产酸芽孢杆菌的分离筛选及对玉米秸秆的微贮效果

发布时间：2018-07-01 08:41

本文选题：玉米秸秆 + 青贮　；参考：《河北农业大学》2015年硕士论文

【摘要】：青贮是秸秆饲料化利用较为成熟的技术,可以最大限度地保存秸秆中的营养物质,为畜牧养殖业提供较为廉价优质的饲草。目前,用于秸秆青贮的微生物添加剂主要是乳酸菌类,然而乳酸菌不耐高温,在生产和贮存过程中易失活,质量不稳定,影响了其使用效果。针对这一问题,本研究拟分离筛选可以发酵产酸的芽孢杆菌菌株,制备微贮菌剂,用于鲜玉米秸秆的贮存。首先从牛粪等样品中共分离得到了芽孢菌菌株200余株,利用变色圈法从中初筛出13株可使溴甲酚紫变黄的产酸菌株。利用平板培养基打孔法对这13株菌进行了复筛,最终获得1株产酸量较高的芽孢杆菌R42-16菌株。该菌株可以发酵糖产生多种有机酸,其中乙酸、乳酸的产量较高,液体发酵条件下分别为220.70μg/m L、174.25μg/m L。根据菌落形态、菌体特征、生理生化试验、以及16S r DNA序列分析,菌株R42-16被鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。对R42-16菌株的生物学特性进行研究发现,该菌株对酸、胆酸盐具有较强的耐受性,耐受p H的范围为1.0~7.0,耐受胆酸盐的范围为0.1%~0.3%;该菌株对蔗糖、果糖的利用率均较高;同时,菌株R42-16对致病大肠杆菌O157:H7具有较强的拮抗活性。通过单因素试验和正交试验,对菌株R42-16的培养基组成和培养条件进行了优化。优化培养基为:玉米粉1.0%、酵母浸粉2.0%、Mn SO4·H2O 0.01%、Ca Cl2·H2O 0.01%;适宜的培养条件为:培养基p H6.0、装液量为50m L/250m L三角瓶、接种量为4.0m L/瓶、发酵时间为48h。在此培养条件下,发酵液中菌株R42-16的活菌数为3.19×109CFU·m L-1,芽孢形成率为93.76%。对菌株R42-16对玉米秸秆的发酵效果进行了评价。设定菌剂施用量分别为8.0×108 CFU/kg(试验组Ⅰ)、1.0×109 CFU/kg(试验组Ⅱ)和1.2×109 CFU/kg(试验组Ⅲ),发酵30d,对各试验组秸秆的营养指标对比如下:与青贮对照组相比,试验组Ⅰ中的乳酸、乙酸含量差异不显著(p0.05),试验组Ⅱ、试验组Ⅲ中两者的含量均显著增加(p0.05),试验组Ⅱ、试验组Ⅲ的乳酸含量分别提高24.38%、25.0%,乙酸含量分别提高292.87%、338.52%。但试验组Ⅱ和试验组Ⅲ之间差异不显著(p0.05)。试验组Ⅰ中的丁酸含量、氨态氮/总氮与对照组相比差异不显著(p0.05),而试验组Ⅱ、试验组Ⅲ与对照组相比,显著降低(p0.05),试验组Ⅱ、试验组Ⅲ的丁酸含量分别降低了47.34%、53.36%;氨态氮/总氮分别降低了36.89%、42.33%。但试验组Ⅱ、试验组Ⅲ之间差异不显著(p0.05)。试验组Ⅰ的粗蛋白含量与对照组相比,差异不显著(p0.05),试验组Ⅱ、试验组Ⅲ与对照组相比,含量显著增加(p0.05),试验组Ⅱ、试验组Ⅲ的粗蛋白含量分别为8.43%、9.11%,与对照组相比分别增加了50.26%、62.39%。但是,各试验组的干物质、粗纤维、中性洗涤纤维、酸性洗涤纤维、酸性洗涤木质素含量等指标差异不显著(P0.05)。针对青贮秸秆暴露在空气中(开窖后)容易产生“二次发酵”的问题,本文从物料温度、p H、气味感官等方面对发酵秸秆进行了有氧稳定性的测定。结果表明,在室温为18℃的条件下,三个试验组和青贮对照组在第1-8天时料温均接近于室温。对照组在第9天时料温开始上升,第10天时料温高于室温1.57℃,此时青贮秸秆开始呈现不稳定状态,第14天时料温高于室温3.09℃,呈现“二次发酵”症状。而此时试验组的料温仅高于室温1.1℃左右。测定秸秆的p H变化发现,在第1-8天时青贮对照组与试验组的p H相差均不大,对照组p H在第10-12天时上升较快,在第12天时已经达到了5.59。一般认为秸秆的p H达到5.0时即发生了“二次发酵”,当p H为5.5时秸秆不能再饲喂。但试验组Ⅰ、Ⅱ、Ⅲ在第15天时的p H分别为4.23、4.03、4.03。表明在秸秆中添加菌株R42-16能够提高发酵玉米秸秆的稳定性。综上所述,菌株R42-16可以作为微生物添加剂应用于玉米秸秆的微贮。
[Abstract]:Silage is a mature technology for straw feed utilization. It can preserve the nutrients in straw to the maximum extent and provide cheap and high quality forage for animal husbandry. At present, the main microbial additives used in straw silage are lactic acid bacteria. However, lactic acid bacteria are not resistant to high temperature and are easily inactive during production and storage, and the quality is not good. In order to solve this problem, this study aims to isolate and screen Bacillus spore producing Bacillus that can ferment acid, and prepare microorganism for the storage of fresh corn straw. First, more than 200 strains of Bacillus are isolated from cow dung and other samples. 13 strains of brominated mresol can be yellowed from the medium of color change circle. The 13 strains of bacteria were screened by plate culture medium drilling method, and 1 strains of Bacillus R42-16 with high acid content were obtained. The strain can ferment sugar to produce a variety of organic acids, of which the yield of acetic acid, lactic acid is higher, the liquid fermentation conditions are 220.70 mu g/m L, 174.25 Mu g/m L. according to colony morphology and bacteria. Characteristics, physiological and biochemical tests and 16S R DNA sequence analysis, strain R42-16 was identified as Bacillus amyloid (Bacillus amyloliquefaciens). The study of the biological characteristics of the strain of R42-16 found that the strain had strong tolerance to acid, cholate, the range of tolerance to P H was 1.0~7.0, and the range of tolerance to cholate was 0.1%~0.3%; The utilization of sucrose and fructose was high. At the same time, the strain R42-16 had strong antagonistic activity to the pathogenic Escherichia coli O157:H7. Through single factor test and orthogonal test, the medium composition and culture conditions of strain R42-16 were optimized. The optimum medium was as follows: corn flour 1%, yeast extract 2%, Mn SO4. H2O 0.01%, Ca Cl2. H2O 0.01%, the suitable culture conditions are: culture medium P H6.0, loading amount of 50m L/250m L bottle, inoculation amount of 4.0m L/ bottle, and fermentation time as 48h. in this culture condition, the living bacteria number of strain R42-16 in the fermentation liquid is 3.19 x 109CFU. M L-1, and the formation rate of sporulation is the evaluation of the fermentation effect of corn straw. The dosage of the bacteria was 8 * 108 CFU/kg (test group I), 1 x 109 CFU/kg (test group II) and 1.2 x 109 CFU/kg (test group III), fermented 30d. The nutritional indexes of the straw were compared as follows: compared with the silage control group, the lactic acid content in the test group I was not significantly different (P0.05), test group II, and test group III The content of the test group was increased significantly (P0.05). The test group II, the lactic acid content of the test group III increased by 24.38%, 25%, and the acetic acid content increased by 292.87%, respectively, 338.52%., but the test group II and the experimental group III had no significant difference (P0.05). The content of butyric acid in the test group I, ammonia nitrogen / total nitrogen were not significantly different from the control group (P0.05), while the test group II, The test group III was significantly lower than the control group (P0.05), the test group II, the test group III butyric acid content decreased by 47.34%, 53.36%, ammonia nitrogen / total nitrogen decreased by 36.89%, 42.33%. respectively, but the test group II, the test group III difference was not significant (P0.05). Test group I of crude protein content compared to the control group, the difference was not significant (P0.05), test Test group II, the test group III compared with the control group, the content increased significantly (P0.05), the test group II, the test group III crude protein content was 8.43%, 9.11%, respectively, compared with the control group increased by 50.26%, respectively, 62.39%., but the test groups of dry matter, coarse fiber, neutral washing polyester fiber, acid washing fiber, acid detergent lignin content and other indicators P0.05. The problem of "two fermentation" was easily produced for the exposure of silage straw to the air (after the cellar). The aerobic stability of the fermented straw was measured from the material temperature, P H and odor sense. The results showed that three test groups and the silage control group were at the 1-8 day time under the room temperature of 18. The temperature of the control group was close to room temperature. The temperature of the control group began to rise at ninth days and the temperature of the material was higher than 1.57 C at room temperature at tenth days. At this time, the silage straw began to appear unstable, and the temperature was higher than room temperature 3.09 centigrade at fourteenth days. The temperature of the experiment group was higher than that of the room temperature at the room temperature of 1.1. The P H determination of straw was found. The difference between the silage control group and the test group P H was not significant at the 1-8 day, and the P H in the control group rose faster at the 10-12 day, and at twelfth days it had reached 5.59. generally believed that when the straw P H reached 5, the "two fermentation" occurred and the straw could not be fed when p H was 5.5. But the P H of the test group I, II, and III was 4. at fifteenth days respectively. 23,4.03,4.03. showed that adding strain R42-16 in straw could improve the stability of the fermented corn straw. To sum up, strain R42-16 could be used as microbiological additive to the micro storage of corn straw.
【学位授予单位】：河北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S816.53

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