| Kou wan lao(KWL, also called as Gua-Nai by researchers in U.S.A.) is a traditional dairy product in China, which is manufactured by coagulating milk with the addition of extracts from fermented rice wine. Its smooth taste and attractive flavor are preferred by many Chinese, but its soft texture, complex manufacture method limit its industrial production. Until now, only handmade production way is used in its manufacture. The aim of this study was to find the optimum conditions to shorten the manufacture time and provide basic information for industrial purposes. The mechanism of gel forming, microbiology of KWL, characteristics of milk clotting enzyme (MCE), relationship between gel texture and sensory evaluation were studies in the thesis. Main results in the thesis are:1. There are different characteristics of MCE in varied Jiu-yao. High correlations was found between MCE produced during fermentation and strains Rhizopus in Jiu-yao. R.liquefaciens Yamazaki isolated from Jiu-yao B4 has higher MCE activity among four Jiu-yao. Three strains, R. minimus Van Tieghem, M. ambiguous Vuillemin, R. equines Constantion et Lucet, were isolated from other Jiu-yao. They showed lower MCE activity than R. liquefaciens Yamazaki.2. It was shown that liquid fermentation was more unsuitable to produce MCE by Rhizopus B4M2. The strain, Rhizopus B4M2, could increase production of MCE in the solid state fermantation. The optimum ratio of bran and glutinous rice to water content as 3:1 can improve MCE activity. Organic nitrogen resource can promote the growth of strain and synthesis of MCE, while inorganic nitrogenresource indicated inhibiting effect.3. The purification procedures, comprising of evaporation, ethanol precipitation, size-exclusion chromatography using Sephadex G-100, and ion-exchange chromatography using DEAE-52-cellulose, conferred 2461-folds purification and 12.9% recovery of MCE. 60% and 71% ethanol precipitation resulted in 51.9% recovery of total MCE, and two components were obtained, whose activities were 14.55 U/mg protein and 18.44 U/mg protein, respectively. MCE was stable at the pH range of 1~7 at 4 ℃. The optimum temperature for MCE activity was 40℃ and MCE was completely inactivated at 55 ℃ after 20 min. The N-terminal amino acid sequence was GTGSVPVTDYQNDVE, which showed higher identity with those from other fungal proteinase. Its molecular weight was 36.6 kDa.4. Clotting time decreased with increasing enzyme concentration and ethanol concentration. Total solid of skim milk prolonged the clotting time. Sugar did not show significant effect on clotting time of skim milk.5. Interaction between colloid casein micelle of skim milk coagulated by Rhizopus MCE was stronger than that by calf rennet and proteinase. The higher viscosity value of gel coagulated by using 5% of Rhizopus MCE was observed under pH 6.27, and pH 6.51. The sugar and total acid in skim milk gel did not show significant effects on hardness of KWL. Addition of 3%~4% ethanol, calcium and MCE of Rhizopus lead to the increase of hardness of KWL. Combination of high calcium concentration, high total acid with low MCE can increase hardness of KWL gel and maximum hardness was 130.2 g.6. The micrograph of environment scan electronic microscopy (ESEM) showed that structure ofKWL was sandwich-like. The thickness of layers became denser with increasing MCE concentration.7. Correlation between sensory evaluation results with instrument ones was studied. The sensory evaluation scores increased progressively with increasing hardness and cohesiveness of KWL. Factor analysis was found to be useful to pick up common factors, which can represent the whole sensory characteristics of KWL gel. Factor cluster showed higher correct classification rate than comprehensive sensory evaluation distinction method when using the Fisher's model.
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