Metabolic Regulation Of Methyl Ketone Synthesis Of Cream Fermented By Lactococcus Lactis

Methyl ketones are important flavor substances of fermented dairy products and intermediate of synthetic flavors. Chemical synthesis of methyl ketone has a single character which is far from natural feeling that milk products needs. Lactococcus lactis subsp. lactis has a significant economic value, because it is an important industrial fermentation agent and host strain. This strain has a positive effect on methyl ketones in most dairy products. Here, we proposed the biosynthesis of methyl ketone by Lactococcus lactis. Our research measured the enzymatic activities of the synthesis pathway under different conditions(fatty acids, fermentation condition, soluble saccharides, metal ions) of Lactococcus lactis subsp. lactis and discussed the metabolic regulatory mechanism of methyl ketone synthesis with its enzymatic levels.The volatile compounds of cream were analyzed by SPME-GC-MS technology. About 21 kinds of volatile substances were detected in milk cream, mostly, the fatty acids, methyl ketones and lactones. The detection results of fermented cream with SPME-GC-MS showed that 15 kinds of flavor compounds were identified. There were 4 kinds of free fatty acids and 4 type of methyl ketones. A comparison of these types and relative content was performed on the volatile compounds data. The results showed that Lactococcus lactis subsp. lactis had a substantial effect on the yield of methyl ketone. Through principal component analysis, relative odor activity value and sensory evaluation, there were 5 kinds of essential flavor compounds of single strain fermented cream of Lactococcus lactis subsp. lactis. The 5 types of key flavor compounds were 2-undecanone, 2-nonanone, nonanal, heptanal and hexanal(ROAV≥1). At the same time, butyric acid, hexanoic acid, 2-tridecanone, and 2-heptanone played important modification roles in the overall flavor(0.1≤ ROAV < 1).The different growth stages of Lactococcus lactis subsp. lactis had different β-oxidation ability and thioesterase activity and the maximum value appeared in stable period. Therefore, the synthesis process of methyl ketones mainly occured in the stable phase. Palmitic acid could inhibit the growth of Lactococcus lactis subsp. lactis and could induce β-oxidation system of Lactococcus lactis subsp. lactis as well. These results proved that fatty acid oxidation appears to be the principal mechanism of resistance. Through detection of acyl-Co A dehydrogenase, enoyl-Co A hydratase, β-hydroxyacyl-Co A dehydrogenase, thiolase and thioesterase activities of Lactococcus lactis subsp. lactis, we identified that Lactococcus lactis subsp. lactis has pronounced β-oxidation,s ability and thioesterase activity. Combined with GC-MS analytical data, plus the combining effects of substrates and nutritional factors on the synthesis pathway of methyl ketone, the main pathway of methyl ketones synthesis of Lactococcus lactis subsp. lactis was incomplete β-oxidation pathway.Palmitic acid C16:0 could induce β-oxidation system of Lactococcus lactis subsp. lactis, but this induction ability was somehow different. Lactococcus lactis subsp. lactis cultured at 30°C; p H(6-7) had higher β-oxidation ability. Glucose, fructose, sucrose, lactose and galactose could keep or inhibit thiolase activity. The activity of acyl-Co A dehydrogenases increased markedly in the presence of Ca2+ and Mg2+, whereas decreased by 1 mmol/L-Fe2+ or 12 mmol/L-Mg2+. Mg2+, Ca2+, Fe2+ and Cu2+ could induce enoyl-Co A hydratase, inhibit β-3-hydroxyacyl Co A dehydrogenase activity and induce thiolase activity. The fad A gene expression of Lactococcus lactis under different culture conditions was significant difference(p<0.05). The expression amount of calcium treatment was 3.02 times as big as normal culture conditions. The expression amount of fructose treatment was 28.71 times as big as normal culture conditions.Midchain fatty acid(Octoic acid C8:0) and long chain fatty acid(Palmitic acid C16:0) could induce thioesterase activity of Lactococcus lactis subsp. lactis. Fructose could induce thioesterase activity and inhibit thiolase activity. Through correlation and regression analysis, there was a significant correlation between thioesterase and enoyl-Co A hydratase activity under cultivation of soluble saccharides and copper ion(p<0.05). Mg2+, Ca2+, Fe2+ and Cu2+ could inhibit thioesterases activity. There was a significant correlation between the content of calcium ion and thiolase activity. The total relative content of methyl ketone of fermented cream was 1.29 times of the control. When fructose, calcium ion and palmitic acid were added to cream, these substances could promote producing methyl ketone from Lactococcus lactis subsp. lactis.In short, since fatty acids, fermentation conditions, soluble saccharides, and metal ions can affect enzymes activities of the incomplete β-oxidation pathway associated with the methyl ketone synthesis, we can change these factors to regulate methyl ketones synthesis. These results could contribute to understand the relationship of flavor compounds and fatty acid metabolism as played by Lactococcus lactis subsp. lactis in dairy products.