Study On The Effect Of Fungi From Silkworm Rearing Environment On The PH Value Of Artificial Diet Products

To illustrate the effect of the fungi on artificial diet in the silkworm rearing environment,this study adopts conventional culture-dependent technology to isolate and identify different fungal species from the silkworm feed and feces under the artificial diet rearing condition,and study the growth of different representive isolated strains in potato dextrose broth medium and different silkworm artificial diet products.Pichia kudravzevii was used to explore the factors and mechanisms affecting the pH of silkworm artificial diet.The summary is as follows.1.This study adopts conventional culture-dependent technology to isolate and identify seven different fungal species from the silkworm feed and feces under the artificial diet rearing condition.These strains were identified as Geotrichum candidum,Candida sp.and Pichia kudravzevii of yeasts,and Aspergillus tamarii,Aspergillus flavus,Neurospora sp.and Aspergillus niger of mould using morphology and ITS sequence.Two strains fungi isolated from the mulberry leaves of the mulberry field in Zijingang Campus of Zhejiang University were used as reference and were identified as Asperigillus aculeatinus and Mucor sp..2.The growth pH range of the isolated strains in PDB medium with different pH,the growth pattern in acid PDB medium,the growth and reproduction on three selected artificial diets(AD,AD1 and AD2)and the changes in feed pH were investigated.The results showed that the isolated strains can grow in a liquid medium with pH 2.00 to 10.00.Notably,yeasts are more suitable for neutral partial acid,so that the pH of the acidic medium changed in a smaller range but molds are more suitable for alkaline conditions,which significantly changed the pH of the acidic medium.In all the three artificial diets,the Candida sp.did not grow;the Neurospora sp.only grew on the AD diet;the other isolated strains could grow on the three diets,the total ATP increased significantly and most reached the highest value on the 5th day.A.niger can decrease the pH value of AD,AD1 and AD2 diets to 4.15 ± 0.32(P=0.021),4.04 ± 0.07(P=0.000)and 4.63±0.32(P=0.005)respectively.The remaining strains all increased the diet pH significantly.A.flavus had the most significant impact,which increased the pH of the AD2 to 8.40±0.22(P=0.002)after 15 days of cultivation.3.Pichia Kudravzevii strain was inoculated into silkworm artificial feed(AD3),which can increase the diet pH to 7.5 in 5 days.When being inoculated to the liquid YPD with different pH and different glucose contents,P.kudravzevii adapted to the change of medium pH,and alkalized culture medium,increasing pH within 6 h and alkalizing 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 was obvious under the low glucose concentration(<0.6%),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 for different periods of culture,and the release of ammonia,the expression of alkalization related genes and the activity of glutamate dehydrogenase in the environment were investigated.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/m3(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),which was extremely significantly higher than that of control group(P<0.01).The expression levels of alkaliation-related genes GDH2,DUR1,2 and ATO3 in liquid medium were firstly increased and then decreased.The GDH2 transcription level was up-regulated at 8 h(pH 3.00),4 h(pH 5.00)and 8 h(pH 7.00),and was extremely significantly higher than that of control group(P<0.01),and then decreased.The transcription level of ATO3 was significantly up-regulated at 4 h(pH 3.00),12 h(pH 5.00)and 8 h(pH 7.00)(P<0.05),and then decreased.DUR1,2 transcription levels at 8 h were extremely significantly higher than those of control group(P<0.01).The alkalization degree after adding antimycin A decreased or even abolished.