| Douchi is one of the earliest flavoring fermented food in the diet of our people, with its high edible value, high nutrional value, good taste, and has certain pharmacological effects and become one of the people’s favorite food. Yongchuan douchi is representative of the product China mucor type Douchi. Its production process includes the selection of soaking, cooking, miscellany, koji making, mixing, fermentation and other parts. With the increase of value on food safety, content of biogenic amines in the Chinese and foreign traditional fermented food is becoming attracting more and more attention, but the research of the biogenic amines in Douchi is still lacking. In order to meet consumers’safety and health requirements, Biogenic amines of Douchi need to be studied. By taking different sampling times of Yongchuan traditional Mucor fermented soybean in starter-making and late fermentation, this study dynamically track the basic nutrition composition and the content of biogenic amies, histidine decarboxylase activities and Microorganism in the entire process. The purpose is to study the biogenic amines content and histamine formation mechanism of traditional Mucor fermented soybean, and to dicuss histamine forming mechanism. This paper came to the following conclusions:(1) The method of detecting the contents of biogenic amine in China traditional fermented Douchi by HPLC was developed. The method was based on a reversed-phase column(Agilent XDB C18column,250mm X4.6mm X5μm) for precolumn derivatization with Benzoyl chloride; Douchi samples by high performance liquid chromatography using gradient elution(methanol and ultra pure water), and detected with spectrophotometric UV-vis detection at254nm. It allows simultaneous determination of8amines in less than25min for putrescine and cadaverine, tryptamine,2-phenyl-ethylamine, spermidine, spermine, histamine and tyramine. Linearity for each amine is observed between1to80 μg/mL.Detection limits range from0.03to O.l0μg/mL. The average recovery of China traditional fermented Douchi for biogenic amines was96.80-106.10%. With the merits of high sensitivity, precision and well repeatability, the method was feasible for the determination of biogenic amines in China traditional fermented Douchi. By this method the kinds and contents of biogenic amine in China traditional fermented Douchi was determined. It was noticed that the biogenic amine kinds and contents varied with different China traditional fermented Douchi. The variation of total biogenic amine in China traditional fermented Douchi was101.07-427.19mg/kg.(2) The results of the basic components’change in the process are as follows:samples’ moisture content decreased from46.85%to33.75%after being soaked, and remained at around45%during the post-fermentation process. The changes of Samples’pH was not obvious in the process of koji-making, and it gradually decreased from6.42of raw material to4.80of finished product. Samples’amino acid nitrogen content increased from0.43g/100g of raw material to1.57g/100g of finished product. Samples’total acid content increased first and then decreased in the process of koji-making, but it increased in the post-fermentation process, and it increased from0.38g/100g of raw material to2.14g/100g of the finished product.(3) Changes in biogenic amines in yongchuan Mucor type douchi in the processing were studied, results show that samples are detected putrescine, cadaverine, tryptamine,2-phenethylamine, spermidine, spermine, histamine and tyramine in the processing, but raw material don’t contain2-phenylethylamine, spermine, histamine and tyramine. The change trend of putrescine and tyramine content is not obvious, respectively, within30.29～55.05mg/kg and36.58～54.17mg/kg;2-phenylethylamine, tyramine, histamine were initially not detected, but increased to54.82,34.44,30.83mg/kg of finished products respectively; Spermine was detected in late fermentation for215days; Spermidine content decreased in the koji making process, reached the minimum12.99mg/kg, then increased gradually, reached22.39mg/kg in the post-fermentation process; Cadaverine decreased slowly in the koji making process, but it decreased rapidly to0in the early post-fermetation stage, and finally reached43.76mg/kg.(4) From the reseach of the Yongchuan Mucor type douchi Formation Mechanism of histamine in the processing. The results show that:histamine was not detected in raw material, but histamine content was12.53mg/kg in the first two days of the koji(S2), and finally reached34.45mg/kg of finished products. The results show that there is a material conversing to histamine. Free histidine content increased first and then decreased and then increased in the koji-making process. The process of the formation of histamine is accompanied by the decrease of free histidine, it shows that free histidine is involved in the formation of histamine. At the same time histidine decarboxylase activity increased in the koji-making process, decreased gradually in the early post-fermetation, and increased gradually in the late fermentation. Verification of histamine precursor experiment, hitamine producing strains were inoculated in culture medium containing histidine and culture medium without histidine, culture medium containing histidine was detected histamine, but culture medium without histidine wasn’t detected histamine, the histidine in culture medium decreased, histidine content fall and histamine content rise, and the number of moles of two substances change is about1:1. It prove that histidine is the precursor of histamine. The results showed that11biogenic amine-forming bacterium strains from Yongchuan Mucor type douchi, which were identified to be Bacillus subtilis(3strains), Bacillus amyloliquefaciens(3strains), Bacillus atrophaeus(2strains), Bacillus methylotrophicus (3strains). So it comes to the conclusion that soybean protein produce free amino acids under protease, then histidine produce histamine under histidine decarboxylase which is produced by bigenic amine-forming strain. |
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