Application Of Micrococcus Luteus KDF2and Its Extracellular Protease KRP2in Sufu

As the typical representative of Chinese traditional fermented soy products, sufu has a history of more than1500years. Although the production of sufu has been industrialized, there still exists problems such as unstable quality and long fermentation time. On the basis of previous work, microbial technology was used to study strain features of Micrococcus luteus KDF2in this research and enzyme technology to study enzymatic property from Micrococcus luteus KDF2.As the study above, Micrococcus luteus KDF2was used as starter culture as well as protease KRP2from Micrococcus luteus KDF2by enzymic method to ferment sufu, and physicochemical indexes were also kept track of during the fermentation process. Finally, the medium and the fermentation process parameters of Micrococcus luteus KDF2were optimized by fermentation engineering technology. This research gave the foundation for fermenting kedong sufu by enzyme and pure bacteria.Strains characteristic of Micrococcus luteus KDF2showed that the optimum growth temperature of Micrococcus luteus KDF2was32℃, and the optimum pH was8.0. Under the condition of7%(w/v) NaCl and13%(v/v) ethanol, Micrococcus luteus KDF2showed strong resistance acceptability.Properties of protease KRP2from Micrococcus luteus KDF2showed that the protease KRP2exhibited maximal activity was at40℃and pH9.0. It showed high stability in the presence of9%(v/v) ethanol and8%(w/v) NaCl. The activity of protease KRP2was enhanced when Ca2+and Mg2+ions were existing.Moisture, total acid, amino nitrogen, peptides, free amino acid and texture of sufu were measured at different fermentation periods when Micrococcus luteus KDF2was used as the starter. The results showed that when the sufu was fermented with M. luteus KDF2for90d, its moisture content, amino nitrogen and total acidity were reached59.3%(≤72.00%),0.5023g/100g of sufu (≤1.2g/100g of sufu) and0.6512g/100g of sufu (≥0.45g/100g of sufu), respectively, which reached the industrial standard of sufu. Peptide content of sufu was detected by HPLC and the primary peaks were found in8.979min,19.977min,20.268min,23.631min and27.962min. The total peak area on90d was45800mAU×s. For the free amino acid, on90d, the total free amino acid was578.3mg/100g of sufu, and total essential amino acids was249.8mg/100g of sufu. The concentration of glutamic acid (umami amino acid) was the highest, and lysine (isbitter amino acid) followed. When sufu was fermented for90d, the hardness and adhesiveness of sufu were200g and32gsec, respectively. Moisture, total acid, amino nitrogen, peptides, free amino acid and the texture of sufu were measured at different fermentation periods when KPR2, the extracellular protease of Micrococcus luteus KDF2, was used in sufu fermentation. The results showed that on the120d of fermentation, the moisture content, amino nitrogen and total acidity were reached70.3%(≤74.00%)0.3618/100g of sufu (≤1.3g/100g of sufu) and2.0286g/100g of sufu (≥0.35g/100g of sufu) respectively, which reached the industrial standard of sufu except the total acidity. The primary peaks of peptide were found in8.979min,19.977min,20.268min,23.631min and27.962min. The total peak area on120d was20297.4mAUxs The total free amino acids on120d was114.8mg/100g of sufu and the total essential amino acids was76.6mg/100g of sufu The concentration of methionine with bitter taste was the highest value above the threshold, followed by lysine with sweet taste. When sufu was fermented for120d, the hardness and adhesiveness of sufu were450g and33gsec, respectively.This research optimized the Micrococcus luteus KDF2culture medium and its fermentation conditions through response surface methodology. The optimized formula of culture medium was as follow:sucrose11.57g/L, peptone:yeast extract1:1and its total concentration32.13g/L, the concentration of dipotassium hydrogen phosphate8.09g/L. Under this condition the total number of colonies of bacteria reached4.79×109CFU/mL. Cultivation of Micrococcus luteus KDF2showed that the optimum temperature of Micrococcus luteus KDF2was30℃, the optimum was pH8.0, the loaded liquid was50mL/250mL and the oscillation frequency was150r/min, and under this condition the final total number of bacteria colonies was5.42×109CFU/mL. Based on the above results, Micrococcus luteus KDF2could be used as a potential starter culture strain during the production of sufu. However, the protease KRP2was not fit to apply in sufu fermentation in the form of single enzyme.