Screening And Identification Of Chitinase Producing Strain And Its Possibility For Inhibiting Moulds Growth Of Corn Stalk

Mechanized bale is a major way to harvest and store corn stalk, but the problem of corn stalk bale mildew is a great risk to dairy cow and other ruminant animals, caused by changeable weathers, high moisture content and mixed mud. Chemical fungicides commonly used such as propionic acid, propionate, calcium oxide are restricted in the applic ation of corn stalk mouldproof by price, addition amount, safety factors. Producing chitinase Bacillus subtilis BS-1, acid producing Lactobacillus plantarum LABP and dominant moulds in corn stalk were used to investigating the microbial method for inhibiting moulds, and provide a reference for the prolongation of storage period and improvement of utilization efficiency.A chitinase producing strain of BS-1 was screened from corn stalk using colloidal chitin medium. At the same time, the dominant moulds were isolated and purified. The identification of strain BS-1 and dominant moulds were carried out through morphological observation and 16 S r DNA or 18 S r DNA sequence analysis. In addition, the chitinase activity of strain BS-1 was assayed using 3, 5-Dinitrosalicylic acid(DNS) colorimetric method after 24 h, 48 h, 72 h, 96 h, 120 h, 144 h and 168 h of fermentation and the inhibition action of strain BS-1 to dominant moulds was detected by oxford cup method. In order to evaluate the utilization of corn stalk, the BS-1 or/and LABP was inoculated in the corn stalk medium, respectively. Factorial design was used in this experiment, the corn stalk with moisture content followed by 15%, 20% and 25%, each moisture content of corn stalk was treated with BS-1, LABP and their compound strains, with 0.5% propionate as positive control, no treatment for blank control. Exposing to the air after vacuum stored for 30 days, aerobic stability, nutrient contents and the number of moulds in corn stalk were detected. The results showed as follows:(1) The strain BS-1 isolated from corn stalk was Bacillus subtilis and four dominant moulds were Mucor circinelloides, Fusarium oxysporum, Aspergillus oryzae, Aspergillus niger, respectively.(2) After 120 h of fermentation at 37℃, the highest chitinase activity of strain BS-1 was obtained to be 3.23U/m L.(3) The fermentation liquid of strain BS-1 showed obvious inhibition to four dominant moulds from corn stalk, the diameter of the inhibition zone was 18.13 mm、18.48mm、17.55mm、15.68 mm, respectively.(4) In the early of corn stalk mildew, DM and starch content decreased significantly(P(27)0.05), and CP, NDF content showed a rising trend, but there was no significantly effect on the ADF(P(29)0.05). The nutritional index variation amplitude increases with the aggravation of mildew, the relative loss rate of starch was the highest especially.(5) When the moisture content of corn stalk was 20%, corn stalk treated with compound strains showed shorter aerobic stabilization time than propionic acid treatment, no significant difference(P(29)0.05), but significantly longer than the control(P(27)0.05). When the moisture content increased to 25%, all treatments have suffered serious mildew, there were obvious visible mildew spots.(6) When the moisture content of corn stalk was 20%, the moulds quantity in the stalk was 104cfu/g after treated with compound strains, while the blank control reached 106cfu/g. when the moisture content increased to 25%, the moulds quantity in the experimental groups and control group were all reached 106cfu/g.The following conclusions are obtained from the above experimental results. The chitinase producing strain BS-1 can effectively inhibit growth of Mucor circinelloides, Fusarium oxysporum, Aspergillus oryzae, Aspergillus niger. When the moisture content of corn stalk was 20%, compound strains with 107cfu/g could inhibit the growth of moulds in corn stalk, the aerobic stability of corn stalk was increased, the loss rate of nutrient contents and moulds quantity were decreased, the effects were similar to 0.5% propionate treatment. There were no significantly effects on control the moulds, when the moisture content increased to 25%.