【作者】 龚梦琳；

【导师】 刘四新； 吴毓炜；

【作者基本信息】 海南大学 ， 食品加工与安全， 2023， 硕士

【摘要】 椰纤果(Nata de coco)是细菌纤维素(Bacterial Cellulose,BC)在食品工业应用时的商业名称。椰纤果的发酵生产采用的是浅盘、堆叠的发酵方式简单粗放,使得生产菌种没有得到标准化管理,事实上生产菌种是易受污染的多菌共存的混杂体系,且接种后的发酵环境随季节、气候变化,导致发酵时共存微生物的种类数量也随之变化无常,这成为目前BC发酵生产很不稳定,有时低产甚至失败的原因之一。本研究以BC工厂的生产菌种为研究对象,对其中的各微生物进行分离纯化、筛选与BC产生菌共培养能促进BC合成的“配角菌”,然后研究它们之间共培养的接种比例、接种顺序和发酵温度等条件,建立起与有效配角菌共存、并可高效促进BC合成、而不受无效或抑制性杂菌影响的共培养混合菌种,最后通过不同条件下的验证,构建出稳定、高效、能使BC发酵生产免受环境波动影响的共培养菌种体系,为BC的可持续工业化生产探索可能的“卡脖子”问题,也是解决工业发酵中的“种业”问题,同时也为课题组深入研究BC合成的代谢调控提供高价值的研究材料和研究模型。主要研究结果如下:(1)生产菌种的发酵特性:对来自工厂不同批次的生产菌种以不同的接种方式进行传代发酵培养,以验证课题组前期研究结果,显示针对混杂不纯的生产菌种确实以膜内菌液接种时的BC产量高于膜下菌液接种方式,初步表明膜内菌液和膜下菌液的菌相组成不同,且对BC合成的主发酵产生重要影响。(2)生产菌种中各微生物的分离纯化及BC高产菌株的筛选鉴定:对先后三批次的生产菌种进行BC菌的分离纯化,并对其中较优的35株菌中形态典型或BC高产的20株菌进行分子生物学鉴定,结果均为椰冻驹形氏杆菌(Komagataeibacter nataicola,简作Kn),它们中如JG15、JG242、JG161为较高产菌株,日均产量均在2.3-2.5 g/L·d以上(于FCW)。分离工作中未见菌体和菌落形态特征与BC产能间存在必然联系。通过干重产量的多次比较试验,选出产能较高且稳定的菌株JG15、JG242,日均产量2.4 g/L·d以上,和产能最低的菌株JG11日均产量1.0 g/L·d以下,进行下一步共培养筛选研究。(3)生产菌种中不产BC微生物的纯化及促进性菌株的初筛:从先后各批次生产菌种中分离筛选得到约200株不产BC的杂菌,先后进行了三轮共培养促进效果筛选试验。最终确定促进效果达显著的杂菌共13株,如GPc4114、GPc6111、FGm1、FGp3等。(4)基于高产BC菌株的促进性配角菌的复筛及鉴定:将所筛获的高产菌株JG15与前述各配角菌分别在新鲜椰子水培养基和实验室预发酵培养基中进行共培养促进效果筛选。最终选出促进效果极显著的一株细菌GPc4114和一株酵母菌FGm1,经对多株主角菌(JG15、JG33、JG242、JG11)进行促进效果验证、都显示能达到显著影响,故选定该两株菌作为后续构建高效菌种体系的配角菌。经种属鉴定:GPc4114为植物乳杆菌(Lactobacillus plantarum),FGm1为酿酒酵母(Saccharomyces cerevisiae)。(5)高效共培养条件优化:基于高产主角菌与高效配角菌在不同接种顺序、不同接种比例、不同培养温度情况下进行共培养发酵试验,结果显示:与细菌共培养的最佳条件是主角菌与配角菌同时接种、接种比例为活菌数比1:10?、发酵温度30oC,共培养促产BC效果最好,约为1.43倍(于FCW);与酵母菌共培养的最佳条件是主角菌与配角菌也是同时接种、接种比例为活菌数比1:10?、发酵温度28～30oC,共培养促产BC效果最好,约为1.39倍。(6)稳定高效的共培养菌种体系构建及验证:对JG15:GPc4114以活菌数1:10?进行同时混合接种、30oC下培养,以此构建成细菌共培养菌种体系;对JG15:FGm1以活菌数1:10?进行同时混合接种、28～30oC下培养,以此构成酵母菌共培养菌种体系。将该两个共培体系在五种不同种类培养基中进行验证,并以工厂的生产菌种FB1为阳性对照,发现构建的稳定高效共培养生产菌种体系的BC产量极显著高于纯种BC菌JG15的单菌发酵,同时与生产菌种FB1产量相当、无显著性差异;在传代稳定性方面,在工厂预发酵椰子水FG3培养基中连续传代五次,发现每代次的BC产能间无显著性差异。更多还原

【Abstract】 Nata de coco is the commercial name of Bacterial Cellulose(BC)when it is applied in the food industry.The simple and extensive fermentation method of shallow dish and stack is adopted in the fermentation production of coconut fruit,which makes the production of bacteria not standardized management.In fact,the production of seed is a hybrid system prone to contamination and multi-bacteria co-existence.Besides,the fermentation environment after inoculation changes with the seasons and climate,resulting in the variety and quantity of coexistence of microorganisms during fermentation also changes.This has become one of the reasons why BC fermentation production is very unstable,sometimes low yield and even failure.In this study,the bacteria produced in BC factory was taken as the research object,and the microorganisms were separated,purified,screened and co-cultured with BC producing seed to promote BC synthesis of "supporting bacteria",and then the co-cultured inoculation ratio,inoculation sequence,fermentation temperature and other conditions were studied.Establish a co-culture mix of bacteria that co-exist with effective supporting bacteria and can effectively promote BC synthesis without being affected by ineffective or inhibitory hybrid bacteria.Finally,through verification under different conditions,establish a stable,efficient co-culture system that can make BC fermentation production free from environmental fluctuations,and explore possible "bottleneck" problems for sustainable industrial production of BC.It is also a solution to the problem of "seed industry" in industrial fermentation,and also provides highvalue research materials and research models for the research group to further study the metabolic regulation of BC synthesis.The main research results are as follows:(1)Fermentation characteristics of production seeds: In order to verify the results of the previous research,the results showed that the BC yield of the mixed and impure production seeds inoculated with the intramembrane bacterial solution was indeed higher than that of the submembrane bacterial solution inoculated with the intramembrane bacterial solution,which preliminarily indicated that the bacterial phase composition of the intrambrane bacterial solution and the submembrane bacterial solution was different.It also has important influence on the main fermentation of BC synthesis.(2)Isolation and purification of microorganisms in production seeds and screening and identification of high yield BC strains: Three batches of production strains were isolated and purified by BC bacteria,and 20 strains with typical morphology or high yield of BC among the 35 strains were identified by molecular biology.The results showed that all strains were Komagataeibacter nataicola(Kn).Among them,JG15,JG242 and JG161 were high yielding strains,with average daily yield above 2.3-2.5 g/L·d(FCW).There was an inevitable correlation between the morphology characteristics of bacteria and colony and BC productivity.Through multiple comparative tests on dry weight yield,strains JG15 and JG242 with high and stable productivity and average daily yield above 2.4 g/L·d were selected,and strains JG11 with the lowest productivity were selected with average daily yield below 1.0 g/L·d for further co-culture screening study.(3)Purification of non-BC producing microorganisms in production seeds and preliminary screening of promoting strains: About 200 strains without BC production were isolated and screened from each batch of production strains,and three rounds of co-culture promoting effect screening tests were conducted.Finally,a total of 13 strains with significant promoting effect were determined,such as GPc4114,GPc6111,FGm1,FGp3,etc.(4)Re-screening and identification of promoting supporting bacteria based on High-yielding BC Strain: The high-yielding strain JG15 and the aforementioned supporting strains were co-cultured in fresh coconut water medium and laboratory prefermentation medium respectively to screen the promoting effect.Finally,a bacterium GPc4114 and a yeast FGm1 with very significant promoting effect were selected.After the promotion effect verification of several leading strains(JG15,JG33,JG242,and JG11),they all showed that they could achieve significant influence.Therefore,these two strains were selected as supporting bacteria in the subsequent construction of efficient strain system.Species identification: GPc4114 is Lactobacillus plantarum,and FGm1 is Saccharomyces cerevisiae.(5)Optimization of high efficiency co-culture conditions: With bacteria cultured best supporting conditions is main bacteria and fungus at the same time,inoculated proportion for the number of living bacterium than 1:10 ? and fermentation temperature30 oC,best trained for BC production effect,about 1.43 times(in FCW);With yeast culture character is the best condition of bacteria and supporting bacteria is also at the same time,inoculated proportion for the number of living bacterium than 1:10 ?,fermentation temperature 28 ～ 30 oC,best trained for BC production effect,which is about 1.39 times.(6)Stable and efficient strains of co-culture system building and validation: For JG15: GPc4114 with the number of living bacterium 1:10 ? mixed inoculation,30 oC culture at the same time,in order to build into a bacterial seeds of co-culture system;For JG15: FGm1 with the number of living bacterium 1:10 ? mixed inoculation,28～30oC culture at the same time,to form yeast sseeds of co-culture system.The two coculture systems were verified in five different kinds of culture medium,and the production strain FB1 in the factory was used as the positive control.It was found that the stable and efficient co-culture production system was significantly higher than the single bacteria fermentation of pure strain BC JG15,and the yield was comparable to that of production strain FB1,but had no significant difference.In terms of the stability of passage,there was no significant difference in BC productivity of each generation after five consecutive passages in factory pre-fermented coconut water FG3 medium.更多还原