| Apple wine is a kind of drink cider which is produced using pure fruit juice as fermented materials, it not only contains the flavor of apple juice, but also possess the fragrance of the wine, which gradually becomes dominant in fruit wine beverage market in our country.Furthermore, industrial development is in conformity with the official macroscopic strategy,i.e. Chinese spirits made from cereal is step by step replaced by the wine made from fruits.China is the biggest apple produced countries in the world, apple wine not only could improve the additional values of apple, but also meet the demand of consumers for low-alcohol cider.Therefore, it is in line with the future development direction of apple industry. However, at present, our cider wine-producing industry lacks professional cider yeast strains, which could produce more aroma compounds, meanwhile, the research level of the regulatory mechanism of aroma compounds in fermentation process is not profound enough for us. In the present research, a cider yeast strain 38 was used as starting strain, which was formed by protoplast fusion and kept by our laboratory, a new genetic engineering strain 38(?BAT2) was obtained by gene knockout using BAT2 gene knockout fragment constructed by PCR method.Subsequently, 0.5 M and 1.0 M leucine or isoleucine was used as the sole nitrogen source of38 and 38(?BAT2) strains in SCD media to carry out the fermentation experiments, the depletion of the two branched chain amino acids in different timepoints was analyzed by pre-column derivatization prior to RP-HPLC(reversed phase high-performance liquid chromatography). Synchronously, the species and yields of aroma components in the fermented products were determined and analyzed by HS-SPME-GC-MS(headspace-solid phase microextraction-gas chromatography-mass spectrometer) in the main consumption phase of the two amino acids, MM2(molecular mechanics 2) force field is applied to analyze the energy flux for metabolism pathway of the fermented products, the activity of aminotransferase in fermentation liquid was simultaneously analyzed by colorimetric method.Meanwhile, the consumption of glucose in media was determined by DNS(3,5-dinitrosalicylic acid) method. The main results and conclusions of this work are as follows:(1) The short flanking homology region combined with PCR method for BAT2 gene knockout is simple, timesaving and cost-effective.(2) The high concentration of isoleucine and leucine in media would not produce toxicity to cider yeast strains. 98% of isoleucine and leucine were decomposed at 12 h of the logarithmic phase of the two strains, the higher original concentration of the added isoleucine or leucine in media, the higher catabolism rate, it is the proportional relationship.The loss of BAT2 gene in 38(?BAT2) strain showed no significant effect on the catabolism rate of the two branch-chained amino acids. The TEM(transmission electron microscope) photos at 12 h indicated that the chondriosome may be the main decomposed location of the added amino acids.(3) During the main consumption phase of the two branch-chained amino acids decomposed by 38 and 38(?BAT2) strain, the results analyzed by simultaneous GC-MS showed that 35 volatile compounds are found in SCD media contained isoleucine, 33 volatile compounds are found in SCD media contained leucine,respectively. The essential differences of species among the volatile aroma metabolites for the two sole nitrogen resources are that2-methylbutyraldehyde, 2-methylbutanol and 2-methylbutyl acetate are derived from isoleucine, yet 3-methylbutyraldehyde, 3-methylbutanol and 3-methylbutyl acetate are derived from leucine. They are important indexity aroma compounds in brewed wine. The yields of 2-methyl-1-propanol in the metabolites increased with the increase addition amounts of isoleucine or leucine in media. Some branch metabolism pathways were found among the volatile metabolites of the fermentation liquid added with the two sole nitrogen resources, i.e.C8 pathway(octanal, octanol, octanoic acid and ethyl octanoate), C9 pathway(nonanal and nonanol),C10(decanal, decanoic acid and ethyl decanoate), C12 pathway(dodecanal,dodecanol and dodecanoic acid), C14 pathway(tetradecanol, ethyl myristate and isopropyl myristate), C16 pathway(hexadecanol, methyl palmitate, ethyl palmitate and isopropyl palmitate). It is found that every branch pathway generally consists of a pair of side-by-side redox reactions, in which one is that aldehyde is oxidized to be acid, the other is aldehyde is reduced to be alcohol. It maintains the balance between NAD+ and NADH. The pathway also generally includes an esterification reaction to store redundant energy and maintain energy balance. Among them, the reactions that fatty aldehydes are converted to fatty acids or alcohols are generally spontaneous processes to release energy. But the esterification reactions between higher fatty acids and alcohols are generally non-spontaneous processes to absorb energy. Some compounds of them are regulated by BAT2 gene combined with the added isoleucine or leucine. During the main consumption phase of the two branch-chained amino acids, the yields of glutamine(Gln) increased with the increasing depletion amounts of isoleucine or leucine, which indicated that the deamination pathway consists of two steps when yeast cells assimilate high concentration of branch-chained amino acids. The first step isthat the amino groups of branch-chained amino acids are transfered to ɑ-ketone glutaric acid and produce glutamic acid molecules, the formed glutamic acid molecules further accept the amino groups of branch-chained amino acids so that plenty of glutamine molecules are produced.(4) The analyses results of branch-chained amino acid aminotransferase activity of the two strains showed that the loss of BAT2 gene had no significant effect on the branch-chained amino acid aminotransferase activity of 38(?BAT2) strain during the main consumption phase of the two branch-chained amino acids.(5) To compare with control group, the time to reach the residue concentration of glucose was postponed for 24 h or so in SCD media added with isoleucine or leucine.The above-mentioned results and conclusions in the present work would provide a theoretical basis for the further study of the aroma regulation of apple cider yeast, and also give an important reference for the development of apple wine industry. |
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