家蚕人工饲料来源毕赤酵母菌的分离及对饲料酸碱度的影响

Isolation of Pichia kudravzevii from Silkworm Artificial Diet and Its Effect on Feed pH

为探明真菌繁殖对家蚕人工饲料酸碱度的影响,采用常规分离纯化技术,从人工饲料育养蚕环境的蚕座上获得1株可碱化饲料的真菌,经形态学和ITS序列鉴定,为库德里阿兹威氏毕赤酵母(Pichia kudravzevii)。该菌株接种至家蚕人工饲料,使饲料pH在5 d内由初始值上升至7.54;接种至不同pH和葡萄糖含量的液体YPD中,其能适应繁殖基质pH的变化,碱化培养基质,使酸性培养基pH在6 h内升高,在24 h内上升到7.86(pH 5.00)和8.26(pH 6.00);培养基质中葡萄糖含量影响碱化,低浓度葡萄糖(<0.6%)培养基碱化现象明显,高浓度葡萄糖(>1.2%)培养基碱化现象消失。菌株接种至pH为3.00、5.00和7.00的不含葡萄糖YPD培养基中培养不同时间,对环境中氨气释放量、碱化相关基因表达及谷氨酸脱氢酶活性进行研究,结果表明,与添加2%葡萄糖相比,固体培养基中氨气释放量在培养24 h(pH 5.00)和4 h(pH 7.00)时极显著上升(P<0.01),随后持续升至最高值280 mg/m^(3)(pH 7.00);谷氨酸脱氢酶活性逐渐升高,在12 h(pH 5.00)和24 h(pH 7.00)时达到最大值53.84 U/mL和24.84 U/mL,极显著高于对照组(P<0.01);液体培养基中碱化相关基因GDH2、DUR1,2和ATO3表达水平呈先升高后降低趋势,GDH2转录水平分别在8 h(pH 3.00)、4 h(pH 5.00)和8 h(pH 7.00)上调最高,极显著高于对照组(P<0.01),随后下降,ATO3转录水平分别在4 h(pH 3.00)、12 h(pH 5.00)和8 h(pH 7.00)显著上调,且最高(P<0.05),然后下降,DUR1,2转录水平均在8 h极显著高于对照组(P<0.01)。添加抗霉素A,碱化程度下降甚至消失。该研究可为解明家蚕人工饲料的保鲜机制及研发保鲜技术提供参考。

To illustrate the effect of the fungi on the changes of pH value of silkworm artificial diet,we isolated a strain of fungus,which can alkalize the diet,from the artificial diet by conventional isolation and purification technology.The strain was identified as Pichia kudravzevii by following morphology and ITS sequencing.It could increase the diet pH to 7.54 in 5 d after being inoculated to artificial diet.When inoculated to the liquid YPD medium with different pH and different glucose contents,P.kudravzevii could adapt to the change of medium pH,and alkalize the culture medium,increasing the pH within 6 h and alkalizing the medium to 7.86(pH 5.00)and 8.26(pH 6.00)within 24 h respectively.Glucose content of the culture medium affected the alkalization.Alkalization of the medium with low glucose concentration(<0.6%)was obvious,while it disappeared under the high glucose concentration(>1.2%).The strain was inoculated into glucose-free YPD medium with pH of 3.00,5.00 and 7.00 respectively for different periods of culture time,and the release of ammonia,the expression of alkalization-related genes and the activity of glutamate dehydrogenase in the environment were investigated.The results showed that compared with the addition of 2%glucose,the release of ammonia in solid medium increased extremely significantly at 24 h(pH 5.00)and 4 h(pH 7.00)(P<0.01),and then continued to reach the highest value of 280 mg/m^(3)(pH 7.00).The activity of glutamate dehydrogenase increased gradually and reached the maximum value 53.84 U/mL and 24.84 U/mL at 12 h(pH 5.00)and 24 h(pH 7.00),respectively,which was extremely significantly higher than that of the control group(P<0.01).The expression levels of alkaliation-related genes GDH 2,DUR 1,2 and ATO3 in liquid medium were first increased and then decreased.The transcription levels of GDH2 were up-regulated to the maximum at 8 h(3.00),4 h(5.00)and 8 h(7.00),respectively,which were extremely significantly higher than those of the control group(P<0.01),and then decreased.The transcription levels of ATO 3 were significantly up-regulated to the maximum at 4 h(3.00),12 h(5.00)and 8 h(7.00)(P<0.05),and then decreased.The transcription levels of DUR1,2 were extremely significantly higher than those of the control group(P<0.01).The alkalization degree after adding antimycin A decreased or even abolished.This research provides a reference for the research and development of preservation and fresh-keeping technology of silkworm artificial diet.