本文選題：玉米秸稈 + 青貯��； 參考：《河北農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：青貯是秸稈飼料化利用較為成熟的技術(shù),可以最大限度地保存秸稈中的營(yíng)養(yǎng)物質(zhì),為畜牧養(yǎng)殖業(yè)提供較為廉價(jià)優(yōu)質(zhì)的飼草。目前,用于秸稈青貯的微生物添加劑主要是乳酸菌類,然而乳酸菌不耐高溫,在生產(chǎn)和貯存過程中易失活,質(zhì)量不穩(wěn)定,影響了其使用效果。針對(duì)這一問題,本研究擬分離篩選可以發(fā)酵產(chǎn)酸的芽孢桿菌菌株,制備微貯菌劑,用于鮮玉米秸稈的貯存。首先從牛糞等樣品中共分離得到了芽孢菌菌株200余株,利用變色圈法從中初篩出13株可使溴甲酚紫變黃的產(chǎn)酸菌株。利用平板培養(yǎng)基打孔法對(duì)這13株菌進(jìn)行了復(fù)篩,最終獲得1株產(chǎn)酸量較高的芽孢桿菌R42-16菌株。該菌株可以發(fā)酵糖產(chǎn)生多種有機(jī)酸,其中乙酸、乳酸的產(chǎn)量較高,液體發(fā)酵條件下分別為220.70μg/m L、174.25μg/m L。根據(jù)菌落形態(tài)、菌體特征、生理生化試驗(yàn)、以及16S r DNA序列分析,菌株R42-16被鑒定為解淀粉芽孢桿菌(Bacillus amyloliquefaciens)。對(duì)R42-16菌株的生物學(xué)特性進(jìn)行研究發(fā)現(xiàn),該菌株對(duì)酸、膽酸鹽具有較強(qiáng)的耐受性,耐受p H的范圍為1.0~7.0,耐受膽酸鹽的范圍為0.1%~0.3%;該菌株對(duì)蔗糖、果糖的利用率均較高;同時(shí),菌株R42-16對(duì)致病大腸桿菌O157:H7具有較強(qiáng)的拮抗活性。通過單因素試驗(yàn)和正交試驗(yàn),對(duì)菌株R42-16的培養(yǎng)基組成和培養(yǎng)條件進(jìn)行了優(yōu)化。優(yōu)化培養(yǎng)基為:玉米粉1.0%、酵母浸粉2.0%、Mn SO4·H2O 0.01%、Ca Cl2·H2O 0.01%;適宜的培養(yǎng)條件為:培養(yǎng)基p H6.0、裝液量為50m L/250m L三角瓶、接種量為4.0m L/瓶、發(fā)酵時(shí)間為48h。在此培養(yǎng)條件下,發(fā)酵液中菌株R42-16的活菌數(shù)為3.19×109CFU·m L-1,芽孢形成率為93.76%。對(duì)菌株R42-16對(duì)玉米秸稈的發(fā)酵效果進(jìn)行了評(píng)價(jià)。設(shè)定菌劑施用量分別為8.0×108 CFU/kg(試驗(yàn)組Ⅰ)、1.0×109 CFU/kg(試驗(yàn)組Ⅱ)和1.2×109 CFU/kg(試驗(yàn)組Ⅲ),發(fā)酵30d,對(duì)各試驗(yàn)組秸稈的營(yíng)養(yǎng)指標(biāo)對(duì)比如下:與青貯對(duì)照組相比,試驗(yàn)組Ⅰ中的乳酸、乙酸含量差異不顯著(p0.05),試驗(yàn)組Ⅱ、試驗(yàn)組Ⅲ中兩者的含量均顯著增加(p0.05),試驗(yàn)組Ⅱ、試驗(yàn)組Ⅲ的乳酸含量分別提高24.38%、25.0%,乙酸含量分別提高292.87%、338.52%。但試驗(yàn)組Ⅱ和試驗(yàn)組Ⅲ之間差異不顯著(p0.05)。試驗(yàn)組Ⅰ中的丁酸含量、氨態(tài)氮/總氮與對(duì)照組相比差異不顯著(p0.05),而試驗(yàn)組Ⅱ、試驗(yàn)組Ⅲ與對(duì)照組相比,顯著降低(p0.05),試驗(yàn)組Ⅱ、試驗(yàn)組Ⅲ的丁酸含量分別降低了47.34%、53.36%;氨態(tài)氮/總氮分別降低了36.89%、42.33%。但試驗(yàn)組Ⅱ、試驗(yàn)組Ⅲ之間差異不顯著(p0.05)。試驗(yàn)組Ⅰ的粗蛋白含量與對(duì)照組相比,差異不顯著(p0.05),試驗(yàn)組Ⅱ、試驗(yàn)組Ⅲ與對(duì)照組相比,含量顯著增加(p0.05),試驗(yàn)組Ⅱ、試驗(yàn)組Ⅲ的粗蛋白含量分別為8.43%、9.11%,與對(duì)照組相比分別增加了50.26%、62.39%。但是,各試驗(yàn)組的干物質(zhì)、粗纖維、中性洗滌纖維、酸性洗滌纖維、酸性洗滌木質(zhì)素含量等指標(biāo)差異不顯著(P0.05)。針對(duì)青貯秸稈暴露在空氣中(開窖后)容易產(chǎn)生“二次發(fā)酵”的問題,本文從物料溫度、p H、氣味感官等方面對(duì)發(fā)酵秸稈進(jìn)行了有氧穩(wěn)定性的測(cè)定。結(jié)果表明,在室溫為18℃的條件下,三個(gè)試驗(yàn)組和青貯對(duì)照組在第1-8天時(shí)料溫均接近于室溫。對(duì)照組在第9天時(shí)料溫開始上升,第10天時(shí)料溫高于室溫1.57℃,此時(shí)青貯秸稈開始呈現(xiàn)不穩(wěn)定狀態(tài),第14天時(shí)料溫高于室溫3.09℃,呈現(xiàn)“二次發(fā)酵”癥狀。而此時(shí)試驗(yàn)組的料溫僅高于室溫1.1℃左右。測(cè)定秸稈的p H變化發(fā)現(xiàn),在第1-8天時(shí)青貯對(duì)照組與試驗(yàn)組的p H相差均不大,對(duì)照組p H在第10-12天時(shí)上升較快,在第12天時(shí)已經(jīng)達(dá)到了5.59。一般認(rèn)為秸稈的p H達(dá)到5.0時(shí)即發(fā)生了“二次發(fā)酵”,當(dāng)p H為5.5時(shí)秸稈不能再飼喂。但試驗(yàn)組Ⅰ、Ⅱ、Ⅲ在第15天時(shí)的p H分別為4.23、4.03、4.03。表明在秸稈中添加菌株R42-16能夠提高發(fā)酵玉米秸稈的穩(wěn)定性。綜上所述,菌株R42-16可以作為微生物添加劑應(yīng)用于玉米秸稈的微貯。
[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.
【學(xué)位授予單位】：河北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S816.53

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