| Biogenic amines are worldwide considered as potential humful substance to human health which are mainly produced by the action of microbial decarboxylases on amino acids.It has been recognized that biogenic amines occur in a wide range of fermented meat product which is rich in protein. The results of some scholars in home and abroad showed that the content of biogenic amines in fermented sausage exceeded the FDA standards, which has become a potential safety problem. Traditional Chinese sausage is one form of spontaneously fermented sausages loved by consumers as special flavor. The species and content of biogenic amines were investigated in traditional Chinese sausage. Biogenic amines-productive dominant bacteria and biogenic amines oxidase -productive dominant bacteria which meet the standard of meat starter were separated and identified. The growth characterization of biogenic amines-productive dominant bacteria and biogenic amines oxidase-productive dominant bacteria was studied as well as influencing factors of producing biogenic amines and reducing biogenic amines by oxidation, accumulation mechanism of biogenic amines was explored at preliminary. Plant extracts can inhibate biogenic amines-productive dominant bacteria were screened and inhibation was realized according to the optimized formulation. Finally, applying L. plantarum-001+S.xylosus as starter, the plant extracts and L. plantarum-001+S.xylosus+the plant extracts, traditional Chinese sausage were produced respectively, and the inhibition of them on endogenous bacteria and biogenic amines was studied. The detailed contents and results are shown as follows:1. Investigation on characterization of biogenic amines and factors influencing their formation in traditional Chinese sausagesFourty two samples were obtained from traditional Chinese sausage seven main production areas. In order to clear the charateristic of biogenic amines and found the relation among biogenic amines, physicochemical properties and microbial distribution, the concentration of biogenic amines, NaCl, and NaNO2, pH, free amino acid, water content, Enterobacteria, Pseudomonas, Lactic acid bacteria, Micrococcus/Staphycococcus were investigated. Four groups of samples were identified on the basis of total amines, by cluster analysis. Group A included sausages showing low amine contents (76.5～220 mg/kg) and accounted for 28.5% of the sausages examined. Group B included sausages with moderate amine contents (220～600 mg/kg) and accounted for 45.2%. Group C included 11.9% of the samples showing high total biogenic amines contents (600～1000mg/kg) and group D contained 14.28% of the samples showing very high levels of biogenic amines (higher than 1000 mg/kg) which is more than the standard of FDA (total amount of Biogenic amines <1000mg/kg). In the other hand, tyramine content was higher than FDA standard (tyramine content≤100mg/kg) in about 57.1% of the samples and histamine content was higer than FDA standard (histamine content<50mg/kg) in about 57.1%of the samples found in this research. The pH values of samples varied from 5.30 to 6.56 and the content of nitrite varied from 0 to 10.31 mg/kg was less than 4 mg/kg in most of the samples. There were very large differences in water contents and NaC1 contents of our samples which ranged from 10.83 % to 42.96% and 2.45% to 10.84% respectively. Great differences in free amino acid content also were found and the range of the amount is 25.50mg/g-211.59 mg/g. No obvious relationship was found between physicochemical index with the contents of biogenic amine. Lactic acid bacteria and micrococcus/ staphycococcus are dominant bacteria in samples and high correlation coefficients were found between the total counts of Enterobacteria and concentrations of total biogenic amines (r= 0.7286). No pseudomonas was found.2. Study on isolation and identification of biogenic amines- productive and biogenic amines oxidase- productive dominant bacteria in traditional Chinese sausageBiogenic amines-productive dominant bacteria from samples showing higher biogenic amines contents and biogenic amines oxidase-productive dominant bacteria from samples showing lower biogenic amines contents were separated. A new method of monolayer cultivation with double-layer color development was established.96 biogenic amines-productive lactic acid bacteria and 58 biogenic amines-productive Enterobacteria were abtained. The 154 biogenic amines-productive bacteria belong to 5 species according to the result of polymerase chain reaction denaturing gradient gel eleetrophoresis (PCR-DGGE) analysis. After sequencing of PCR-products of 16S rDNA fragments and alignmenting in genebank, the 5 biogenic amines-productive bacteria are Enterococcus faecium, Enterococcus faecalis, Enterobacter cloacae, Escherichia coli and Enterobacter aerogenes. Biogenic amines-productive gene were determined by special primers. Biogenic amines oxidase-productive dominant 11 lactic acid bacteria and 2 Staphylococcus xylosus were abtained, after sequencing of PCR-products of 16S rDNA fragments and alignmenting in genebank, then analyzed by DNAMAN, the 13 bacterial belong to L.plantarum 001, L. plantarum 002 and Staphylococcus xylosus.3.Study on influencing factors of biogenic amines-productionEnvironmental factors such as pH, oxygen content, temperature and addtion level of Nacl, sugar and effects of biogenic amines-productive dominant bacteria between each other were studied in order to find production mechnasim of biogenic amines by biogenic amines-productive dominant bacteria. The results showed that the growth rate and production ability of biogenic amines of biogenic amines-productive dominant bacteria were inhibited at initial pH 5.00, and production ability of biogenic amines was lower at aerobic environment than anaerobic ones. Production ability of phenylethylamine by Enterococcus faecium and Enterococcus faecalis was higher under mixed cultivation than single cultivation significantly while production ability of tyramine was higher significantly in mixed cultivation than single cultivation only in logarithmic growth phase and the difference disappeared after stationary phase. Production ability of putrescine and cadaverine by Enterobacter cloacae, Escherichia coli and Enterobacter aerogenes was higher significantly under mixed cultivation between each other than under single cultiveation at 12～44 hours and the difference disappeared after 44 hous. Temperature, pH, addtion level of NaCl and sugar have significant effect on growth rate of biogenic amines-productive dominat bacteria and the effect degree of pH and temperature is higher by four factors quadratic rotation experiment. There were significant interaction effect of pH and temperuture on production of phenylethylamine, tyramine, cadaverine and putrescine except for production phenylethylamine of Enterococcus faecalis, and the effects were increased with the increased of temperature. There were sigificant interaction effects of addition level of sugar and temperature on production of putrescine by Escherichia coli and the same as production of cadaverine by Enterobacter aerogenes, the effects were also increased with the increased of temperature.4. Study on influencing factors on oxidation effectiveness of biogenic amines oxidaseOxidation kinetics of biogenic amines and the effects of pH on oxidation of biogenic amines as well as the effects between each other of biogenic amines oxidase-productive dominant bacteria were studied.The results showed that biogenic amines oxidase was produced in logarithmic growth phase and pH has significant effect on oxidation of biogenic amines at the same time. The oxidation rate of biogenic amines was higher in culture medium with initial pH 7.50 and 0.5% glucose than initial 6.00 and 1% glucose. No significant synergistic effect was found by mix culturation between each other of L.plantarum·001, L. plantarum·002 and Staphylococcus xylosus. But the oxidation ability was higher when L. plantarum ·001 and L. plantarum·002 cultured with Staphylococcus xylosus than L. plantarum ·001 and L. plantarum·002 cultured individually.5. Study on screening plant extracts with inhibation on biogenic amines-productive bacteria and optimizing compositionThe essential oil of cinnamon, clove, garlic, aniseed and tea polyphenol were screened as significant inhibition on biogenic amines-productive bacteria, and optimum combination the five plant exact for inhibition biogenic amines-productive bacteria was evaluated by RSM. The results showed essential oil of cinnamon, polyphenol, garlic, aniseed and tea polyphenol have significant inhibition on biogenic amines-productive bacteria and the antibacterial effects of cinnamon essential oil were more positive than that of tea polyphenols, clove essential oil, aniseed essential oil and garlic essential oil orderly. Sigificant interaction effects were found between cinnamon essential oil and clove essential oil. The highest cell concentration in experimental group was 4.84 log(cfu/ml) while it is 9.581og(cfu/ml) in the control. The results of regression analysis showed that the optimum proportion of the combination of essential oil of cinnamon, clove, garlic, aniseed and tea polyphenol was 0.02673%,0.0400%,0.0252%,0.0254% and 0.245% respectively when the lowest cell concentration was 2.318 log(cfu/ml)。6. Study on effect of starter and plant extracts on microecology distribution and biogenic amines in traditional Chinese sausageFour types of traditional Chinese sausages differing in starter and plant extracts(starter:L. plantarum 001+Staphylococcus xylosus; plant extracts:the compistion as 5; starter+ plant extracts; control) were produced with raw meat of three manufacturers (group A, B and C) and addition biogenic amines-productive bacteria (group D) respectively. Biogenic amines were determined by HPLC and microecology distribution was studied by conventional culturation combination with PCR-DGGE. The results showed that starter and plant extracts have significant inhibition on endogenous Enterobacteria and pseudomonas and synergistic effect of starter and plant extracts on the inhibition was found. And the inhibation of the plant extracts was stronger than the starter. The content of Enterobacteria and pseudomonas was more than 5.5 log(cfu/g) and 4.2 log(cfu/g) respectively in group B in which inhibition of the starter and plant extracts on biogenic amines production were also found but the total amount of biogenic amines was high. The starter and plant extracts can control biogenic amines production and synergistic effect of them was found by multiple comparison. |
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