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JR kiwi-'31.}. 111 IS: ~-- .3...— mummnmnn'm W fuse-1‘s 3 1293 006,61 [gisw ..- .‘ '.0' IO 4' . .‘fi‘ ~I‘J‘ I‘d“ H mm __; " a 5' a f: R " ','~ f2~ ,, ,1,- «iz, :«~ ; 33w!» ‘ ,_‘ ._ . ..- .. n u’, -., . 0 .~ ,.I‘\v Qan6«-;a ~,‘-'....L a. axe—aug- WBEE figfue‘] This is to certify that the thesis entitled 7’9 ’4 ”/LK flittb «cf presented by SYLVESFEA NGOI/ [CWYENEHLO '7? 0?954-9/ has been accepted towards fulfillment of the requirements for ”263 7672 § degree in M 5/1165 , 4! Major professor / Date é/Q/Ké 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution MSU RETURNING MATERIALS: Piace in book drop to LJBRARJES remove this checkout from -_ your record. FINES will be charged if book is returned after the date stamped below. eLiW ( :93 Guinea; “311 CHARACTERISTICS OF AN ORIENTAL-TYPE CULTURE AS APPLIED TO A MILK PRODUCT BY Sylvester Ngozi Onyeneho A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1986 ABSTRACT CHARACTERISTICS OF AN ORIENTAL-TYPE CULTURE AS APPLIED TO A MILK PRODUCT BY Sylvester Ngozi Onyeneho A Chinese wine cake was used to ferment steamed glutinous rice and produce a liquid filtrate. The fil- trate, "sweet-leavening," contained 11.06% ethanol and exhibited proteolytic and milk-clotting activities. This filtrate was added to a milk-based mix to obtain a sweet- set gel product, "Gua-nai." The organisms in the wine cake were isolated and identified as Amylomyces rouxii, Rhizopus oryzae, Aspergillus oryzae and a yeast, Endomygopsis burtonii. The pH of the fermentation medium fell from 6.6 to 3.81 and titratable acidity rose to 0.66% in six days. A gradual disappearance of the filamentous molds left only the yeast in the fermentation medium. The isolated organisms were used to reconstitute the wine cake and subsequently employed in Gua-nai manu— facture. This product was compared with Gua-nai made with commercial wine cake and the differences between them were not significant. To Adaugo, Emeka and Abumchi and to the Glory of God Almighty ACKNOWLEDGMENTS I would like to express my sincere appreciation to my major professor, Dr. J.A. Partridge for his eternal patience, helpful criticisms and valuable suggestions during the course of my study and preparation of this manuscript. I am also very grateful to Dr. J.R. Brunner for his helpful suggestions with regard to experimental procedures and his effort in reviewing this manuscript; to Dr. J.J. Pestka for his guidance‘and for supplying some of the materials used in this work; to Dr. C.M. Stine for his suggestions and serving in my guidance committee and to Dr. E.S. Beneke, Department of Botany and Plant Pathology for his help in confirming the identities of the isolated molds. I am also indebted to my friends Mucio Furtado and Khimji Nakrani for their assistance and to Charles Lin for translating the Chinese labels on some materials used in this study. The technical assistance of Ursula Koch is highly appreciated. Financial Support provided by the Federal University of Technology, Owerri, Nigeria is gratefully acknowledged. Lastly, I am unable to express adequately my iii gratefulness to my wife Kate Uchechi and to my father Joseph and members of my family for their patience, encouragement and support during this period of graduate study. iv TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . . . vii LIST OF FIGURES . . . . . . . . . . . . . . . . viii INTRODUCTION . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . . . . 3 Fermented Rice Foods and Beverages . . . 3 Ethanol Production By Fermentation . . . 8 Flavor Development During Fermentation . ll Milk-clotting Enzymes From Molds . . . . 12 Formation of Milk Curd . . . . . . . . . 18 MATERIALS AND METHODS . . . . . . . . . . . . . 20 Gua-nai Manufacture . . . . . . . . . . . 20 Isolation of Microorganisms From Rice Culture . . . . . . . . . . . . . 23 Identification of Isolates . . . . . . . 24. Evaluation of Pure Isolates for Ability to Ferment Rice . . . . . . . . . . . . 25 Microbial Changes During Rice Fermentation . . . . . . . . . . . . . 26 Reconstitution of the Rice Culture . . . 26 Analysis of Filtrate From Rice Fermentation . . . . . . . . . . . . . 30 Ethanol Determination . . . . . . . . . . 30 Assay For Milk-clotting Activity . . . . 31 Assay For Proteolytic Activity . . . . . . Compositional Analysis of Gua-nai . . . . Consumer Taste Panel . . . . . . . . . . . RESULTS AND DISCUSSION . . . . . . . . . . . . . Isolation and Characterization of Micro- organisms From the Chinese Wine Cake . . Physical Changes During Rice Fermentation. Gua-nai Manufacture . . ... . . . . . . . pH and Titratable Acidity Changes During Rice Fermentation . . . . . . . . Microbial Changes During Rice Fermentation . . . . . . . . . . . . . . Ethanol Production During Rice Fermentation . . . . . . . . . . . . . . Evaluation of Culture Filtrates for Milk- clotting and Proteolytic Activities . . Commercial vs. Reconstituted Culture in Gua-nai Manufacture . . . . . . . . . . SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . RECOMMENDATIONS FOR FURTHER RESEARCH . . . . . . BIBLIOGRAPHY O O O O O O O O O O O O O O O O O 0 vi 32 33 34 36 36 47 49 51 54 59 64 71 75 78 79 LIST OF TABLES Table 1. Descriptive Chart for A. rouxii . . . . . . 2. Descriptive Chart for R. oryzae . . . . . . 3. Descriptive Chart for A. oryzae . . . . . . 4. Descriptive Chart for 5. burtonii . . . . . 5. Microbial Changes During Rice Fermentation 6. Ethanol Production During Rice Fermentation 7. Milk-clotting and Proteolytic Activities of Cultural Filtrates . . . . . . . . . . 8. Effect of pH on Milk-clotting Activity . . 9. Compositional Analysis of Gua-nai . . . . . 10. Consumer Taste Panel Results . . . . . . . vii Page 37 39 42 44 56 62 65 67 72 73 LIST OF FIGURES Figure 1. Flow Sheet for Gua-nai Manufacture . . . . . . 2. Flow Sheet for Rice Culture Preparation . . . 3. Freeze-dried Culture Preparation . . . . . . . 4. Gua-nai Taste Panel Score Card . .1. . . . . . 5. Amylomyces rouxii . . . . . . . . . . . . . . 6. Rhizopus oryzae . . . . . . . . . . . . . . . 7. Aspergillus oryzae . . . . . . . . . . . . . . 8. Endomycopsis burtonii . . . . . . . . . . . . 9. Steamed Glutinous Rice--Before and After Fermentation . . . . . . . . . . . . . . . . 10. Changes in pH Accompanying the Fermentation of Rice by the Various Cultures . . . . . . 11. Changes in Titratable Acidity Accompanying the Fermentation of Rice by the Various cultures 0 O O O O O O O O I O O I O O O O O 12. High Performance Liquid Chromatograms Showing Ethanol Analysis of Culture Filtrates when (A) Reconstituted Culture and (B) Commercial Chinese Wine Cake Culture Were Used to Ferment Steamed Glutinous Rice . . . . . . viii Page 22 28 29 35 38 41 43 45 48 52 53 60 INTRODUCTION One of the most attractive features of milk is that its nature makes it readily adaptable for modification. Milk can be changed into different physical forms, liquid or solid and its bland flavor can be modified in the final product with flavors elaborated by microorganisms or by natural or artificial flavor additives. Moreover, the highly nutritive protein fractions of milk and milk products rightfully contribute to the supply of the world's protein needs. The dairy industry has been very active in develop- ing products capable of stimulating the curiosity of the consumer. The great success of these products, such as yogurt, is attributable to their organoleptic qualities. To remain dynamic and progressive, the dairy industry has to be formulating innovative new products. In pursuance of these objectives, a new sweet-curdled milk product was developed at the Michigan State University's Dairy plant early in 1985. This product called "Gua—nai" (Guan's milk) is a sweet-set gel product from pasteurized whole or skim milk. The sweet-curdling process was achieved with the "sweet-leavening"--a culture system of oriental origin. Gua-nai has distinct flavor and aroma, a smooth 2 surface, is milky in color and approximates yogurt in mouth feel. The present study was undertaken to isolate and characterizetjuamiCroorganisms in the Chinese wine cake and monitor the microbial changes that occur during rice fermentation. In addition the liquid from the rice fermentation was to be analyzed and the oriental rice culture was to be reconstituted with the isolated micro- organisms. REVIEW OF LITERATURE ,Fermented Rice Foods and Beverages Steinkraus (1983) reviewed the literature pertaining to alcoholic foods and beverages in which starch hydrolysis and fermentation are accomplished by amylolytic molds and yeasts. He stated that, in all cases, saccharification of the starchy substrate is due primarily to amylases produced by filamentous fungi. He emphasized that the essential amylase producers range from Aspergillus oryzae, used in Japanese sake manufacture, to molds of the genera Mucor and Rhizopus, as well as Amylomypes rouxii and others that play varying roles in the fermentation. He stressed that deliberate transfer of fermenting substrate from one batch to the next is a very common way of inoculating new fermentations. Several methods are employed in inoculating Indonesian tempe--a white, mold-covered cake produced by fungal fermentation of dehulled, hydrated, and partially cooked soybean cotyledons. Among these is the use of a "ragi-tempe" in which the essential mold belonging to the genus Rhizopus is grown on rice flour and dried as a small, round flattened cake. This cake contains the essential 4 molds and yeast necessary for the alcoholic fermentation (Ko, 1977 and Yeoh, 1977a). According to these authors, variations exist in the traditional methods of preparing ragi. Generally, it is made by mixing rice flour with dry powdered ginger, pepper, chilis, garlic, cane sugar, or other additives. The mixture is moistened with water and inoculated with dry powdered ragi from previous batches. It is then incubated for several days at ambient tempera— ture followed by dehydration to preserve the cakes until needed (Saono et al., 1974). Studies on microorganisms isolated from ragi and Chinese yeast cake show that the essential organisms in ragi tape to be molds of the Amylomyces rouxii Calmette- type (formerly known as Chlamydomucor oryzae) (Ellis et al., 1976) and yeasts of the Endomycopsis burtonii (Ko, 1972). KC (1972) used pure cultures of A, rouxii and E. fibuliger and manufactured acceptable tape ketan. Kodama and Yoshizawa (1977) described the manufac- ture of Koji--a concentrate of fungal amylases, proteases and other enzymes. This is obtained by overgrowing steamed rice with selected strains of Aspergillus oryzae. They stated that rice is soaked, drained, steamed, cooled and inoculated with spores of A, oryzae and incubated at 30°C for 6 days or until there is abundant sporulation. At the conclusion of the incubation period, the fungal mycelium covers the grains. The final product Koji, 5 contains enzymes, vitamins and other nutrients. Yoshizawa (1977) also stated that, during Koji manufacture, alpha— amylase (liquefying amylase) and amyloglucosidase (saccharifying amylase) are elaborated and hydrolyze the rice starch to dextrins, glucose and maltose. Dwidjoseputro and Wolf (1970) isolated the following microorganisms from ragi used for tempe fermentation: Rhizopus oryzae, R. arrhizus, R. oligpsporus, Mucor rouxii. Other molds isolated include Trichosporon ppllulans, Aspergillus niger and Fusarium g2, Saono et a1 . (1977) described the use of ragi in the manufacture of Indonesian tape-ketan. Glutinous rice is washed, soaked for one hour or longer, steamed until well cooked and sticky, spread on a woody bamboo tray and cooled to room temperature. Powdered ragi is then spread on the rice which is placed in containers to ferment. The product is a sweet/sour alcoholic paste. Cronk et a1 . (1977) studied the biochemical changes that occur in the substrate during tape fermentation. They found that A, rouxii produced about 5.6% (vol/vol)ethanol after 96 hours of incubation. The highest concentration of ethanol, 8% (vol/vol) was produced by A. rouxii in com- bination with the yeast Endomycopsis burtonii after 144 hr. They also observed that the mold alone reduced the initial pH from 6.3 to about 4.0 in 48 hours. Ragi is also used in the fermentation of the Malaysian rice wine "tapai" 6 which is produced from glutinous rice. At the conclusion of fermentation, the liquid phase is clear yellow and very sweet before the addition of Brandy (Wong and Jackson, 1977). Japanese sake, as described by Murakami (1972), is a clear, pale yellow, rice wine with an alcoholic content of 15 to 16 percent or higher. It possesses a character- istic aroma, little acid, and a slight sweetness. In sake manufacture, rice of the short-grained variety is washed and steeped in water before steaming. Starch sacchari- fication is achieved through the use of Koji--a concentrate of fungal amylases and other enzymes produced by Aspergillus oryzae (Kodama and Yoshizawa, 1977). According to these workers, sake manufacture is a good example of complex microbial interaction in which A. oryzae is the source of amylase and proteases for saccharification of the starch and the hydrolysis of the rice protein. A yeast inoculum of Saccharomyces sake is introduced during the fermentation process for ethanol production. Several other rice wines have been described which involve the fermentation of steamed rice by filamentous molds or in combination with a variety of yeasts. These include the Phillipine "Tapuy" which is highly acidic but sweet, aromatic and alcoholic (Uyenco and Gacutan, 1977b; Tanimura et al., 1978a); Primitive "Thai" rice wine, a cloudy, effervescent, yellowish, alcoholic drink 7 (Steinkraus, 1977); Indian "ruhi" a strongly alcoholic beverage (Dahiya and Prabhu, 1977). The inoculum consists of molds of the genera Rhizopus and M2225 as well as yeasts and lactic acid bacteria. Ethanol concentrations in the order of 14 percent (vol/vol) are obtained. Dahiya and Prabhu (1977) described another Indian wine of low alcoholic content called "madhu," a product of rice fermentation by the same microbes as used to produce "ruhi." "Ang-kak," the Chinese red rice wine is a product of fermentation by various strains of Monascus purpureus. It is used for coloring various foods such as fish and Chinese cheese (Hesseltine, 1965). Rice is inoculated with Monascus possessing saccharifying and proteolytic activity (Pichyangkura, 1977). He also stated that Monascus secretes rubropunctatin and monascorubin (red pigments); and rubropunctamine and monascorubramine (purple pigments). Monascus produces ethanol and organic acids on suitable media. "Lao—chao" is another fermented rice product in which steamed glutinous rice is inoculated with a culture containing filamentous fungi and a yeast. Wang and Hesseltine (1970) identified the fermenting organisms as Rhizopus oryzae, R. Chinensis, Chlamydomucor oryzae and a yeast, Endomycopsis. According to these authors, starch is the most important constituent in the fermentathmm 8 which is hydrolyzed by amylase and other glucosidases produced by the organisms yielding substances which are used by the yeast to produce ethanol. They stated that ‘3. Chinensis is known to produce large amounts of lactic acid, alcohol, and carbon dioxide. Thus, a mixture of esters can be formed without yeast fermentation. They stated that Lao-chao is soft, juicy, sweet and slightly- alcoholic and has a unique place in the diet of new mothers. Ethanol Production by Fermentation The term 'fermentation' describes the breakdown of carbohydrate materials under anaerobic conditions (Porter, 1980). It involves the mechanism of oxidation-reduction with organic compounds rather than molecular oxygen serving as terminal election acceptors. According to Ribereau-Gayon and Peynaud (1970), alcoholic fermentation goes through four essential phases: formation of trioses from glucose; dehydrogenation of trioses into pyruvic acid; decarboxylation of pyruvic acid into acetaldehyde; and the reduction of acetaldehyde to ethanol. Any raw material composed of hexose sugars can serve as a substrate for ethanol fermentation (Kosaric et al., 1980). Production of ethanol by yeast fermentation has two main applications: Manufacture of a variety of alcoholic 9 beverages; and alcohol production for industrial use (Nancy, 1980), but the manufacture of alcoholic beverages comprises one of the largest industrial applications of microbial activity. Ethanol is a well known cell poison and narcotic and Ramalingham and Finn (1977) showed that yeast growth and sugar conversion during alcoholic fermen- tation are inhibited by the formation of ethanol but §:.cerevisiae remains viable and metabolically active in media containing self-produced ethanol that can reach a concentration as high as 20% v/v (Rose, 1980). Harrison and Graham (1972) stated that the fermentation rate and the maximum level of ethanol production decreased with increased concentration of sugar. Ethanol inhibition is the rate-limiting factor in yeast or other microbial alcoholic fermentations (Stucley and Pamment, 1982). Gray (1941) defined ethanol tolerance as the concentration of ethanol in the growth medium that reduced the rate of sugar utilization. Different organisms vary in the amount of ethanol they produCe and the level they tolerate. Nojiro and Ouchi (1962) stated that almost all yeasts used in making alcoholic beverages have an ethanol tolerance of 20 to 30 percent. They further noted that ethanol toler- ance increases with age of the yeast but the ability to ferment is greater in younger yeasts. Production of alcohol has not been limited to yeasts only. Cronk et al . (1979) studied the production 10 of higher alcohols during Indonesian tape—ketan fermenta- tion. They employed Amylomyces rouxii as the principal mold alone or in combination with yeasts. They observed that A. rouxii alone produced nearly the same quantity of alcohol as produced when in combination with Endomycopsis burtonii. Pichyangkura and Kulprecha (1977) examined 52 samples of mold bran "Loog-pang" used as a starter for pro- duction of alcoholic rice wines. They found out that molds involved belong to the genera Amylomyces, Rhizopus, Mucor, Aspergillus and Absidia. Ko (1977) produced "ragi" (starter) containing pure cultures of single microorganisms. He used A. rouxii to ferment steamed glutinous rice and was able to produce typical "tape-ketan," a sweet-sour alcoholic paste. The product has an ethanol concentration of 5.0 percent. Dahiya and Prabhu (1977) noted that molds belonging to the genera Rhizopus and M3235 are involved in the pro- duction of "ruhi." This is a strongly alcoholic beverage of Indian origin made by the fermentation of boiled rice. According to these authors, starch is hydrolyzed to sugar which, in turn, is fermented to ethanol. The ethanol content of "ruhi" is 12-14 percent (vol/vol). Batra and Millner (1974) stated that Mucor rouxianus in addition to its saccharifying ability, can also utilize and ferment several sugars to ethanol; lactose and sucrose 11 being the exception. Flavor Development During Fermentation Flavor is a term used to describe the impressions obtained by the senses of taste and smell. Marshall et a1. (1984) stated that organoleptic qualities are the con- sequences of multiple fermentations. He further said that the selection of the multi-strain or more often multi- species inoculum is necessary to obtain a good fermentation product. Clarification of the many pathways leading to the biosynthesis of important flavor compounds have been care- fully compiled by Collins (1972). He stated that whatever the route of carbohydrate catabolism, the important products are pyruvate, acetyl coenzyme A and acetyl phos- phate. These are the key intermediates and their subse- quent metabolic routes are of interest. Collins and Bruhn (1970) showed that each starter has its own way of meta- bolizing these intermediates to produce flavorsome metabolites. The taste and aroma of a product are generally con- sidered the main factors in determining its overall flavor, and are determined by its chemical composition. Bills et a1 . (1965) and Lindsay et al . (1965) explained that formulation of mixed lactic starters can be based on their ability to produce flavor components such as diacetyl and 12 acetaldehyde as well as their ability to produce acid. The development of quantitative gas chromatographic analysis of cultures has resulted in improved evaluation of flavor volatiles (Davies and Law, 1984). In the manu- facture of tape-ketan, the hydrolysis of steamed rice starch to maltose and glucose and the fermentation of sugars to ethanol and organic acids lead to ester formation that provide an attractive flavor and aroma.(Ko, 1977; Yeoh, 1977b). Moreover, Saono et al . (1977) noted that if yeasts of the Hansenula type are present in tape- production, then acid and ethanol are esterified producing highly aromatic tape. Cronk et al., (1979) showed that fusel oils (higher) alcohols) in concentration of 200—300 mg per liter con- tribute to the flavor, smoothness and body of wines. They also contribute to smoothness and flavor of tape and tape- like products. They also noted that ethanol was detected only in rice fermented with Hansenula species in combina- tion with Amylomyces rouxii. Milk-Clotting Enzymes from Molds The main objective of milk coagulants is the con- version of fluid milk to a curd. Coagulation can be catalyzed by many different proteolytic enzymes, the quantity and variety of which is dependent upon its acti- vity and specificity (Green, 1984). Ramification of this 13 phenomenon influence flavor and texture characteristics in the final product. Davis and Law (1984) stated that enzymes important in this regard are acid proteases pro- duced from mold culture filtrates or animal stomachs. They noted that this group consists of endopeptidases having acid pH optimum. Investigations on the milk- clotting enzymes of mold origin have included various fungi, particularly members of the order Entomophthorales (Oringer, 1960; Whitehill 'et a1 ., 1961).. Knight (1966) surveyed the production of a rennin—like enzyme by molds and implicated species of Mucor, Rhizopus, Aspergillus and Penicillium among others. A great number of the members of the order Mucorales are known to produce important "microbial rennets." Arima and Iwasaki (1964) patented a procedure for producing a strong milk-clotting enzyme by culturing Mucor pussilus Lindt on a medium of wheat bran and water at 30°C. The enzyme system produced by this organism is known to exhibit characteristics similar to that of commercial rennet, the coagulant traditionally employed in the manufacture of cheese. These workers also employed fungi of the genus Rhizopus successfully in microbial rennet production. Gutcho (1974) stated that Mucor rouxii produces a coagulating or curdling enzyme when grown on a suitable culture medium preferably on rice. Charles, Gertzmann and Melachouris (1970) prepared a milk-clotting enzyme product 14 using a culture of Mucor miehei NRRL 3420. They used a medium containing appropriate nutrients. This enzyme product possessed a desirably high ratio of clotting activity to proteolytic activity and was used to coagulate milk in a cheese-making process. Tuasaki et a1 . (1967a; 1967b) showed that the enzyme from M. pussilus is an acid protease having a pH optimum of 3.5 for digesting casein. They further stated that its milk-clotting and proteolytic activities are similar to that of commercial calf rennet; also the enzyme was more stable to heat and more resistant to changes in pH than calf rennet. Thompson (1972) showed that rennet from Mucor miehei was of great commercial significance when she manufactured cheddar cheese with it. She discovered that there was absence of bitterness in the presence of excess rennet. Edelsten and Jensen (1970) showed that rennet from M. miehei was composed 0f at least three distinct components with coagulating properties; each exhibiting characteristic temperature dependence. This View was strengthened by the findings of Sternberg (1971) who demonstrated the existence of more than one proteolytic enzyme in "rennet" produced by Mucor miehei. An enzyme having high milk-clotting activity was isolated from Rhi20pus oligosporus (Wang et al., 1968). This isolate which resembles commercial rennet was stable at 40° or below. Wang et a1 . (1974) demonstrated that 15 strains of Rhizopus and Chlamydomucor oryzae grown on rice flour had milk-clotting, amylolytic, and antibiotic activi- ties. Wang and Hesseltine (1965) observed two proteolytic enzyme systems in the culture filtrates of Rhizopus oligo- sporus; one had a pH optimum at 3.0, the other at 5.5. Both enzyme systems had activities at 50-55°C and were fairly stable at pH 3.0-6.0. Sardinas (1968) demonstrated similarities between calf rennet and the enzyme system from a member of the Pyrenomycetes. This milk-clotting enzyme from Endothia parasitica was similar to commercial rennet in molecular weight, isoelectric point, milk-clotting power, amino acid composition and stability to changes in pH. He also showed that this enzyme system was a mild protease essential for good aged cheddar cheese production. Nadasky (1972) used the E, parasitica enzyme preparation to manufacture Edam cheese and confirmed its similarity to calf rennet. Aspergillus niger no. 58 was employed in the pro- duction of milk-clotting enzyme system. The electro- phoretic separation of a partially purified sample of this enzyme by Osman et a1 . (1969) showed four protein compon- ents, but the milk-clotting enzyme fraction constituted the major portion of the preparation. This portion also exhibited the highest milk-clotting activity and the lowest proteolytic action. The enzymic action was optimal at 50°C and pH 5.8. 16 Chu et al. (1973) partially purified Byssochlamyo- peptidase A, a rennin-like enzyme produced by Byssochlamys £2133, This enzyme system exhibits a pH optimum at 2.9 and a temperature Optimum of 60°C. It was stable at 40°C between pH 3.0 and 6.85. Its milk-clotting activity was dependent on pH and appeared to be maximum at pH 6.2 or above. They did not observe evidence of extensive proteo- lysis during the milk-clotting process. As the result of studies on the hydrolyzing enzymes which are produced by culturing members of Basidiomycetes, a few milk-clotting enzymes were identified and were employed as substitutes for rennet (Mukai and Kawai, 1971). These workers patented a method for producing an enzyme with comparatively weak proteolytic action and a strong milk—clotting power, an exceptional property essential to the manufacture of well-aged Cheddar cheese (Sardinas, 1968). Moreover, this enzyme system can be produced on an industrial scale at low cost utilizing molds belonging to the genera Irpex, Formitopsis, Coriolus or Lenzites cultured in an aqueous nutrient medium (Mukai and Kawai, 1971). The presence of lipases has been one of the major disadvantages of using fungal coagulants as substitutes for calf rennet. However, Kobayashi et al. (1982) have shown that the use of affinity chromatography can eliminate this problem from these "microbial rennets." Passing 17 microbial extract over immobilized pepstatin derivative can permit fast, easy purification of several acid proteases in large quantity and high yield (Kobayashi et al., 1982). Without purification, lipases present in microbial enzyme preparations produce defects in the final product. For instance, the long-chain (C12-C16) fatty acids released by a Mucor miehei lipase produced an un- pleasant "soapy" flavor defect in English Cheddar cheese (Law and Wigmore, 1984). Kikuchi and Toyoda (1970) demon- strated that curd produced by fungal rennets was softer and more easily shattered at cutting and had a bitter taste, particularly when crystalline enzyme preparations were used. These authors used rennet from M, pussilus Lindt to produce Cheddar, Gouda, and Edam cheeses, concluding that the characteristic bitterness was an inherent characteristic of the principal proteolytic enzymes. Organon (1971) explained that this defect was caused by the presence of non-specific enzymes in the fungal rennets of Mucor, Rhizopus, Endothia, Monascus and Colletotricum which can be eliminated by adsorption to silicates. Kikuchi and Toyoda (1970) stated that, bitter flavor not withstanding, fungal rennets could be successfully used in place of calf rennet in cheese manufacture if some para- meters like cutting time, setting temperature and cooking method were slightly modified. Another drawback with using mold enzymes as 18 coagulants results from their high heat stability. The most heat-stable of the generally used coagulants, that from Mucor miehei is now available in oxidized form (Phelan and O'Brien, 1982). In this form, its cheese- making properties are not affected. However, it is rendered labile to heat, and is unlikely to survive normal whey processing temperatures. If these highly heat-stable fungal enzymes are not modified they could resist pasteurization and can cause extensive proteolysis in the final product and possibly produce bitter peptides (Dulley and Kitchen, 1972). Formation of Milk Curd The coagulating properties of a milk can be defined empirically by the onset of coagulation (clotting time) and in the case of cheese, the time when the curd reaches the correct firmness for cutting (cutting time) (Green, 1984). The clotting time can be measured most simply by visual observation. However, a number of instrumental methods are also available which progressively indicate different results from the visual method (Ernstrom, 1974). When clotting time was measured visually, viscometrically and by damping a pendulum it increased with each method. Presumably, because the end points occurred at different extents of casein aggregation (McMahon and Brown, 1982). Milk coagulation occurs in two discrete stages, the 19 enzymic initiation phase being essentially complete before aggregation starts. The initiation phase comprises the specific proteolysis of micellar—bound k-casein to para—k- casein by an enzyme. This process has been followed by using High Performance Liquid Chromatography (HPLC) to separate and measure the heterogenous macr0peptide re- leased from an 8 percent (W/V)-T.C.A. extract (VanHooydonk and Olieman, 1982). Determination of the homogenous pmoduct,para-k-casein was by quantitative polyacrylamide gel electrophoresis (Chaplin and Green, 1980). The aggregation phase which can be followed quantitatively by Turbidimetry (Dalgleish, 1981); or by electron microscopy (Green and Morant, 1981); occurs by a random diffusion—controlled mechanism (Green, 1984). The later stages of aggregation, leading to the assembly of the curd, have been studied microsc0pically (Green et al., 1978). These workers found that initially, the casein micelles linked to form chains which gradually joined together to form a loose network. De Koning (1981) stated that the clotting reaction depends on pH, temperature and calcium-ion concentration of the milk. MATERIALS AND METHODS Gua-nai Manufacture The manufacture of Gua-nai involves three distinct stages (see Figure 1). Rice Fermentation Two hundred grams of Enriched Festive Sweet Rice (Pacific International Rice Mills, Inc., Woodland, CA) was washed twice with distilled water, soaked with four hundred milliliters of distilled water at room temperature for one hour, and steam-cooked for thirty minutes. It was cooled to 35°C and mixed with one gram pulverized rice ball culture. The inoculated rice was loosely packed in a lOOOmL Pyrex beaker, covered with cheesecloth, secured by a rubber band and incubated at 37°C. The fermenting rice was stirred once daily to insure that the grain surfaces were moist. On the third day, the fermenting rice was diluted with one hundred milliliters of distilled water and re- incubated for three additional days. The resulting effer- vescent, yellowish liquid was strained through cheese cloth 20 21 and refrigerated temporarily prior to usage. Treatment of Milk Five pounds (22709) pasteurized whole milk was mixed with 1139 granulated sugar, 113g corn syrup solids, 90.09 non-fat dry milk and 9.09 gelatin. The mixture was heated inaa100°C waterbath with constant stirring until all ingredients dissolved. This milk base was cooled to 4°C. Sweet-Curdling of Milk The clear filtrate from rice fermentation was adjusted to pH 4 . 23 with 0.01N NaOH. Ten percent of the filtrate was added to the milk base and mixed thoroughly. The mixture was poured into cups, covered with lids and incubated at 40°C for one and a half hours. Formation of firm curd marked the end of the sweet-curdling process. The resulting product, Gua-nai was refrigerated at 4°C. In order to eliminate the influence of any living organism, the culture filtrate was passed through a bacteriological filter (0.45 p pore size). The resultant filtrate was added to the milk base in the same proportion as used above and given the same treatments. Sweet Rice Wash Soak (1 hr) Steam-cook (30 min) Cool to 35°C Inoculate with Rice Culture Incubate 37°C (3 days) Dilute with Water Incubate 37°C (3 days) FILTER Adjust pH Pasteurized Whole or Skim Milk Increase Total Solids Heat 100°C Cool to 4°C MIX l Retail Cups | Incubate 40°C, 2% hr I Refrigerate 4°C Gua-nai Figure 1. Flow sheet for Gua-nai manufacture. 23 Isolation of Microorganisms From Rice Culture One gram Shanghai wine production cake, known in our laboratory as the rice ball culture (Ming Fan Rice Cake Factory, Shanghai, China) was macerated with 99 mL of 0.1% peptone water in a Warring blender to obtain a slurry of 10'2 dilution. Serial dilutions, using a sample size of 1.0 mL were prepared with dilution bottles containing 9.0 mL of 0.1% peptone water (10.0g peptone in 90 mL 2 5 dilutions distilled water). Pour plates of 10- to 10- were made in duplicates using 0.1 mL of sample and 1% melted, sterile Potato Dextrose Agar (Difco). One set of plates was incubated at 35°C and the other at 37°C for 48 hours. [30°C did not support adequate growth in a pre- liminary isolation.] Yeast colonies were observed in the preliminary isolation. Therefore, 1.35 mL of 1:10 dilution of tartaric acid was added to 100 mL of the Potato Dextrose Agar imme- diately before pouring into plates containing 0.1 mL of -2 to 10"5 10 dilutions of the rice ball slurry in duplicate and incubated as described above. The final pH of the medium was 3.5. This treatment was necessary to inhibit mold growth, and stimulate yeast growth. At the end of the incubation period, all discernible colonies exhibiting different morphological characteris- tics on plates of PDA (Plain and Acidified), were 24 transferred into plates containing PDA after microscopic examination. The individual cultures were transferred to new PDA plates by streaking every 3 days until pure cultures with- out contamination were obtained. Stock cultures were stored at 4°C and recultured every two weeks. Identification of Isolates The filamentous isolates were coded with the letters A, B, and C. Each was examined microscopically in drops of distilled water and with lactOphenol. Spore measure- ments were made with a stage micrometer. Temperature tolerance was determined by incubating each isolate at room temperature, 30°C, 35°C, 37°C and 40°C. The results of the above and distinguishing morphological features were tabulated. Identification of these isolates according to genera and species were made as described in Compendium of Soil Fungi (Domsch, Gams and Anderson, 1980; Ellis, Rhodes and Hesseltine, 1976). Identification of isolates in doubt was confirmed using known cultures, by comparing their spore and morphological characteristics and tempera- ture tolerance. The cream-colored, buttery colonies (Code D) were similarly examined followed by a determination of its Gram characteristics. Taxonomic studies of this yeast were conducted in accordance with the methods described by 25 Lodder (1970). Prior to these studies, the yeast was pre- cultured in liquid malt extract medium for 24 hours at 37°C. Cells were harvested by centrifugation and washed twice with sterile distilled water and used as inoculum for the biochemical experiments. All the experiments con- ducted and results obtained were tabulated. Evaluation of Pure Isolates for Ability to Ferment Rice Prior to this investigation, each pure isolate, stored at 4°C, was transferred to new Potato Dextrose Agar slant and incubated at 37°C for 8 days. Spore suspensions were prepared by adding 3 mL distilled water to each slant and shaking vigorously for one minute. One milliliter of spore suspension from each pure culture was added to two hundred grams of steamed, cooled, sweet rice and mixed thoroughly. The mixture was loosely packed in a 600 mL Pyrex beaker, covered with cheesecloth, secured with a rubber band and incubated at 37°C with daily stirring to maintain moist surface. The fermenting rice was diluted with 100 mL distilled water on the third day and re-incubated for another three days. Samples of culture liquid were withdrawn daily from the third day to monitor pH and total acidity. After six days, the liquid was strained through cheesecloth and refrigerated for subsequent analysis. 26 Microbial Changes During Rice Fermentation To determine the mycoflora of the fermenting rice, streak plates of each fermentation were made on Potato Dextrose Agar daily from the first day. All samples were examined microscopically, using the procedure described earlier. Reconstitution of the Rice Culture The method employed was a modification of the pro- cedure of Tanimura et al. (1978a) for making Philippine bubod starter. Preparation of Inocula The isolated mold species were maintained on slants of Potato Dextrose Agar (PDA) at 4°C. Before this experi- ment, each organism was transferred to a frech PDA slant and incubated at 37°C for 8 days. Spore suspension suit- able for inoculation were prepared by adding 3.0 mL sterile distilled water to a slant and shaking the cultures vigorously for one minute. Preparation of Rice Culture Glutinous rice flour (Erawan Marketting Co.,L¢d., Bangkok, Thailand) was employed in this experiment. One 27 hundred grams of rice flour was mixed with one gram of glucose (Difco). 100 mL distilled water at 50°C was added and mixed thoroughly to produce a thick paste. The liquid content for each organism from each agar slant was poured into a 600 mL Pyrex beaker containing the rice flour- glucose paste and mixed thoroughly. The beaker was covered with cheesecloth and secured with a rubber band. The beakers were incubated at 37°C for 6 days with daily stirring. After the incubation period, the cultures were mixed in various ratios by weight. A ratio of A. rouxii: E. burtonii: 3, oryzae: A. oryzae = 1:1:10:100 was found to be similar to the commercial wine cake in fermentation ability. The mixture was dried by storing at 32°C for 6 days. (Figure 2) Freeze-Dried Culture Ten grams of Malt Extract (Difco) were mixed with one gram glucose and suspended in 300 mL distilled water. The suspension was heated in a 90°C waterbath until all ingredients dissolved. The cotton-plugged 600 mL Pyrex flasks containing this solution was autoclaved at 121°C for 30 minutes at 15 psi. The medium was cooled to 30°C. One mL spore suspension of each pure isolate was added to the medium, mixed and incubated at 37°C with daily shaking. After 6 days, the content of each flask was strained through cheesecloth and freeze-dried. (Figure 3) 28 Sweet Rice Flour + Glucose (10:1 W/W) Mix with 50°C distilled Water (1:1 W/V) Inoculate (3 mL spore suspension of each organism) Incubate 37°C, 6 days Mix A. rouxii: g, burtonii: A. oryzae: A, oryzae = (1:1:10:100 W/W of fermented rice flour/glucose) Dry in Storage 32°C, 6 days Figure 2. Flow Sheet for Rice Culture Preparation. 29 Malt Extract + Glucose (10:1 W/W) 300 mL distilled Water (Heat to 90°C) Autoclave (121°C, 15 psi, 30 min) Cool to 30°C Inoculate (1.0 mL spore suspension) Incubate (37°C, 6 days) Strain through cheesecloth Freeze-dry Figure 3. Freeze-dried Culture Preparation. 30 Analysis of Filtrate From Rice Fermentation RE The pH of the liquid from rice fermentation by pure and mixed cultures were determined with a Chemtrix-type 60A digital pH/mv meter equiped with an Orion (Model 91-63) pH electrode designed for surface measurements. The mean of three different readings is reported. Titratable Acidity The titratable acidity of each culture filtrate was determined with the Nafis apparatus as described by Atherton and Newlander (1981), using 0.1N sodium hydroxide as titer, 9.00 mL sample size and two drops of phenolph- thalein as indicator. Ethanol Determination Sample Preparation for Analysis The samples of fermented rice liquid were diluted 1:1 with 3.0% perchloric acid and passed through a sample preparation filter which removed all particles larger than 0.45 pm. Standard solutions of ethanol were injected prior to each run in a high performance liquid chromatograph to obtain a calibration curve in order to correlate peak height versus concentration. 31 The ethanol concentration of each sample was de- termined by high performance liquid chromatography (HPLC) using the following conditions: Column: HPX-85H Alcohol Analysis Column (BIO-RAD Laboratories, Richmond, CA) Sample Injected: lO‘pL Eluent: 0.01N H2804 Flow Rate: 0.3 mL/min, l6"/hr Temperature: 85°C Detector: Differential Refractometer 8X (Waters 401) Assanyor Milk-Clotting Activity Twelve grams of low heat non-fat dry milk were homogenized in 100 mL 0.01M CaCl ~2H20 and equilibrated 2 for 1 hour at 25°C. The pH was adjusted to 6.2 with 1N HCl. For assay of the milkéclotting activity of each culture filtrate, 10 mL of the substrate was added to 1.0mL of filtrate of a suitable dilution in a test tube and placed in a 40°C waterbath. The end point recorded was the time in seconds required for curd fragments to become visible to the naked eye. This was done by observing the tubes at intervals. The mean of 3 readings is reported. Milk-clotting activity (MCA) was defined as 2400 -——-——— X dilution factor where "t" is the time in t(sec) MCA = seconds necessary for the formation of curd fragments (Kawai and Mukai, 1970). 32 Assay of Proteolytic Activity The substrate used in this assay was Azocoll (Calbiochem-Behring Corp., La Jolla, CA). Azocoll is an insoluble powdered cowhide to which a bright-red dye is attached. The cowhide contains the usual myriad assortment of peptide linkages._ When a proteolytic enzyme breaks any of these linkages, the bound dye is released into the suspending medium. This assay is based on the rate at which dye is released which is an indication of proteolytic activity of the enzyme. The soluble products produced during Azocoll degradation have a principal absorption maximum of 520 nm. Fifty milligrams of Azocoll was suspended in 5.0 mL of 0.1M phosphate buffer pH 7.0, containing 1.0 mL of each culture filtrate. This was incubated for 15 minutes in a 37°C waterbath. The digestive action was stopped by filtering through a Whatman no. 4 filter paper. The fil- trate was measured in a Spectronic 21 (Bausch and Lomb) spectrophotometer at 520 nm, and the absorbance recorded. The blank consisted of all the ingredients used minus the culture filtrate. Prior to performing these determinations, a standard curve was prepared with crystalline pepsin (Pentex Inc., ,Kankakee, IL) using Calbiochem-Behring procedure for color development, by pipetting selected pepsin dilutions to cover the range desired. The diluent was also 0.1M phosphate 33 buffer. Absorbance was plotted against pepsin concentra- tion. Compositional Analysis of Gua-nai Total Protein The total protein in Gua-nai made from commercial and reconstituted cultures were determined by a semimicro- kjeldahl procedure described by AOAC (1975). Sample size used was 0.3g. Fat Content The fat content of the Gua-nai samples was deter- mined by the Babcock test as described by Atherton and Newlander (1981). Sample size used was 9.0g. Total Solids The total solid content was determined by the vacuum oven method (AOAC, 1975). The sample size was 3.09. Alcohol Ethanol content was determined by HPLC as earlier described. 34 [E The pH of the Gua-nai samples were determined as described earlier. Titratable Acidity Nine gram sample of Gua-nai was weighed into Erlen- meyer flask and 9.0 mL distilled water added and shaken. 17.6 mL of this sample was pipetted into 125 mL Erlenmeyer flask and 6 drops of phenolphthalein indicator added. It was then titrated with 0.01N NaOH to the pink-red end point. Titratable acidity as percent lactic acid was calculated as follows: % Acid (as lactic) = (mL NaOH){§)(0.090) X 100 Consumer Taste Panel Samples of Gua-nai prepared with commercial and re— constituted rice cultures were presented to consumer taste panels. The testing method used was the Triangle test as described in the Laboratory Methods for Sensory Evaluation of Food (Elizabeth Larmond, 1977). A sample of the Con- sumer Score Sheet is included (Figure 4). 35 Date: Name: The samples you have been given are made with milk, sugar and a natural coagulator obtained by the fermentation or rice. The only difference is that one batch was made with commercial culture while the other was made with cultures isolated from the commercial culture and subsequently re- constituted. Two of these samples are identical, the third is different. 1. Taste the samples in the order indicated and identify the odd sample. Code Check odd sample 2. Indicate the degree of difference between the duplicate samples and the odd sample. Slight Moderate Much Extreme 3. Acceptability: Odd sample more acceptable Duplicates more acceptable 4. Comments: IFigure 4. Gua—nai Taste Panel Score Card RESULTS AND DISCUSSION Isolation and Characterization of Microorganisms From the Chinese Wine Cake Four microorganisms were isolated from the Chinese wine cake and taxonomic studies of the pure isolates re- vealed the presence of 3 groups of fungi: two members of the Mucorales, one species of Aspergillus,and one yeast species. Table 1 describes the morphological characteristics typical of the isolate coded A. These characteristic features are in good agreement with those of the mold species described by Ellis et al. (1976) as Amylomyces rouxii(former1y Chlamydomucor oryzae). The most outstand- ing features of this organism are the formation of abortive sporangia (Figure 5a) and proliferation of chlamydospores (Figure 5b). The organism coded A was thus identified as Amylomyces rouxii Calmette. The isolate coded B has the morphological character- istics listed in Table 2. These characteristics are identical to those of the mold species described by Domsch et a1. (1980) as Rhi20pus oryzae Went. This organism is distinguished by reduced rhizoids, short, broad 36 Sylvester N. Onyeneho Investigators. , TABLE 1 37 Culture Code: A DESCRIPTIVE m, 1.85/8. C H A RT Source; Chinese :Wj'ne Cake Organism- Amylomyces ”rouxii * EEEEEEEEEEEEEEEEEE, ._I. MORPHOLOGICAL CHARACTERISTICS I. HIPHAE - Erect, 3—6.5mm high. Forms dense white weblike mycelial growth; non-septate and coenocytic 2. Praummrron - Hyaline (Of hyphel elements) 3. RHIZOIDS - Mostly absent (some observed in older cultures) h. STOLONS(runner3)- Absent 5. SIMPLE ASEXUAL SPORES Chlamydospores: Present in large numbers e. Shape: Thick-walled, cylindrical to globose to oval (mostly cylindrical); single or in chains b. Size: Variable, but mostly (ll-45) X (9-33) pm 0. Position: Aerial and Substrate hyphae Mostly Intercalary d. Temperature: Optimum 37°C; maximum 40°C 6. SPECIALIZED assxmu. sronrs : Zygospores absent Sporengiospores - Present but most abort a. Produced in a sporangium b. Sporangium: Mostly abortive Size: 120—150 pm diameter Color: Dark brown, brown to black Shape: Oval or globose Columella Smooth, 50-100 um diameter Shape: Mostly subglobose, continuous with sporangiophore SPORANGIOSPORES - Smooth and gray Size: Variable (13-45) X (8-10) pm Shape: Oval to irregular, some Y-shaped S OR'NG H0 : Erect arise from aerial Hyphae P' A 10? RES Unbranched, 0.5-2.5 mm tall Hyaline to brown 38 FIGURE 5. Amylomyces rouxii (a) showing abortive sporangia, X500, (b) showing intercalary chlamyclospores, X500. Investigatona. Sylvester N. Onyeneho TABLE 2 :9 B Culture codgo ‘ DESCRIPTIVE mm: 1985,86 C HA RT Source; Chinese Fine Cake organisthizopus oryzae Went EEEEEEE:: 4Ajpi MORPHOLOGICAL CHARACTERISTICS 1 I. HIPHLE: Large, broad, non-septate and branching; coenocytic, colonies fast growing on PDA, dark-brown to gray 2. PIGHENTATIUN - Hyaline (Of hyphal elements) 3. RHIZOIDS: Present but reduced 4-8 branches h. STOLONS(runnerg): Present and hyaline 5. SIMPLE ASEXUAL spears Chlamydosporegg Present in large numbers, especially in older cultures e. Shape: Globose to oval, hyaline b. Size: 12-33 pm diameter c. Position: Mostly Intercalary, singly or in chains 6. SPECIALIZED ASEXUAL SPORES Sporangiospores: Present a. Produced in a sporangium b. Sporangium: Terminal Size: 35-75 pm diameter Color: Dark brown Shape: Globose to subglobose Columelle Shape: Hemispheric or globose SPORANGIOSPORES Size: ‘Variable, but mostly 3-20 pm diameter Shape: Biconial or subglobose SPORANG : Arise in fascicles from the node of the. . IOPHORES stolon directly opposite the tuft of rh1201ds; -Sometimes forked 4O sporangiophores, somewhat smaller spores (7-9 pm) and numerous mycelial gemmae (chlamydospores) (Figures 6a, b, c). The isolate coded B was thus identified as Rhizopus ogyzae Went. The morphological features of the isolate coded C are contained in Table 3. These are the same as those of the mold species Aspergillus oryzae described by Domsch et a1. (1980). It is characterized by septate mycelia, long conidiOphores, mostly uniseriate phialides, and globose to elliptical condia. Consequently, the isolate coded C was identified as Aspergillus oryzae Cohn (Figure 7a, b). The morphological, cultural and physiological studies of the yeast species isolated from the wine cake and coded D revealed the characteristics listed in Table 4. In accordance with the scheme of Lodder (1970), these characteristics were in good agreement with those of the yeast strain Endomycopsis burtonii. This yeast exhibited weak fermentation ability of sugars and could not utilize nitrogen sources. It forms pseudomycelium, rarely forms true septate mycelium and does not form a ring or pellicle. The isolate coded D was consequently identified as Endomycppsis burtonii (Figure 8). Several authors have reported the presence of similar organisms in the starters used for many oriental fermentations. Wang and Hesseltine (1970) noted that the FIGURE 6. Rhizopus or zae, showing (a) Rhizoids, (b) stolons and (c) sporangiophores bearing sporangia, X500. 42 -TABLE 3 InVestigatons. Sylvester N. Onyeneho_ Culture God. :_‘ C DESCRIPTIVE . 1.85/8. DATE: C H A RT Source: A. Chinese Winei Cake (Myonkm: A8pergillus oryzae E MORPHOLOGICAL CHARACTERISTICS I. HYPHAE: Septate hyphae, initially forming greyish-white colonies ' with wrinkled edges, later turning green to brown 2. PIGMENTATION3 In culture, color changes from yellow to green, to (of hyphal elements) brown with green shades 3. RHIZOIDS: Absent 1;. STOLONS(runner-s) : Absent 5. SIMPLE ASEXUAL spores: Absent 6. SPECIALIZED ASHUAL SPORES CONIDIA : Numerous a. Size: Variable mostly 4-10 Pm diameter, some 5-6 Pm b. Shape: Globose, subglobose or elliptical c. Arrangement: Uniseriate, some Biseriate sterigmata d. No. of cells: Numerous conidia are released CONIDIOPHORE: Colorless Conidial heads are globose to radiate or columnar Vesicles are large 50-70 Pm diameter, subglobose, clavate or flask-like TEMPERATURE: Optimum 35-37°C, slow growth with small colonies at 30°C * Sclerotia was not found 43 (a) and (b) showing conidial heads, yphae an As ergillus oryzae septate X500. FIGURE 7. 4 4 E TABLE 4 DESCRIPTIVE CHART Investi gators. Culture Code :____ D Source: .Chinese Wine Cake_ MORPHOLOGICAI. CHARACTERISTICS PHYSIOLOGICAL CHARACTERISTICS Sylvester N. Onyeneho Organism: WW Cell Shape: Mostly oval Arrangement: Single, pairs, GARBOHYDRATES FERK- UTlle. , chains and cluster 5913013039 - - Slze: (4x8) Pm ‘ slums: +(weak) + Spores: Present, hat-shaped Fructo e "' Lactose - - Sexual characteristics 51% + f ) :_ IGORODKOWA AGAR) weak Formed after long in- Haltose + (weak) + Asci: cubation Raffinose + (we—3k) + .Ascosnores- 2-3 er ascus ' Shhpe- Oval t: hat-shape'§ITROGEN_SgURCES UTILIZATION. i - Variable 4x8 Otagaiungitrate " g ze ' ( ) Pm Potassium Nitrite - J. L _ CULTURAL CHARACTERISTICS Esp lffigsine - reak I and PD e Colonies: Gelatin Liquefaction .. Color: Yellowish-white Texture: Tough Vit.-free medium 4. Elevation: Raised -- Margin : Wrinkled Esculin splitting ... I‘EUID MALT Ex CT Optimum temperature 37°C Follicle:— Absent Maximum temperature 40°C Ring r-Absent ,. ... .2 ., Sedimentt— + after long in- Gram,s .easticn + Cell morpholggfiatlon Lipase agar + Pseudomycelium; Present Budding : Present, Casein agar .. True micelium: ngrmigal d 50 / sen an " lucose w w agar + (seldom) septate I" 8 Ethanol 1 as 5912 C + source) FIGURE 8 . 45 Endomycopsis burtonii--showing (a) single cells and asci with ascospores after 7 days on Gorodkowa Agar, (b) and (c) true septate mycelia after 15 days in liquid malt extract, X1000. 46 major species present in "Chiu-yueh" or "Pey-yueh" the starter used in the fermentation of steamed glutinous rice to produce Chinese Lao-chao were Rhizopus oryzae, Rhizopus Chinensis, Chlamydomucor oryzae and a yeast Endomycopsis. Pichyangkura and Kulprecha (1977) have isolated A, rouxii, Aspergillus oryzae and RhiZOpus oryzae from "long-pang," a starter utilized in the production of tape-like products and for production of alcoholic rice wines. Park et a1. (1977) reported the presence of Aspergillus oryzae, Rhizopus oryzaeenuiyeasts in natural "nuruk" used in the fermentation of Korean "Yakju" and "Takju" while Yoshizawa (1977) noted that the major mold in "Koji" used for Japanese "sake" was Aspergillus oryzae. In addition, Ko (1977) isolated A, rouxii and a yeast Endomycopsis burtonii from "ragi" starter culture. These evidences show the occurrence of similar microorganisms in the starters employed in most oriental fermentations. Most of these involve the saccharification of the rice starch by the amylases produced by the fila- mentous molds and the utilization of the sugars by the yeasts to produce alcohol (Steinkraus, 1983). Guan and Brunner (1985) employed the same Chinese wine cake used in the present study to produce Gua-nai. These authors reported that the predominant mold in this oriental culture was Rhizopus oryzae but, although no mold count was conducted in the present study, visual observation 47 of the culture plates indicate that Amylomyces rouxii was the predominant mold in the wine cake. Again they reported the presence of Saccharomyces in the oriental culture, but the present study has revealed that the yeast was not Saccharomyces but a weakly fermenting strain, Endomycppsis burtonii. In general, 3 molds A. rouxii, A, oryzae and A. oryzae and one yeast A. burtonii are present in the Chinese wine cake instead of one mold, A. oryzae and Saccharomyces as was earlier reported (Guan and Brunner, 1985). Physical Changes During Rice Fermentation During the course of fermentation of steamed glutinous rice with the Chinese wine cake, several physical changes occurred, including the following: a) Appearance of mycelia of the filamentous molds after 24 hours. The rice grains were covered with fine, white mycelia which later turned brownish-black after spore production. The mycelia disappeared after the first stirring and never appeared again because the fermenting medium was stirred daily. b) Textural changes of the rice occurred. After 24 hours, the rice became soft and juicy. c) Appearance of a clear, effervescent, pale-yellow liquid after 72 hours (Figure 9). 48 FIGURE 9. Steamed glutinous rice--(a) before and (b) after fermentation with the Chinese wine cake. 49 This fermentation is similar to that of Chinese Lao-chao (Wang and Hesseltine, 1970). The starter, sub- strate and conditions used appeared to be related to those employed in this investigation. These authors reported that a mixture of esters was responsible for the fruity aroma of the product, and that lipases of the yeast broke down the lipids of rice to fatty acids, which then react with alcohol to form a mixture of esters, providing the pleasant odor. No data on pH, titratable acidity, milk— clotting and proteolytic activities were reported that could provide a basis for comparison. However, they noted that there was accululation of 1-2% ethanol in 2-3 days. The fermentation of Philippine "tapuy," a highly acidic, but sweet, aromatic alcoholic rice wine, is also similar to the one being reported here. Although the microbiology of "tapuy" fermentation has not been investi- gated thoroughly, Steinkraus (1983) stated that, because of the close relationship between "tape ketan" and "tapuy," it is highly likely that at least one filamentous fungus and one yeast of the Endomycopsis type are required for a typical product. Gua-nai Manufacture The first reported Gua-nai manufacture was by Guan and Brunner (1985). These authors used the same Chinese wine cake as was employed in this study. They reported 50 that 1.5% MSNF and 5-10% sucrose were added to whole or skim milk and 8% (v/v) of filtered cultures was used to inoculate the milk-based mix at 40°C for 1-2 hours. Since the present study was also aimed at improving the manu- facturing process, the following ingredients were used and found to produce a more firmly curdled Gua-nai: whole milk, 5% sucrose, 5% CSS, 4% NFDM and 0.4% gelatin. Cool- ing the pasteurized milk-based mix to 4°C before inocu- lating with 10% (v/v) filtered-culture and incubating the mix in individual containers at 40°C for 1-2 hours was found to produce Gua-nai with smoother surface, firmer gel and a more fruity flavor. Gua-nai produced with this method was kept at 4°C for 30 days and did.not differ in flavor and mouth feel from a newly manufactured product. This indicates that this improved formula could produce Gua-nai with reduced syneresis and longer self-life (:>30 days) as against 10 days which was reported earlier (Guan and Brunner, 1985). Adjusting the pH of the culture filtrate with 0.01N NaOH to 4.23 reduced the incubation time to 1-1% hours. It is recommended that the pasteurized milk-based mix be cooled to 4°C before inoculating with the culture filtrate. This is due to the observation that, if the culture filtrate was added when the temperature of the mix was 40°C, the curdling process was rapid and syneresis 51 often followed. pH and Titratable Acidity Changes DuringARice Fermentation The changes in pH and titratable acidity during the fermentation of steamed glutinous rice are presented in Figures 10 and 11 respectively. The initial pH value of all the fermentation was 6.6. This was the pH of the rice during the soaking process. After 2 days of fermentation when a measurable quantity of liquid had accumulated, there were sharp drOps in pH coupled with concomitant rise in the titratable acidity (measured as lactic acid). For the commercial culture the pH was 4.43 after 3 days and.fell to 3.81 in 6 days. The reconstituted culture had a slightly lower pH value than the commercial type. The opposite trend was followed by these two cultures in titratable acidity changes. In general, a similar pattern was followed by all the cultures used as inocula--a gradual fall in pH and a gradual increase in titratable acidity. The pH of rice fermented by A, rouxii decreased to 4.45 in 3 days and fell to 3.65 in 6 days. Cronk et al. (1977) have re- ported similar observations during the fermentation of Indonesian "tape-ketan." They noted that when A, rouxii alone was used to ferment steamed glutinous rice, the pH of the rice decreased sharply from 6.3 to 4.0 in 2 days; and then gradually increased to 4.5 with continued pH 52 5 ‘ ‘ m - Wine Cake Reconstituted Cake I , ‘ I A. rouxii III“ . m I new It. I” Ia————.——-—T~ , 1_ 2 3 4 5 6 Incubation Period (days) , Figure 10. Changes in pH accompanying the fermenta- tion of rice by the various cultures. 53 1.5- Wine Cake Reconstituted Cake A, rouxii % T.A. (as lactic acid) H. .3. oryzae I p: A. oryzae ’ 1 B T l l: ‘ A I J 1 2 3 4 5 6 Incubation Period (days) .5- a: -' ;;I I ' I m“ -.....— ‘Hmh—_—- ._._1 mug 1 A Figure 11. Changes in titratable acidity accom- panying the fermentation of rice by the various cultures. 54 fermentation. This phenomenon, however, was not observed in the present investigation. The observed trend was a continuous drop in pH values with length of incubation. The result presented in this report compares favorably with the findings of Uyenco and Gacutan (1977b). They reported that the pH of "tapuy" starts at about 6.5 and falls to 3.8 by 48 hours, and titratable acidity (as lactic acid) is 0.32% at 48 hours and reaches 0.59% by day 7, but Tanimura et al. (1978a) reported that the pH of "tapuy" varied from 3.3% to 4.9%. In general, the results of this study follow the same trend as in other rice fermentations utilizing other oriental-type cultures as inocula: sharp drop'hiinitial pH and gradual decrease to almost half the original value accompanied by gradual rise in titratable acidity. Microbial Changes During Rice Fermentation During the course of rice fermentation with the commercial Chinese wine cake, streak plates of the liquid from the fermenting rice was made on Potato-Dextro-Agar daily. This was to determine the mycoflora at each stage of the fermentation. Microscopic examination of the iso- lates were made and the morphological features typical of each organism reported earlier were noted. Taxonomical studies showed the presence of all 4 organisms during the first 3 days of fermentation, followed 55 by gradual disappearance of the filamentous molds. The order of disappearance was A. oryzae>A. rouxii>Asp. oryzae. At the completion of fermentation, only the yeast EndomyCOpsis was present in the final culture (Table 5). It has long been known that fungi with coenocytic, rapidly extending mycelia, tend not to be antagonistic in mixed culture and are readily replaced by the fungi paired against it (Boddy and Rayner, 1983 ). There appears to be a correlation between the types of interaction in which a fungus becomes involved and its mycelial organization and life strategy. There are relatively few single strain culture fermentations of foods and the present investiga- tion is not one of them. An organism that initiates a fermentation will develop until its by-products of growth inhibit further growth and fermentation. During this initial period of growth, other organisms develop and in turn are followed by other more tolerant species. Accord- ing to Pederson (1971), this succession of growth of different species is a natural sequence of growth and the usecflfmixed starters or inocula is based on these facts. Boddy and Rayner (1983 ) noted that septate Ascomy- cotina, Deuteromycotina and Mucorales often Operate antagonistically via antibiosis, the coenocytic front being non-combative is readily replaced by the fungi paired against it. In this investigation, the coenocytic molds in the culture: Rhizopus oryzae and Amylomyces rouxii were 56 Table 5. Microbial Changes During Rice Fermentation Organism Days 1 2 3 4 5 A, rouxii + + + + - A. oryzae + + + - - A. oryzae + + + + + E. burtonii o o o o o + Mycelial form present - Mycelial form absent 0 Yeast present 57 the first to disappear from the fermentation medium. Cooke and Rayner (1984) explained this phenomenon by observing that mycelial contact between colonies causes deadlock and replacement which is often preceded by lysis. Hyphal contact has also been reported to cause inhibition. According to Ikediugwu et al. (1970) this inhibition in- volves mutual or unilateral death of hyphae or hyphal com- partments due to intimate contact with one another. The increase in acidity also appears to have had a mortal effect on the filamentous molds. Figures 10 and 11 show that pH and titratable acidity decreased and increased respectively with time. The pH fell from 6.6 to 3.67 in 6 days while titratable acidity rose to 0.66% within the same period. According to Alexopoulos (1979), the optimum pH for the growth of many molds is 6.0. It therefore appears that the level of acidity in the fermentation medium was not conducive for the continued growth of the filamentous molds, hence they disappeared from the medium. This effect of pH on mycelial growth was demonstrated by Cochrane and Cochrane (1971). They found that when conidia of Fusarium solani were incubated in a complete medium at pH 6.0 they germinated and produced normal branching mycelium. However, when the spores were incu- bated in the same medium at pH 4.0, normal germination was prevented and instead, chlamdospores were formed. They further showed that spores pregerminated at pH 6.0 formed 58 chlamydospores at pH 4.0 in 4-12 hours. In this study the pH fell from 6.6 to 3.67 and could have induced the molds to form chlamydospores in the medium. Yeasts, on the other hand have optimum pH between 3.5 and 3.7 (Phaff and Starmer, 1980). The pH of the fermenting medium in this study was within this range toward the end of the incubation period. At this period, only the yeast A. burtonii and large spherical bodies were observed when a wet mount of the fermenting medium was examined under the microscope. The outcome of this study points to the fact that pH might have played a substantial role in the elimination of the filamentous molds from the medium. Streak plates of the fermenting medium were examined within 3 days. Within this period only yeast colonies appeared when culture filtrate was streaked on PDA at the end of the fermentation period. However, on prolonged incubation, filamentous molds began to appear once again, overgrowing the yeast colonies. This phenomenon tends to show that what was actually eliminated from the fermenting medium were the vegetative, mycelial cells while the fungal spores remained. Smith and Berry (1974) reported that certain members of the Mucorales, especially Mucor rouxii exist in nature as normal mycelial fungi but do have the potential to develop into budding spherical cells. They reported that most of the factors that cause yeast-type 59 morphology in Agggglgpp. also favor a fermentative meta- bolism. Two members of the Mucorales involved in this study are A. oryzae and A. rouxii. These two molds are noted for their ability to produce large amounts of inter- calary chlamydospores. According to Smith and Berry (1974) chlamydospores are produced by many fungi to enable the fungus to survive unfavorable conditions such as low temperatures and lack of nutrients. Vance and Garraway (1984) supported this view by stating that at the onset of harsh environmental conditions, such as nutrient depriva- tion, certain fungi form resistant structures such as chlamydospores and sclerotia which contain stored nutrients and may survive for years under unfavorable conditions. Since the mycelial growth disappeared before the end of rice fermentation, it appears that nutrient depletion induced the filamentous molds to form chlamydospores. Microscopic examination of the culture filtrate confirms this because thicker walled spherical bodies larger than normal yeast cells were observed among the yeast cells. Ethanol ProductiOn During Fermentation Analysis of the culture filtrates by high performance liquid chromatography revealed levels of ethanol production during rice fermentation. Figure 12 shows typical Chromatograms of the filtrates from rice fermented with the commercial culture (the wine cake), containing 11.1 percent 60 Column: HPX-85X Alcohol Analysis (BIO-RAD Laboratories) Sample Injected: 10 L Eluent: 0.01N H2804 Flovaate: 0.3 mL/min Temperature: 85°C Detector: Differential Refracto- meter 8X Chart speed: 0.5 in/min ‘= 12.8% [:’Injection W We. 4 6 8 10 0 2 4 6 8 10 12 Retention Time (min) 0 [ Injection N . 1 FIGURE 12. High Performance Liquid Chromatograms showing ethanol analysis of culture filtrates when (A) Reconstituted culture and (B) Commercial Chinese wine cake culture were used to ferment steamed glutinous rice. 61 ethanol. Ethanol production using the various inocula are presented in Table 6. It is evident that the alcohol content increased with the time of incubation. Again, the filamentous molds could produce appreciable quantities of ethanol (7.2%-10.8%) within the period of fermentation. However, when these pure mold cultures were used in com- bination with the yeast as inocula, more ethanol (9.3%- 11.3%) was produced. The yeast alone could not ferment the rice within the period of fermentation. The variation in ethanol yields among the mold species tested showed that different species vary in their ability to ferment rice and produce ethanol. In this investigation, the reconstituted culture produced more ethanol than the commercial culture. This_cou1d1xadue to the fact that the commercial culture had been in a dry, dormant condition while the reconstituted culture was made with isolated, pure and actively growing cultures. Presumably, the dormant culture would not initially be as active as the pure, fresh growing cultures (Cotter, 1981). Among the 3 filamentous molds isolated from the commercial culture and tested for their ability to ferment rice and produce ethanol, A, rouxii produced the highest concentration of ethanol: 8.0% and 10.8% in 3 and 6 days respectively. This was followed by A. oryzae while Aspergillus ogyzae produced the least. Gray (1945) 62 Table 6. Ethanol Production During Rice Fermentation Ethanol (%) Inoculum 3 days 6 days Commercial culture 9.3 11.1 Reconstituted culture 9.6 12.8 A, rouxii 8.0 10.8 A, oryzae 5.2 9.6 A, oryzae 4.0 7.2 A. rouxii and Endomycopsis --- 11.3 A, oryzae and Endomycopsis --- 11.1 A. oryzae and Endomycopsis --- 9.3 63 explained that the capacity to convert sugar to ethanol is dependent upon various factors, which include substrate, environmental conditions, the microorganism involved, among others. He further explained that the efficiency to produce ethanol depends upon the ability of the micro- organisms to utilize fully the source of carbon or raw materials to produce ethanol. Cronk et a1. (1979) noted that during the production of higher alcohols in the fer- mentation of Tape-Ketan, A, rouxii alone produced nearly the same quantity of alcohol as it did in combination with Endomycopsis burtonii. Again, these authors explained that ethanol production during the initial phase of fermentation was shown to be due primarily to the mold A, rouxii. Dwidjoseputro and Wolf (1970) reported the involvement of A. oryzae, A, oryzae and A, rouxii (Chlamydomucor oryzae) in the fermentation of Indonesian foodstuffs. These authors reported that, in general, the filamentous fungi bring about saccharification of starch in the starting materials, while the yeasts ferment the resulting sugars with the production of small quanities of alcohol, and of esters, which impart desirable flavors to the products. Uyenco and Gacutan (1977b) reported that during the fermentation of "tapuy," ethanol levels were 5.63% and 16.01% in 2 and 7 days respectively. However, Tanimura et al. (1978a) reported that ethanol content in the same product ranged from 13.5% to 19.10% v/v. 64 In this investigation, ethanol content when the commercial culture was used as inoculum was 9.3% in 3 days and rose to 11.1% in 6 days when the fermentation process was stopped. It is evident that a similar pattern was followed in this study as in other fermentations in which mixed cultures of molds and yeast are employed as inocula-- increase in ethanol concentration with time of incubation. Guan and Brunner (1985) reported an alcohol content of about 5% in the culture filtrate when the wine cake was used to ferment steamed glutinous rice. However, 11.1% (v/v) ethanol was obtained in this study. This disparity could be due to the variation in the dilution step of the fermentation procedure. These authors diluted the medium with 2 volumes of water on the third day while half volume of water was used in this study to dilute the medium. Evaluation of Culture Filtrates for Milk-Clotting and Proteolytic Activities All the fermentation liquors obtained by using the various inocula individually to ferment steamed glutinous rice exhibited both milk-clotting and proteolytic activi- ties. The variation in these activities are presented in Table 7. These results indicate that milk-clotting activity appears to be invariably accompanied by proteo- lytic activity. This observation agrees with the findings of Olson and Bottazi (1977). These authors reported that Table 7. Milk-Clotting and Proteolytic Activities of 65 Culture Filtrates. Inoculum Milk-clotting Proteolytic AQA Activity(MCA)* Activity(PA)° PA Comm. culture 10.7 0.10 112.6 Reconstituted culture 11.4 0.09 127.0 A. rouxii 26.7 0.08 333.8 'A. oryzae V 16.0 0.17 97.0 A, oryzae 8.9 0.10 105.0 0.60 mg/ml Pepsin 10.0 0.10 105.0 * MCA = ° Absorbance measured at 520 nm 2400 t(sec) X Dilution Factor 66 enzymatically induced coagulation of milk involves modifi- cation and aggregation of some of the milk protein frac- tions, the caseins which exist primarily as sperical particles or micelles. Green (1972) and Jolles (1975) noted that the release of peptides during hydrolysis of milk proteins yields para-k—casein containing micelles that are more hydrophobic and have a lower net negative charge, and concluded that both conditions favor aggregation of casein micelles and clotting of milk. However, Cherryan et al. (1975b) and Fuente and Alais (1975) noted that clotting time of enzyme-modified casein micelles occurs in the presence of calcium ions and is extremely temperature and pH dependent. This dependence of milk-clotting by enzymes on pH was observed during this study. The pH of the filtrate from rice fermented with the wine cake was adjusted with 0.01N NaOH to various higher levels and then tested for ability to clot milk. The result shows that the filtrate clotted milk and formed gels without any sign of syneresis in 2 pH ranges: 4.23-4.26 and 6.16-6.20. However, with the former pH range, firm gels were formed within 90 minutes while the later pH range took about 150 minutestx>form firm gels (Table 8). This observation indicates the possibility of existence of two enzyme systems in this culture filtrate, active at different pH ranges. More work needs to be done in relation to this concept. 67 Table 8. Effect of pH on milk-clotting activity when Chinese wine cake was the inoculum. pH Range Milk-Clotting Incubation Period Activity (MCA)* (Sweet-curdling at 40°C in min) 4.2 - 4.3 22.0 60 - 90 6.1 - 6.2 17.4 90 - 150 * MCA = 2400 X Dilution Factor t(sec) 68 When each of the culture filtrates was used to sweet-curdle milk during "Gua-nai" manufacture, firm gels were formed within 3 hours at 40°C. However, the filtrate from the A. oryzae fermentation produced a gel structure which was followed by syneresis. Data in Table 7 show that A. oryzae demonstrated the highest proteolytic activity with comparatively low milk-clotting activity. Available evidence confirms this finding. Kawai and Mukai (1970) noted that A. oryzae produces an enzyme with high proteolytic activity in addition to milk-clotting activity. They noted that cheese produced by this enzyme would have soft curd texture or have an off-flavor, especially a bitter taste. Arima, Iwasaki and Tamura (1967) stated that all strains of Rhizopus produced proteolytic enzymes and clotted milk within 30 minutes. They also noted that peptonization of the curd was observed. Among the 3 filamentous molds isolated from the commercial culture, Amylomyces rouxii demonstrated the highest milk-clotting activity with a comparatively low proteolytic activity. With an MCA/PA ratio of 333.8, this fungus can be seen as a potential source of a rennet sub- stitute. According to Richardson et a1. (1967), for a microbial enzyme to serve as a rennet substitute, it is necessary that it should not only have high milk-clotting activity but also low proteolytic activity. These authors stated that the ratios of milk-clotting activity and 69 proteolytic activity (MCA/PA) of 3 rennets on the market: Hansen's calf rennet, Mucor pusillus rennet (Meito's rennet)and Endothia parasitica rennet (Pfizer's rennet), were measured and adopted as the index of screening test for rennet substitutes. Tam.and Whitaker (1972) noted that rennin is ideal for cheese production because of its limited and specific proteolytic activity on k-casein and because of its high ratio of clotting activity to general proteolytic activity. They noted that only those enzymes which have limited proteolysis on the casein coagulum are considered suitable for the production of cheese since limited hydrolysis of casein contributes to ripening. However, Mukai and Kawai (1970) stated that to use MCA/PA ratio as the index for screening tests, the testing pH should be taken into consideration. They noted that since the pH of fresh milk, when rennet preparations are added during cheese manufacture is 6.2-6.3, then MCA/PA ratio at pH 6.0 will be the important index of organisms. The result of this investigation points to the possibility of existence of a good enzyme for cheese making in the fermentation filtrate when the Chinese wine cake is used as inoculum. The existence of proteolytic enzyme in Aspergillus oryzae has been shown by cultural studies. Tsugo and Yamauchi (1959) reported that A, oryzae produced a potent milk-clotting enzyme. Arima et al. (1967) also showed that 19 strains of Aspergillus, including A, oryzae 70 they tested, demonstrated milk-clotting acitivity. In this investigation, all the filamentous molds isolated produced milk-clotting and proteolytic enzymes when cultured in steamed glutinous rice, but at varying levels. When different concentrations of pepsin solution were tested for milk-clotting and proteolytic activities, it was observed that 0.60 mg/mL pepsin produced about the same milk-clotting and proteolytic activities as 1 mL of the filtrate obtained when the commercial culture was used to ferment steamed glutinous rice. This indicates that the concentration of protease in one milliliter of the filtrate when the wine cake was used as inoculum was equi- valent to approximately 0.6 mg/mL of pepsin. To evaluate the ability of the microbial free sample to clot milk, the liquid from rice fermented with the commercial culture was passed through microbiological filters (0.8 um and .45 um). The resultant filtrate was tested for milk-clotting activity and for production of Gua-nai. No difference was detected in the milk-clotting activities of both filtered and unfiltered liquids. Moreover, when the filtered liquid was used to sweet-curdle the milk-based mix, Gua— nai resulted at the incubation temperature and time. This result clearly eliminates the role of any microorganism in the milk-clotting process and in the sweet-curdling of 71 milk during Gua-nai manufacture. Therefore, whatever is responsible for clotting the milk must be present in the filtered liquid, most probably an enzyme system. Commercial vs. Reconstituted Cultures in Gua-nai Manufacture To provide information concerning the ability of the reconstituted culture to produce acceptable Gua-nai, both the commercial and the reconstituted cultures were each used as inoculum to ferment steamed glutinous rice. The fermented liquid from each was used separately to pro- duce Gua-nai. These products were compared by compositional and consumer panel analyses. Table 9 shows the results of the compositional analysis of both samples. These results show that the gross compositions of both products did not differ considerably. Again, the Triangle test (Larmond, 1977) was em- ployed to design the consumer taste panel. The panelists evaluated the products to identify the odd samples for the degree of difference between the samples, and for overall acceptability. The results of these are presented in Table 10. Out of a total of 51 panelists, l4 correctly identified the odd sample which was made with the commer- cial culture as against 37 who could not correctly identify it. Statistical analysis (Triangle test, difference analysis) shows no significant difference between the 2 72 Table 9. Compositional Analysis of Gua—nai Characteristic Commercial Reconstituted Culture Culture Total Solids (%) 79.5 80.3 Fat (%) 1.5 1.5 Total Protein (%) 3.1 3.1 Alcohol (%) 1.1 1.1 pH 6.3 6.3 T.A. 0.33 0.32 73 Table 10. Consumer Taste Panel Results 1. Total Responses - 51 2. Identification of odd sample: Correct - 14 (27%) Incorrect - 37 (73%) 3. Degree of Difference: Slight - 38 (74.5%) Moderate - 10 (19.6%) Much - 3 (5.9%) Extreme - 0 (0.0%) 4. Acceptability: Odd sample more acceptable - 27 .(52.9%) Duplicate samples more acceptable 23 (45.1%) No response - 1 (2.0%) 74 samples. Three quarters of the panelists indicated that there was only a slight difference between the 2 samples. Regarding the overall acceptability, more consumers preferred the odd sample which was made with the commercial culture, but the difference was not significant. Both flavor and texture of the samples were most frequently cited at the reasons for preference. The result of this study shows the very close identity of both the Chinese wine cake (commercial culture) and the rice culture reconstituted in the laboratory with the pure microorganisms isolated from the commercial culture. SUMMARY AND CONCLUS ION Chinese wine cake was utilized to ferment steamed glutinous rice. The liquid filtrate from the fermented rice was added to a milk-based mix and produced a sweet- set gel product, "Gua-nai." The filtrate contained 11.06% ethanol and exhibited both proteolyticanuimilk-clotting activities. Thus, it was concluded that the liquid filtrate contained an enzyme system. The pH optima for this filtrate were in two ranges: 4.23 - 4.26 and 6.16 - 6.20. Therefore it was concluded that there might be more than one enzyme system existing in this culture filtrate. The microorganisms present in the wine cake were isolated and identified as Amylomyces rouxii, RhizoEus oryzae, Aspergillus oryzae and a yeast, Endomycopsis burtonii. Since these were present in the original culture, they were therefore tested individually for their ability to ferment steamed glutinous rice and produce a liquid filtrate which was examined for ethanol content, and proteolytic and milk-clotting activities. Results of these screening tests confirmed previous reports regarding ethanol production, and production of milk-clotting and 75 76 proteolytic enzymes by all the filamentous molds isolated in this study. However, A. rouxii produced the highest quantity of ethanol (10.8%) and exhibited the highest milk-clotting and lowest proteolytic activities. Because of its high MCA/PA ratio, (333.8), it was concluded that this mold could be a potential source of "microbial rennet." The yeast strain was unable to ferment rice and so, no liquid filtrate was available for analysis. How- ever, when each of the molds was individually combined with the yeast, relatively higher ethanol concentrations were obtained within the same period. During the process of rice fermentation with the Chinese wine cake as inoculum, the pH of the medium fell from 6.60 to 3.81 and the titratable acidity rose to 0.66% in 6 days. The same pattern of drOp in pH and rise in T.A. was followed when the molds were individually used as inoculum to ferment glutinous rice. Within this period, there was gradual disappearance of the filamentous molds and finally, only the yeast was left in the medium. (Because of earlier reports and the results obtained in this study, it was concluded that this phenomenon was as a result of antibiosis or rise in total acidity or both. The order of disappearance of the filamentous molds was A. oryzae, A, rouxii and A, oryzae. The pure isolates were combined and employed in the reconstitution of the wine cake. There was some doubt 77 as to whether the reconstituted culture was exactly the same as the commercial wine cake. Therefore both the commercial culture and the reconstituted cultures were employed in Gua-nai manufacture, and the resultant products compared by both compositional and a consumer taste panel analyses. The results did not show any significant differences between the two products. Thus, it was concluded that the reconstituted culture could successfully produce acceptable Gua-nai. RECOMMENDATIONS FOR FURTHER INVESTIGATIONS Although some of the physical and biochemical changes taking place during the fermentation of steamed glutinous rice with the Chinese wine cake have been investigated, many questions pertaining to the overall reaction are yet to be answered. Therefore, the following are some of the aspects of the study that are suggested for further investigation: 1. Purification and characterization of the enzyme systems in the culture filtrates. 2. Identification and monitoring of the levels of various sugars,especially glucose, during the course of rice fermentation. 3. Elaboration of the chemical flavor changes occurring during fermentation of rice. 78 BIBLIOGRAPHY Alexopoulos, C. J. and C. W. Mims. 1979. Introductory Mycology, New York, John Wiley and Sons. 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