羊肚菌液体深层发酵条件及多糖提取工艺的研究
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摘要
羊肚菌是一种珍贵的食药两用真菌,营养丰富,且羊肚菌多糖具有增强肌体免疫力、抗疲劳、抗肿瘤、抗衰老等诸多功效。目前,就有关羊肚菌液体深层发酵方面,仅研究了发酵条件对菌丝体生物量的影响,而未考虑对发酵液中胞外多糖产量的影响,在菌丝体多糖和胞外多糖提取过程中,各关键因素对多糖提取效率的影响也没有进行研究。
本研究以羊肚菌(Morchella esculenta)为材料,采用液体深层发酵技术,研究不同培养基和发酵条件对菌丝体生物量和胞外多糖产量的共同影响,并对羊肚菌的液体深层发酵工艺进行优化。研究了在菌丝体多糖和胞外多糖提取过程中,各关键因素对提取效率的影响,并对多糖提取工艺进行优化,以期为羊肚菌发酵产品,特别是发酵多糖的进一步研究提供理论基础和科学依据。主要研究结果有:
1.以发酵获取的菌丝体生物量和胞外多糖产量为指标,通过一次单因素筛选试验和两次正交试验,对碳源、氮源及无机盐进行筛选,最终确定了羊肚菌液体深层发酵的最优培养基配比为:玉米粉4.0%、葡萄糖1.0%、黄豆粉2.0%、酵母粉0.3%、KH_2PO_4 0.2%、MgSO_4 0.1%、CaSO_4 0.1%。
2.在确定最优发酵培养基的基础上,进行不同温度、起始pH、装液量、接种量、摇床转速的单因素试验,通过液体摇瓶培养,研究了各因素对菌丝体生物量和胞外多糖的影响。确定了羊肚菌摇瓶培养的最优条件为:24℃、起始pH 5.8、摇瓶装液量100ml/250ml、接种量10.0ml、摇床转速140r/min。
3.羊肚菌发酵液中菌丝体生物量与胞外多糖产量呈正相关,与氨基氮含量呈一定负相关,与还原糖含量及发酵液pH有一定的相关性。发酵第108h,菌丝体生物量最大,胞外多糖产量接近最大值,还原糖含量和蛋白质含量接近最低值,可以确定菌丝体生长、增殖已达到最佳状态,确定108h左右为发酵的终点时间。
4.在羊肚菌发酵菌丝体多糖提取过程中,对浸提比、浸提时间、浸提温度和醇析浓度这四个因素进行正交设计,确定了发酵菌丝体多糖的最佳提取工艺为:浸提温度为80℃,提取时间150min,浸提比1∶40,醇析浓度为75%,即加3倍水体积的95%乙醇沉淀后,沉淀物经无水乙醇反复洗涤后,真空冷冻干燥,得菌丝体粗多糖。在羊肚菌发酵胞外多糖提取过程中,对浓缩倍数、浓缩温度、醇析浓度和提取液pH值这四个因素进行正交设计,确定了胞外多糖的最佳提取工艺为:首先将离心去除菌丝体的滤液的pH调至6.0,80℃浓缩至原体积的1/3,冷却后加入预冷的95%乙醇使其终浓度为75%,放置4℃冰箱至少24h,4500r/m离心收集沉淀。沉淀经无水乙醇反复洗涤后,真空冷冻干燥,得胞外粗多糖。
Morchella escuIenta is one kind of valuable edible and medicial fungus,its nutrition is rich,and the polysaccharide from Morchella esculenta has a wide variety of medicinal activities,such as enhancing body immunity,anti-fatigue and anti-tumor, anti-aging and many other effects.On the area of the submerged fermentation of Morchella esculenta,it studied the impacts of the fermentation conditions on the mycelia biomass,but the impacts of the fermentation conditions on the exopolysaccharide were not studied.And the impacts of many key factors were not studied in the extraction of intracellular polysaccharide and exopolysaccharide.
In this study,the experiment studied the materials of Morchella esculentar,using submerged fermentation technology.The impacts of different fermentation conditions on mycelia biomass and exopolysaccharide yield were studied.The process of submerged fermentation of Morchella esculentar was optimized.The impacts of many key factors were also studied in the extraction of intracellular polysaccharide and exopolysaccharide.The process of extraction of polysaccharide and exopolysaccharide was optimized,too.It was hoped to provide a theoretical and scientific basic for the further studies of fermentation products,especially the exopolysaccharide from submerged fermentation of MorcheIla esculentar.The results were as follows:
1.The optimal ratio of carbon sources,nitrogen sources and inorganic salts,was identified through a single factor screening test and the two orthogonal tests, accessing to the mycelia biomass and exopolysaccharide yield.The optimal medium ratio of the submerged fermentation was as follows:corn powder 4.0%,glucose 1.0% soybean meal 2.0%,yeast extract 0.3%,KH_2PO_4 0.2%,MgSO_4 0.1%CaSO_4 0.1%.
2.The impacts of different factors on the mycelia biomass and exopolysaccharide yield,as temperature,pH,liquid volume and inoculum amount, rotation speed,,were studied on the basic of the optimal fermentation medium. Results of the test showed that the optimal culture conditions was as follows:24℃, initial pH 5.8,the liquid volume 100ml/250ml,inoculating amount 10.0ml,rotation speed 140r/min.
3.It was found that the mycelia biomass was positively correlated with the exopolysaccharide yield,was negative correlated with the content of amino nitrogen, and had some relevance with the residual sugar concentration and the pH.In the 108h, mycelia biomass reached the maximum value,the exopolysaccharide yield reached the maximum value nearly,and the content of residual sugar and protein reached the minimum value nearly.It could be identified that the growth of mycelia had reached the best condition,and determined 108h around for the end time of fermentation.
4.Four factors,extraction ratio,time,temperature and ethanol concentration, were studied in the process of polysaccharides extraction from mycelia of Morchella esculenta.The suitable process was determined as follows:80℃,150min,add water of 40 times of mycelia,and then precipitated at a final 75%ethanol at 4℃overnight. The pellets were harvested by centrifuge,washed several times with 95%ethanol,and the polysaccharides from mycelia could be gained.
Four factors,concentration ratio,temperature,ethanol concentration and pH, were studied in the process of exopolysaccharide extraction.The suitable process for exopolysaccharide extraction was determined as follows:pH of ferment liquid was changed to 6.0,concentrated to 1/3 volume at 80℃and then precipitated at a final 75%ethanol at 4℃overnight.The pellets were harvested by centrifuge,washed several times with 95%ethanol,and the exopolysaccharide could be gained.
本研究以羊肚菌(Morchella esculenta)为材料,采用液体深层发酵技术,研究不同培养基和发酵条件对菌丝体生物量和胞外多糖产量的共同影响,并对羊肚菌的液体深层发酵工艺进行优化。研究了在菌丝体多糖和胞外多糖提取过程中,各关键因素对提取效率的影响,并对多糖提取工艺进行优化,以期为羊肚菌发酵产品,特别是发酵多糖的进一步研究提供理论基础和科学依据。主要研究结果有:
1.以发酵获取的菌丝体生物量和胞外多糖产量为指标,通过一次单因素筛选试验和两次正交试验,对碳源、氮源及无机盐进行筛选,最终确定了羊肚菌液体深层发酵的最优培养基配比为:玉米粉4.0%、葡萄糖1.0%、黄豆粉2.0%、酵母粉0.3%、KH_2PO_4 0.2%、MgSO_4 0.1%、CaSO_4 0.1%。
2.在确定最优发酵培养基的基础上,进行不同温度、起始pH、装液量、接种量、摇床转速的单因素试验,通过液体摇瓶培养,研究了各因素对菌丝体生物量和胞外多糖的影响。确定了羊肚菌摇瓶培养的最优条件为:24℃、起始pH 5.8、摇瓶装液量100ml/250ml、接种量10.0ml、摇床转速140r/min。
3.羊肚菌发酵液中菌丝体生物量与胞外多糖产量呈正相关,与氨基氮含量呈一定负相关,与还原糖含量及发酵液pH有一定的相关性。发酵第108h,菌丝体生物量最大,胞外多糖产量接近最大值,还原糖含量和蛋白质含量接近最低值,可以确定菌丝体生长、增殖已达到最佳状态,确定108h左右为发酵的终点时间。
4.在羊肚菌发酵菌丝体多糖提取过程中,对浸提比、浸提时间、浸提温度和醇析浓度这四个因素进行正交设计,确定了发酵菌丝体多糖的最佳提取工艺为:浸提温度为80℃,提取时间150min,浸提比1∶40,醇析浓度为75%,即加3倍水体积的95%乙醇沉淀后,沉淀物经无水乙醇反复洗涤后,真空冷冻干燥,得菌丝体粗多糖。在羊肚菌发酵胞外多糖提取过程中,对浓缩倍数、浓缩温度、醇析浓度和提取液pH值这四个因素进行正交设计,确定了胞外多糖的最佳提取工艺为:首先将离心去除菌丝体的滤液的pH调至6.0,80℃浓缩至原体积的1/3,冷却后加入预冷的95%乙醇使其终浓度为75%,放置4℃冰箱至少24h,4500r/m离心收集沉淀。沉淀经无水乙醇反复洗涤后,真空冷冻干燥,得胞外粗多糖。
Morchella escuIenta is one kind of valuable edible and medicial fungus,its nutrition is rich,and the polysaccharide from Morchella esculenta has a wide variety of medicinal activities,such as enhancing body immunity,anti-fatigue and anti-tumor, anti-aging and many other effects.On the area of the submerged fermentation of Morchella esculenta,it studied the impacts of the fermentation conditions on the mycelia biomass,but the impacts of the fermentation conditions on the exopolysaccharide were not studied.And the impacts of many key factors were not studied in the extraction of intracellular polysaccharide and exopolysaccharide.
In this study,the experiment studied the materials of Morchella esculentar,using submerged fermentation technology.The impacts of different fermentation conditions on mycelia biomass and exopolysaccharide yield were studied.The process of submerged fermentation of Morchella esculentar was optimized.The impacts of many key factors were also studied in the extraction of intracellular polysaccharide and exopolysaccharide.The process of extraction of polysaccharide and exopolysaccharide was optimized,too.It was hoped to provide a theoretical and scientific basic for the further studies of fermentation products,especially the exopolysaccharide from submerged fermentation of MorcheIla esculentar.The results were as follows:
1.The optimal ratio of carbon sources,nitrogen sources and inorganic salts,was identified through a single factor screening test and the two orthogonal tests, accessing to the mycelia biomass and exopolysaccharide yield.The optimal medium ratio of the submerged fermentation was as follows:corn powder 4.0%,glucose 1.0% soybean meal 2.0%,yeast extract 0.3%,KH_2PO_4 0.2%,MgSO_4 0.1%CaSO_4 0.1%.
2.The impacts of different factors on the mycelia biomass and exopolysaccharide yield,as temperature,pH,liquid volume and inoculum amount, rotation speed,,were studied on the basic of the optimal fermentation medium. Results of the test showed that the optimal culture conditions was as follows:24℃, initial pH 5.8,the liquid volume 100ml/250ml,inoculating amount 10.0ml,rotation speed 140r/min.
3.It was found that the mycelia biomass was positively correlated with the exopolysaccharide yield,was negative correlated with the content of amino nitrogen, and had some relevance with the residual sugar concentration and the pH.In the 108h, mycelia biomass reached the maximum value,the exopolysaccharide yield reached the maximum value nearly,and the content of residual sugar and protein reached the minimum value nearly.It could be identified that the growth of mycelia had reached the best condition,and determined 108h around for the end time of fermentation.
4.Four factors,extraction ratio,time,temperature and ethanol concentration, were studied in the process of polysaccharides extraction from mycelia of Morchella esculenta.The suitable process was determined as follows:80℃,150min,add water of 40 times of mycelia,and then precipitated at a final 75%ethanol at 4℃overnight. The pellets were harvested by centrifuge,washed several times with 95%ethanol,and the polysaccharides from mycelia could be gained.
Four factors,concentration ratio,temperature,ethanol concentration and pH, were studied in the process of exopolysaccharide extraction.The suitable process for exopolysaccharide extraction was determined as follows:pH of ferment liquid was changed to 6.0,concentrated to 1/3 volume at 80℃and then precipitated at a final 75%ethanol at 4℃overnight.The pellets were harvested by centrifuge,washed several times with 95%ethanol,and the exopolysaccharide could be gained.
引文
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