Microbial composition during Chinese soy sauce koji-making based on culture dependent and independent methods
Koji-making is a key process for production of high quality soy sauce. The microbial composition during koji-making was investigated by culture-dependent and culture-independent methods to determine predominant bacterial and fungal populations. The culture-dependent methods used were direct culture and colony morphology observation, and PCR amplification of 16S/26S rDNA fragments followed by sequencing analysis. The culture-independent method was based on the analysis of 16S/26S rDNA clone libraries. There were differences between the results obtained by different methods. However, sufficient overlap existed between the different methods to identify potentially significant microbial groups. 16 and 20 different bacterial species were identified using culture-dependent and culture-independent methods, respectively. 7 species could be identified by both methods. The most predominant bacterial genera were Weissella and Staphylococcus. Both 6 different fungal species were identified using culture-dependent and culture-independent methods, respectively. Only 3 species could be identified by both sets of methods. The most predominant fungi were Aspergillus and Candida species. This work illustrated the importance of a comprehensive polyphasic approach in the analysis of microbial composition during soy sauce koji-making, the knowledge of which will enable further optimization of microbial composition and quality control of koji to upgrade Chinese traditional soy sauce product.
Soy sauce is a dark, salty sauce made from fermenting boiled soybeans and roasted grain. It is a basic ingredient in Chinese, Japanese and other Asian cooking, and is increasingly popular in Western cuisine. The annual production of soy sauce is about 5 million metric tons in China, accounting for over 50% of the world production. Generally, the fermentation of soy sauce is classified into two ways: low-salt solid-state fermentation and high-salt liquid-state fermentation. Both processes have a similar principle that involves four main steps: (1) Preparation of raw materials; (2) Koji-making; (3) Brine fermentation; and (4) Sauce extraction and blending. Among these steps, koji-making plays a significant role (Sano et al., 2007).
During koji-making, enzymes from moulds bring about the saccharification of starch and decomposition of protein in the raw materials that support yeast and bacterial growth essential to the main brine fermentation (Wicklow et al., 2007). Thick layer aeration is mostly used for koji-making. The material is mixed with wheat flour or bran, and then inoculated with mould for about 28 h, which Aspergillus oryzae is generally used. During the incubation, it is necessary to stir the mixture when the product temperature increases to 37 °C, in order to promote mycelium growth of mould ( Luh, 1995). Spores begin to form after 18 h, and after 26–28 h, the culture mixture becomes greenish yellow in colour as a result of mould growth and sporulation, which is also shown in fungal enumeration curve.
Microbial composition during Chinese soy sauce koji-making based on culture dependent and independent methods
Koji-making is a key process for production of high quality soy sauce. The microbial composition during koji-making was investigated by culture-dependent and culture-independent methods to determine predominant bacterial and fungal populations. The culture-dependent methods used were direct culture and colony morphology observation, and PCR amplification of 16S/26S rDNA fragments followed by sequencing analysis. The culture-independent method was based on the analysis of 16S/26S rDNA clone libraries. There were differences between the results obtained by different methods. However, sufficient overlap existed between the different methods to identify potentially significant microbial groups. 16 and 20 different bacterial species were identified using culture-dependent and culture-independent methods, respectively. 7 species could be identified by both methods. The most predominant bacterial genera were Weissella and Staphylococcus. Both 6 different fungal species were identified using culture-dependent and culture-independent methods, respectively. Only 3 species could be identified by both sets of methods. The most predominant fungi were Aspergillus and Candida species. This work illustrated the importance of a comprehensive polyphasic approach in the analysis of microbial composition during soy sauce koji-making, the knowledge of which will enable further optimization of microbial composition and quality control of koji to upgrade Chinese traditional soy sauce product.
Soy sauce is a dark, salty sauce made from fermenting boiled soybeans and roasted grain. It is a basic ingredient in Chinese, Japanese and other Asian cooking, and is increasingly popular in Western cuisine. The annual production of soy sauce is about 5 million metric tons in China, accounting for over 50% of the world production. Generally, the fermentation of soy sauce is classified into two ways: low-salt solid-state fermentation and high-salt liquid-state fermentation. Both processes have a similar principle that involves four main steps: (1) Preparation of raw materials; (2) Koji-making; (3) Brine fermentation; and (4) Sauce extraction and blending. Among these steps, koji-making plays a significant role (Sano et al., 2007).
During koji-making, enzymes from moulds bring about the saccharification of starch and decomposition of protein in the raw materials that support yeast and bacterial growth essential to the main brine fermentation (Wicklow et al., 2007). Thick layer aeration is mostly used for koji-making. The material is mixed with wheat flour or bran, and then inoculated with mould for about 28 h, which Aspergillus oryzae is generally used. During the incubation, it is necessary to stir the mixture when the product temperature increases to 37 °C, in order to promote mycelium growth of mould ( Luh, 1995). Spores begin to form after 18 h, and after 26–28 h, the culture mixture becomes greenish yellow in colour as a result of mould growth and sporulation, which is also shown in fungal enumeration curve.
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