSO4) Gypsum disposal Active carbon (Decolorization treatment) Filtration Mother liquor recycle Concentration Crystalization Centrifugation Drying Sieving Packaging

21. 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.

22. LACTIC ACID PRODUCTION 15 % maize sugar 10% CaCO3 0.5% malt sprout 74.4 % H2O O.25 (NH4)2PO4 Shake flask culture (Lactobacillus) Production fermenter Batch fermentation (1 week) Slurry + Lime Filter Press Evaporator

23. Calcium lactate + H2SO4 Acid conversion tank Filter (remove CaSO4) Cake slurry tank Evaporator Filter Press Storage Lactic acid [50%]