| Due to the various excellent performance of bacterial cellulose, it has been widely concerned, while its research and application are also very extensive. At present, there are various raw materials for industrial production at home and abroad. But the production of BC is the highest when coconut water is the main raw material,and the use of coconut water is the most common in the Southeast Asian countries. In China, Hainan Province is an important birthplace and production base for BC research and production. BC is also known as coconut fiber (that is nata de coco). At present, BC is mainly used in the field of food and cosmetics. Production practice shows that raw coconut water is usually placed naturally (it produces natural fermented, referred to as "pre-fermenting") for a few days and then used for BC production, which production is higher than the direct use of fresh coconut water. However, due to the natural fermentation process of coconut water without any control in the production, environmental conditions have affected the results of coconut water pre-fermentation, such as temperature, humidity, microbial flora conditions ,fermentation time and so on, which makes the BC yield and success rate ups and downs,and sometimes even causes "zero harvest" which makes the production costs rise. This is a serious impact on the healthy development of Hainan BC industry. Moreover, at present,Hainan BC production enterprises are also subject to cheap labor in Southeast Asia and rich raw materials to bring the serious impact of low-priced products. Therefore, it is an important proposition to study the metabolic regulation of BC, and how to select the strain/ strain combination, which plays a key role in the natural pre-fermentation. So as to replace the existing "laissez-faire" with "dominant bacteria pre-fermentation", so as to solve the practical problems of instability and quality of BC fermentation production,which is of great theoretical and practical significance to promote the healthy development of BC industry.In this study, the experiment was conducted to select the origin of coconut water,pre-fermentation time and pre-fermentation environment to explain the determinism and universality of pre-fermentation treatment to BC synthesis and to promote the difference.The analysis of conventional physical and chemical components was carried out to explore the essential differences in the pre-fermentation process. Then, the yeast and lactic acid bacteria were screened by the method of isolating the bacteria, and the sieving strain was used to simulate the natural pre-fermentation, and the strains with significant promotion effect and stable effect were initially selected. Finally, according to the general all-factor design, the strain was simulated by natural combination, and the best strain combination was screened according to BC yield. The main results are as follows:(1) Coconut water natural pre-fermentation to promote the BC synthesis of the universality and certainty of the studyThe coconut water from different habitats at home and abroad can promote BC synthesis significantly at different environments after different times of natural pre-fermentation. And coconut aquatic products, natural pre-fermentation environment and pre-fermentation time, these three conditions on the BC production growth have a greater impact. When the fresh coconut water was pre-fermented in the laboratory environment for 1-3 days, the coconut water of each origin was most significant for BC. YN had the highest increase in BC production at the second day of pre-fermentation, reaching 10.8 times of the control group (fresh coconut water). TN samples were the least to increase BC production after pre-fermentation and reached the maximum at the first day, which was 3.29 g / L and 5.1 times of that of the control group (TN). When YN was pre-fermented in the environment of coconut processing factory, the BC yield reached the highest value of 14.48 g / L on the first day and 25.0 times of that of the control group (YN), which indicated that BC production was the best choice in the coconut processing factory environment. Thus, it can be seen that the promotion effect of coconut water natural pre-fermentation treatment on BC synthesis was a common and definite fact, but the degree of promoting effect was also affected by the three factors of coconut aquatic product, pre-fermentation time and pre-fermentation environment influences.(2) Study on the correlation between pre-fermented coconut water components and BC high yieldA total of 10 samples of YN, YF-F1, YF-F2, YF-L, HN, HF, BN, BF, TN and TF were analyzed for the composition, including soluble solids, reducing sugar content, pH and total acid content of the determination, the results showed that the overall trend were basically the same.The types and contents of seven organic acids in YF-F1, YF-F2, HF and BF samples were determined by high performance liquid chromatography, which were performented for 0-9 d. The results showed that the contents of D-galacturonic acid, succinic acid and fumaric acid were significantly or extremely significant negatively correlated with BC production.Volatile components were tested for the pre-fermentation broth of the key points (peak or inflection point) for each group's sample yield changed by gas phase mass spectrometry.Through the comparative analysis of ingredients found that different origin of fresh coconut water aroma ingredients are very different. No aldehydes were detected in YN and no alcohol was detected in TN. In addition to HF 2 d and TF 1 d, the types of compounds after pre-fermentation were increased. And all the test samples contained a phenolic substance, that is 2,4-tert-butyl phenol. 2, 4-tert-butyl phenol content showed a downward trend in the fermentation process. The yield of lauric acid in YF-F1 1d with higher BC yield was about 10.75 g / L higher than that of YF-F1 3 d. The content of lauric acid in YF-F2 2d was about 13.31 g / L higher than that of YF-F2 for 4 days. But it is speculated that there is some evidence that there is some correlation between high levels of lauric acid and high efficiency synthesis.(3) Isolation, Screening and Identification of Pre - fermented Coconut WaterAccording to the results of BC production, YF-F1 had the highest effect on BC synthesis. And it was suggested that it was related to its biological strain. Therefore, the yeast and lactic acid bacteria in YF-F1, which were pre-fermented for 1 to 8 days of natural fermentation, were isolated and purified by traditional means. According to the colony morphology and morphology of the isolates, 51 strains of yeast and 12 lactic acid bacteria were isolated.(4) Screening and identification of single strain for high efficiencyThe yeast and lactobacillus strains were screened for natural pre-fermentation and then used for BC fermentation. According to the promotion of BC production and the role of the promotion of the stability of the efficient promotion of strains to screen. 18 strains of yeats were selected from 51 strains of yeasts. The results showed that there were 12 strains of Saccharomyces cerevisiae, 1 strain of Torulaspora delbrueckii, 2 strains of Clavispora lusitaniae, 1 strain of Candida tropicalis and 2 strains of Pichia galeiformis. In the 12 strains of lactic acid bacteria selected to promote the BC yield is more obvious and stable 5 strains of bacteria identification. The results showed that there were 4 strains of Lactobacillus brevis and 1 strain of Lactobacillus crustorum. According to BC production,it can be seen that the promoting effect of yeast fermentation broth on BC synthesis is generally higher than that of lactic acid bacteria pre-fermentation broth. The results showed that Saccharomyces cerevisiae, Torulaspora delbrueckii and Clavispora lusitaniae had the most significant effect on BC, and the BC yield was mostly greater than 3.00 g. The results showed that the yield of BC was only 2.00 g / L, and the difference of promoting effect between lactic acid bacteria was not significant.(5) Study on the effect of mixed bacteria simulated natural pre-fermentation on BC yieldThe effect of the single strain pre-fermentation broth on BC promoting and the promotion of stability were the primary screening criteria, as well as taking into account the characteristics of species diversity. Five strains of yeast and three strains of lactic acid bacteria were screened out among the genera. According to the general full-factor design experiment, the promotion effect of each experimental group was evaluated according to the effect of BC production promotion and the stability of the promotion. The results showed that there was no significant difference between the two groups in the fermentation of BC, and the degree of promotion was similar to that of single lactic acid bacteria, and less than the corresponding single yeast fermentation broth for the main fermentation of the BC yield. In terms of BC production in the strain combinations, the best combination was strain 117-1 (Clavispora lusitaniae) and strain 64-1 (Lactobacillus brevis) with a BC yield of 3.36 g / L, and it is 1.26 times the yield of single lactic acid bacteria 64-1 BC,which is 70% of BC production in single yeast strain 117-1 and 71% of positive control group (FCW).It can also be seen that this study mainly adopts the "strong + strong" combination of yeast and lactic acid bacteria. However, the promotion effect on BC production did not improve the effect of positive control group (natural pre-fermented coconut water) on BC production. Therefore, in order to improve the BC production, the combination of high efficiency to promote the role of screening still need to be further explored. |
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