Isolation And Screening Of Tannic Acid And Phytic Acid Degradation Microorganism And Optimization Of Conditions For Solid State Fermentation In Double-low Rapeseed Meal

A series of experiments were conducted to isolate and screen microorganism withthe capacity of degrading tannic acid and phytic acid from soil, to evaluate thecapacity of isolates on degradation tannic acid and phytic acid in double-low rapeseedmeal, and to elucidate the optimum moisture, pH, temperature and minerals ofsolid-state fermentation in double-low rapeseed meal. Results of studies werepresented as follows:1. Enrichment-culture technique was used to isolate and screen efficientmicroorganisms that degraded both tannic acid and phytic acid from soil. As a result,109isolates of microorganisms were isolated, of which42isolates （including13isolates of bacteria,22isolates of yeasts and7isolates of fungi） exhibit phytic aciddegradation potential and18isolates （including6isolates of bacteria，1isolate ofyeast and11isolates of fungi） exhibit tannic acid degradation potential. Among theseisolates, there are5isolates of fungi （M-6、M-3、 M-1、3.951、3.316） and1isolateof yeast （2.120） possessed the capacity of degradation in both tannic acid and phyticacid.2. Studies were conducted to evaluate the capacity of isolates M-6、M-3、M-1、3.951、3.316and2.120on tannic acid and phytic acid degradation in rapeseed meal,during the solid-state fermentation process （temperature30℃, moisture100%, pH6.6-7.0）, and one non-inoculated group was taken as the control.All groups wererandomly assigned to triplicate.Each treatment was collected on the1st,2nd,4th and7th day, seperately. The concentration of tannic acid and phytic acid was detected inall72treatments before and after the fermentation. Results indicated that all3Aspergullus niger significantly degraded tannic acid and phytic acid compared to the control group （P<0.05）. And the highest degradation was occurred in Aspergullusniger M-6fermentated1d （P<0.05）.3. The experiments were designed to optimize the conditions of the solid-statefermentation on tannic acid and phytic acid degradation in Double-low rapeseed meal.Seven moisture levels （50%,75%,100%,150%,200%,250%and300%）, sixtemperature levels （10℃,15℃,20℃,25℃,30℃and35℃） and eight pH levels （3,4,5,6,7,8,9and10） were set up in solid-state fermentation procedure, respectively.Each moisture, temperature and pH level was assigned to triplicate. The resultsshowed that the tannic acid and phytic acid degradation of M-6fermented in themoisture200%, temperature25℃and pH6for1d was significantly higher than allother treatments （P<0.05）. Further more, the effect of glucose （20g/l，1ml）, urea （10g/l,1ml） and minerals mixtures （NaNO₃2g/l, MgSO4·7H₂O1g/l, KH₂PO₄2g/l, NaCl1g/l, FeSO₄2g/l, MnSO40.6g/l and CuSO40.4g/l,1ml） were supplemented inAspergullus niger M-6fermentation for1d. The results indicated that thesupplementation of urea enhanced detoxification of tannic acid significantly （P<0.05）,while the glucose and minerals significantly reduced the concentration of phytic acid（P<0.05）. And, the percentages of tannic acid and phytic acid in Double-low rapeseedmeal decreased from1.32%to0.28%and2.81%to0.63%separately, after1dfermentation of Aspergullus niger M-6at25℃，pH6，moisture200%and additionwith urea, glucose and minerals mixture. Besides, Aspergullus niger M-6couldincrease the content of crude protein from37.16%to43.48%and the digestible crudeprotein in vitro from75.32%to79.15%in the optimal fermentation.4. The study was carried out to characterize the M-6by hyphae and clone., and theisolate M-6was characterized as Aspergillus niger. Comparisons of sequences of theITS1-5.8S-ITS2ribosomal DNA region of the most similar species strongly supportedthis conclusion. The activities of tannase and phylase of M-6at different pH （4,5,6and7） and temperatures （20℃,25℃,30℃and35℃） were studied. The resultshowed that the activities of tannase and phylase are5.97±0.12U/ml and4.47±0.03U/ml, separately, which are significantly higher at pH5than other treatments （P<0.05）. And the activities of tannase and phylase at30℃are5.13±0.03U/ml and4.33±0.12U/ml, separately, which are significantly higher than other temperatures（P<0.05）.