Study On Debittering Technology With Microorganism Of Grapefruit Wine

As a typical tropical and subtropical fruit, grapefruit has beautiful color, unique aroma.crystal clear and crisp flesh, tasty, unique flavor,It also has high nutritional value and health function.In addition to be fresh food, the grapefruit can also be processed into a variety of products. Grapefruit wine obtained by fermentation,As low alcohol wine,it has natural flavor of grapefruit, abundant nutrition and health value.Grapefruit wine is more and more favored by contemporary people. However, not many more consumers can accept the slightly heavyer bitter of grapefruit wine. Therefore, the research on debittering technology of grapefruit wine can help improving the development of grapefruit wine.In this paper, debittering technology with microorganism of grapefruit wine was studied.soil from rotten grapefruit accumulation and grapefruit trees, rot grapefruit peel and the Aspergillus niger strains preserved in laboratory were chosed as strains sources.High yield naringinase producing strain was screened from nature.In order to further improve the performance of enzyme production, mutation breeding of it has been studied.And then,the optimal fermentation condition of high yield naringinase producing strain was also researched.The crude enzyme liquid was abtained from the fermentation and was applied to the debittering experiment of grapefruit juice for producing grapefruit wine. From the tests,the optimum debittering conditions was determined. Accordingly, the study on debittering technology with microorganism of grapefruit wine can be achieved by these experiments.There sults were showed as follows:1.In this experiment,128 fungi was isolated and purified from soil of rotten grapefruit accumulation and grapefruit trees, rot grapefruit peel.These fungi and 23 strains of Aspergillus niger which preserved in aboratory were chosen by by semisolid high-level screening. There were 53 strains appearring obvious transparent solution circles in top tubes.36 of these strains has the transparent solution circles which thickness can be measured. After shake flask screening for the 36 strains, a relatively high naringinase activity of strains M53 was determined. The enzyme activity of this production was 507.07μg/mL.min. M53 strain of colony characteristics and morphology were initially identified as Aspergillus niger strain. (Aspergillus niger M53).2.The combination of UV radiation 2-4min and 0.2% LiCl is mutagenic treatment conditions. Under these conditions, the wild-type strain M53 was mutation breeded. The mutant strains were identified by naringin screening plate.31 mutant strains which colonies grew well and transparent zone diameter/colony diameter larger than the original strain M53(1.21)were picked. These mutants were screened by shake flask.23 of these mutant strains which naringinase activity are higher than strain M53 were selected and saved. The mutant strain M53-7 has higher naringinase activity was abtained,which enzymatic activity is 721.08μg/mL.min, higher than the original strain as 214.01 units. The increasing rate of its naringinase activity is 42.21%.The genetic stability study showed that the mutant enzyme more stable performance of mutant M53-7. It can be regarded as a strain in the follow-up study.3.The result showes that the best carbon source is sucrose, the best inorganic nitrogen sources is NH4NO3, the best organic nitrogen source is soybean meal. The addition of these inorganic salts were that CaCl₂0.1%,MgSO4-7H2O 0.5%, ZnSO₄0.1%.According to the results of single factor tests, orthogonal experimental was designed to determine the best ratio of the main factors sugar, soybean meal, NH4NO3,K2HPO₄, which is affecting the enzyme activity in medium.The expriment was test accorded to four factors and three level orthogonal design table. Results showes that optimal concentration of sugar, soybean meal, NH4NO3,K2HPO4, are 0.5%,3.0%,1.0%,0.1%.4.Tthe optimal fermentation conditions were abtained in the study of fermentation conditions On the mutant strain M53-7.It is that addition of inducer naringin is 0.05%,medium initial pH is 5.0, culture temperature is 28℃; inoculation is 0.3 mL,medium loading capacity is 30mL/250mL, addition of Tween-80 is 0.15%, time of shake flask is 96h in the improved fermentation medium and the optimum fermentation conditions. Under the optimum culture conditions for enzyme production fermentation, the enzymatic activity of mutant strain M53-7 maintain in 872.28μg/mL.min. The performance of the mutant strain M53-7 to produce enzymes is more stable.5.Use naringinase crude extrac from shake flask of mutant M53-7 to dibetter grapefruit juice. The result of this preliminary study shows that best debittering effection was achieved when crude enzyme solution is 8U/mL, pH is 3.5, treatment is 50℃,time of treatment is 60min. Under these conditions, the debittering rate achieved 47.12%. This treatment can both remove a large number of bitter substances contained in grapefruit juice and maintain the unique flavor of grapefruit wine in subsequent fermentation.Innovation of this paper:Soil from rotten grapefruit accumulation and grapefruit trees, rot grapefruit peel and the Aspergillus niger strains preserved in laboratory were chosed as strain sources. The strain M53 which has high naringinase activity was screened out of these strain sources. After UV and LiCl compound mutation on strain M53,a mutant strain M53-7 was selected, which enzyme production has been more significant improvement The best fermentation conditions of mutant strain M53-7 was established in this paper. And the debittering effection of grapefruit wine was preliminary studied with mutant strain M53-7's fermentation crude extract.Conclusion:The mutant strain M53-7 was abtained by natural selection and mutation breeding.lt has high cell growth speed, abundant mycelium production, short cycle of enzyme production and more stable performance of enzyme production.It is an excellent industrial production strain source. When grapefruit juice was bittered by crude enzyme extract of mutant strain M53-7's fermentation, a large number of bitter substances of grapefruit wine can be effectively removed, and the unique flavor of grapefruit wine also can be maintained in latter fermentation.