| Diacetyl is an important flavor compound in dairy products because of its cream flavor. It is better to generate it by naturally occurring LAB "cell factory" than produced by chemical methods. It is necessary and meaningful to develop domestic excellent wild resources into the natural cell factories for high-yielding diacetyl. To study the non-transgenic diacetyl producing GARS bacteria is urgent, because most of the researches on high-yielding diacetyl by L.lactis were through the transgenic methods or given the favorable conditions. In addition, there is few reseraches about diacetyl produced by E.faecalis in current, so it is very necessary to make a comparative study between E.faecalis and L.lactis. In the era of bioinformatics, genomics model, metabolic model, and many other high-throughput precise regulation means came into being, so it is very helpful to explore the mechanism of high-yielding diacetyl through the contrasitive analysis of in silicon research with general biochemical regulation.This topic includes four parts:the selection of original strains, the premise, reason and optimization of high-yielding diacetyl. Firstly, we determine the wild strains, commercial strains, engineering strains and mutagenic strains (another classified method are L.lactis and E.faecalis); Secondly, pyruvate accumulation from citric acid and amino acids was researched. Thirdly, we study the enzyme activities (internal factors) and pH, O2(external factors) in order to investgate how they effect the diacetyl metabolism; Finally, the genomic model to guide the high-yielding diacetyl was applied to summarize the best diacetyl-producing program of representative starting strains.There were totally16strains selected, including13wild strains with diacetyl yield greater than0.08g·L-1from116strains saved in KLDS, and a commercial strain, a restriction-induced NZ9000and a NTG mutagenic mutant. The diacetyl production ability of them and diacetyl content of seven different manufacturers products were detected.On the basis,6strains were selected as a representative through the citric acid using selective medium and plasmid extraction, CIT specific primer PCR method, and comparing with each other. It was conclued that the citric acid utilizing level of three L.lactis strains were higher than that of E.faecalis’by measure the consumption of citric acid and generation of diacetyl and acetoin during 100min, and the strongest in the latter KLDS4.1003is closer to the former KLDS4.1002. In addition, KLDS4.0325and KLDS4.1004were sent to measure plasmid genome and draw restrictive physical maps, respectively, in order to profound analysis of the plasmid, and corresponding with the metabolic data.The influence of amino acids on pyruvate accumulation by16strains from the10kinds of amino acids was studied. Effect of Asp and Ala was the most obvious, although they are invalid on the5E.faecalis strains. In order to generate maximum diacetyl, the KLDS4.0325,4.1001and4.1002that with the highest using efficiency of citric acid were studied. Pyruvate accumulation of all the3strains were highest and lowest at pH8.0and4.5, respectively. Meanwhile, the values of other6points were similar. It can be seen that pyruvate accumulation of3strains have a certain influence by pH value. Making reference to the accumulation of citric acid, pH6.5,7.0,7.5, this3points for both accumulation of pyruvate were the most favorable, considered Asp separately, the degree of degradation pH5.0was second.The key of the accumulation of pyruvate is the ALD enzyme, there has been found two natural ALD-strains, KLDS4.0316and4.0325belonging to the Lactococcal lactis, and both are CIT+at the same time, so there is no need to do the NTG mutagenesis. KLDS4.0326and KLDS4.0346,the wild types of E.faecalis are ALD-naturally, so the other2strains KLDS4.1004and KLDS4.1005should be made the NTG mutagenesis. After the mutagenesis KLDS4.1005didn’t show positive mutation strains, while KLDS4.1004which is derived from KLDS4.1003obtained good performance, called the induced strain KLDS4.1003, both strains’absorption efficiency of citric acid are equivalent, after5h the mutagenesis strain’s absorption efficiency of citric acid is higher than that of parent strain, finally after10h the citric acid was run out, indicating that the CIT and ALS enzyme activity are not inhibited in the mutation, and the difference between the two strains is only in ALD activity. Moreover, the growth rate of KLDS4.1003was greater than that of the parent strain (only at3,4,8h three points are similar), which proves that the NTG mutagenesis did no have negtive impact on the growth of mutagenesis strain.The pH value as macro-control factor were discussed to study more about the enzymes except ALD. The pH4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0were selected. The results indicated that the enzyme activity of LDH, DR, ALD in L.lactis were lower than that of E.faecalis, which from a certain degree to explain why the former producing more diacetyl. The maximum growth rate and maximum cell count of these four strains were determined, and each pH point maximum yield of diacetyl and the corresponding acetoin amount were measured. Results showed that maximum growth rate and cell count of the L.lactis generally slower than that of the E.faecalis, respectively. However, diacetyl production is higher, which indicated that a combination of factors to infer the activity and the number of cells was greater than the growth rate. Overall, pH6.5,7.0on two types of bacteria are research priorities,7.0was appropriate because of the enzyme activity and6.5 because of the number of bacteria more. One representative each choose two strains that KLDS4.0325and KLDS4.1003at pH6.5,7.0, respectively, a total of6time points of4,8,12,16,20,24h detecting bacterial count and diacetyl production changes the number of bacteria. Results indicated that under the limited spatial and nutritional conditions, too much microbes will lead to reduced production of diacetyl.In order to do the depth research and provide the basis for the future development of starter culture, which could produce high level of aroma compounds. Whole genome data of KLDS4.0325was determined and compared with representative strains of L.lactis.Throughout the whole subject, it included two contrasts that the first one was among wild bacteria, commercial bacteria, mutagenic bacteria and engineered bacteria and the second one was between L.lactis and E.faecalis. In summary we thoroughly and macroscopicly analyzed metabolism of high-yielding diacetyl.The innovation in the subject:a. According to the metabolic sequence of diacetyl, we used a method of comparison and analysis, as same time we studied it with gradually advancing. Finally we found out KLDS4.0325and KLDS4.1003which possessed great ability of producing diacetyl to act as natural "cell factories" with Chinese wild bacteria characteristics.b. We made the research for co-accumulation of pyruvate with metabolism of citric acid and amino acid. Meanwhile we draw restrictive maps for CIT+plasmids and determined plasmid genome, then considered macroscopic impact of accumulating pyruvate with pH.c. Taking account of the growth of bacteria, enzyme activity and considering the effects of pH and O2, We comprehensively analyzed internal and external causes for high levels of diacetyl produced by the original strain from the view of natural molecular means instead of genetical molecular means.d. Determining whole genome data of KLDS4.0325and comparing it with representative strains of L.lactis, we made the depth research and provided the basis for the future development of starter cultures which could produce high level of aroma compounds. |
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