新型饲料添加剂二十八烷醇的开发及应用效果研究
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摘要
本论文对米糠蜡中二十八烷醇的提取工艺进行了优化,并对二十八烷醇作为一种新型饲料添加剂的毒理学和在畜禽动物上的应用效果进行了研究。
通过对溶剂法从粗糠蜡中制取精糠蜡的工艺、微波水解法分离出精糠蜡中的高级脂肪醇的工艺和高真空蒸馏工艺提取二十八烷醇工艺中主要影响因素进行单因素试验和正交试验研究得出,溶剂法由粗糠蜡制取精糠蜡的最优工艺条件为：以溶剂比1：8(m/V)的乙酸乙酯,在温度70℃下热回流萃取1.5h；微波水解法皂化精糠蜡的最优工艺条件为：微波功率700w,以料碱比1：2(m/V)的NaOH溶液,微波皂化精糠蜡12min;高真空蒸馏工艺在蒸馏温度220～240℃,真空度11.3Pa的条件下可获得含量为53.72%的二十八烷醇。同时,建立了毛细管柱气相色谱法对二十八烷醇进行定性定量分析的测定方法。
将含二十八烷醇的受试物应用于饲料中进行了急性毒性、遗传毒性和30d喂养试验研究,旨在为二十八烷醇在饲料中的应用提供安全性依据。采用霍恩氏法,对40只(20±2)g雌雄各半清洁级ICR小白鼠和40只(200±20)g雌雄各半清洁级Wistar大鼠进行急性经口毒性试验；选用TA97、TA98、TA100、TA102四种经生物特性鉴定合格的菌株,进行加与不加大鼠肝S9的标准平皿掺入法Ames试验;选用50只体重25～30g雌雄各半的清洁级ICR小白鼠进行小鼠骨髓细胞微核试验；选用25只体重25-35g清洁级雄性ICR小白鼠进行小鼠精子畸变试验；采用饲料掺入法,选用80只刚断乳的体重(85±10)g,雌雄各半的Wistar大白鼠,进行30d喂养试验。结果表明：含二十八烷醇的受试物对ICR小白鼠、Wistar大鼠的急性经口LD50＞10.0g/(kg·BW),属实际无毒物质；遗传毒性试验表明受试物无致突变作用；大鼠30d喂养试验中未见动物生长、健康状况、生化、血液学指标和器官组织形态的异常变化,推测二十八烷醇用于动物体的最大无作用剂量大于40mg/kg。
为了探索二十八烷醇对畜禽动物的饲用有效性,本文对二十八烷醇在断奶仔猪和蛋鸡上的应用效果进行了系统的研究。选取105头21日龄、初始体重为(5.70±1.41)kg的健康“杜×长×大”断奶仔猪,随机分为对照组、泰妙菌素组(泰妙菌素33g/kg)和二十八烷醇组(二十八烷醇8mg/kg),每组7个重复,每个重复5头猪,进行饲养试验,试验期42d,研究二十八烷醇对仔猪生长性能、养分消化率、腹泻情况、血液指标和激素分泌量及机体GLUT-4、GS和AMPK基因的表达量的影响。结果表明,整个试验期内,二十八烷醇组显著提高仔猪的日增重和饲料转化率(P<0.05),对仔猪的腹泻程度有明显改善(P<0.05),对血液生化和抗氧化指标也有显著影响(P<0.05),并可提高日粮中干物质和粗蛋白质的表观消化率(P>0.05)；与泰妙菌素组相比,二十八烷醇组仔猪的血液指标显著优于泰妙菌素组(P<0.05),日增重、饲料转化率、营养成分消化率和腹泻程度指标与泰妙菌素组效果无显著差异(P>0.05)。试验前后,断奶仔猪血液中生长激素、胰高血糖素、肾上腺素三种激素含量的增加量比对照组和泰妙菌素组均有显著提高(P<0.05),肝脏和肌肉中组织中GLUT-4、GS、AMPK三种基因表达量也比对照组和泰妙菌素组有明显增加(P<0.05)。研究结果表明,二十八烷醇对断奶仔猪的生产性能有显著地促进作用,可减少饲料中抗生素的添加,通过促进机体GLUT-4、GS和AMPK基因的表达量的增加,促进体内多个有关能量代谢的酶类物质的合成,调节机体能量代谢。
研究了日粮中添加不同比例的二十八烷醇对蛋鸡生产性能、蛋品质、血液指标和粪便菌群的影响。选用采食正常、产蛋率和体重相近的健康的46周龄、海兰褐产蛋鸡384只,随机分成4组,每组8个重复,每个重复12只鸡。对照组饲喂基础日粮,3个试验组OCTO1、OCTO2、OCT03组饲粮分别在对照组日粮基础上添加8、16、24mg/kg二十八烷醇。试验预饲期1周,正饲期6周。结果表明：1)与对照组相比,OCT02和OCT03组显著提高了蛋鸡的产蛋率(P<0.05)和蛋重；2)试验结束时,3个试验组均显著提高了鸡蛋的蛋白高度和哈夫单位(P<0.05),各试验组间无显著差异(P＞0.05)；OCT02和OCT03组还可促进蛋壳强度的提升(P<0.05)；二十八烷醇对鸡蛋的蛋黄直径、蛋黄颜色、蛋壳厚度影响不明显(P>0.05);3)OCT02和OCT03组显著降低了蛋鸡血清中总胆固醇(TC)、甘油三酯(TG)和低密度脂蛋白(LDL-C)的含量(P<0.05),不影响高密度脂蛋白(HDL-C)的含量(P>0.05);4)OCT02和OCT03组显著降低蛋鸡粪便中大肠杆菌数量(P<0.05),提高乳酸杆菌数量(P＜0.05);5)OCT02与OCT03组对各指标的影响无显著差异,从成本角度综合考虑,蛋鸡日粮中二十八烷醇的添加量以16mg/kg为宜。研究结果表明,二十八烷醇可提高蛋鸡产蛋率、改善鸡蛋品质、降低血脂、调节肠道菌群平衡,对蛋鸡生产性能的提高具有显著地促进作用。
综上所述,本研究为二十八烷醇在饲料领域的应用提供了科学基础和理论依据,对倡导绿色养殖具有重要的实用价值,同时,对促进农副产品的高效利用有积极的推动作用。
In this paper, extraction process in rice bran wax of octacosanol was optimized and toxicology and application effect of octacosanol were studied as a new feed addictive.
The extraction process in rice bran wax of octacosanol was studied, which may obtained high-purity octacosanol. Purified bran wax was obtained from rough bran wax by solvent extraction method and four factors of the variety of solvent, extraction time and extraction temperature on the yield and purity of bran wax were studied. Then the mixture of senior fatty alcohols in purified bran wax was isolated by microwave hydrolysis method and the effect of microwave power, saponification time, alkali liquor types and the ratio of material to alkali on saponification yield were studied. High vacuum distillation process was used to extract and purify of octacosano. At last, the process was optimized by single factor test and orthogonal test. The results showed the optimal process conditions of purifing the rough bran wax was using acetic acid ethyl ester in solvent ratio1:8(m/V) under the temperature70℃and extraction1.5h. The optimal process conditions of microwave hydrolysis method of saponification purified bran wax was under microwave power700w, with alkali ratio1:2(m/V) of NaOH solution, microwave saponification12min. High vacuum distillation process was in the distillation temperature220～240℃and under the condition of vacuum11.3Pa. Thus octacosanol content of53.72%can be obtained. In addition, the determination method of octacosanol was established;
In order to confirm the security of octacosanol in feed the safety toxicology evaluate was proceeded which for providing a theoretical basis of security for the application of octacosanol in feed. The HuoEnShi method was adopted to test the acute oral toxicity which was with40ICR mice of clean level whose weight were (20±2)g and40Wistar rats of clean level whose weight were (200±2)g,and ICR mice and Wistar rats were all male and female half. TA97, TA98, TA100, TA102strains passed biological characteristics identification were choosed in Ames test and the integrated method of the studard AGAR was conducted with add or not the rat liver S9. In bone marrow cell micronucleus test50ICR mice of clean level weight were25-30g and male and female were half were tested. And in mice sperm malformation test25male ICR mice of clean level weight were25-35g were choosed. The feed mixing method was adopted in30days feeding test of Wistar rats.80weanling Wistar rats, weight(85±10)g and male and female half, were divided into four groups averagely and used for high, medium and low dose test groups and control group. The results showed that the feed addictive containing octacosanol was nonpoisonous in reality with oral LD50>10.0g/(kg·BW) to ICR rats and Wistar mice. The results of Ames test, mice bone marrow micronucleus test and mice sperm malformation test were all negative active, which mean no inducing effect of pathologocal changes and mutation. After30days feeding experiment to Wistar mice, there were no abnormal changes in the terms of health status, biological chemical indexes, hematology indexes, as well as organ and tissue morphology. It was included that feed additive containing octacosanol can be used in animal feed safely.
In order to explore the effectiveness of octacosanol to livestock and poultry, the application effect of octacosanol to weanling piglets and laying hens were researched systematically. The effect of octacosanol on growth performance, nutrient digestibility, diarrhea, blood index and hormone secretion content of weanling piglets were stuied. Selected a total of10521-day-old piglets were randomly divided into three groups:Control group; tiamulin group (tiamulin33mg/kg) and octacosanol group (octacosanol8mg/kg). Each group set up7repetition with5pigs and feeding period was42days. The results showed that in the whole experiment period octacosanol group had significant enhanced on ADG and G/F (P<0.05) and improved on diarrhea degree (P<0.05); the blood biochemical and antioxidant index also had a significant differences (P<0.05) and the digestibility of dry matter and crude protein had a significant improved (P <0.05) with control group. Compared with tiamulin group, Octacosanol group showed greater superiority in blood parameters (P<0.05). Although there is no significant difference in ADG, G/F, nutrient digestibility and diarrhea degree (P>0.05), the effect is better than that of tiamulin group. The content of Growth hormone, Adrenaline and Glucagon had significant improved compared with control group and tiamulin group (P＜0.05). In the liver and muscle tissues the gene expression quantity of GLUT-4, GS and AMPK were significantly increased in octacosanol group (P<0.05) which indicated octacosanol could regulate the body's energy metabolism. So this research proved that octacosanol could improve growth performance, immunity and anti-stress ability of weanling piglet through increasing the gene expression quantity of GLUT-4, GS and AMPK which involved in energy metabolism.
The effect of different proportion octacosanol in laying hens feed to hens on laying performance, egg quality, serum parameters and fecal microorganism was conducted. Fourty six-week old laying hens (n=384) were fed with a diet containing0,8,16,24mg/kg octacosanol fors6weeks. We observed that comparing with control feeding octacosanol to laying hens significantly increased the rate of laying eggs and egg weight (P<0.05) and the egg albumen height and haugh unit had significantly increased because octacosanol (P＜0.05). OCT02and OCT03group also could promote the ascension of eggshell intensity (P<0.05). There was no observed adverse effects of the octacosanol on egg yolk diameter, egg yolk color and shell thickness(P>0.05). The levels of TC, TG and LDL-C in serum significantly reduced (P<0.05) after feeding octacosanol were found and the content of HDL-C was not affected by octacosanol (P>0.05). Compared with control group, OCT02and OCT03group could significantly reduce the number of e. coli in laying hens manure (P<0.05) and increase the number of lactobacillus (P<0.05). The results found OCT02and OCT03group of no significant difference (P<0.05). So it could be confirmed that adding16mg/kg octacosanol into laying hens feed was asvisable.
To sum up, this research for the application of octacosanol in the field of feed provides a scientific basis and theoretical basis and has important practical value to advocate green farming. At the same time, it may promote the efficient utilization of subsidiary agricultural products.
通过对溶剂法从粗糠蜡中制取精糠蜡的工艺、微波水解法分离出精糠蜡中的高级脂肪醇的工艺和高真空蒸馏工艺提取二十八烷醇工艺中主要影响因素进行单因素试验和正交试验研究得出,溶剂法由粗糠蜡制取精糠蜡的最优工艺条件为：以溶剂比1：8(m/V)的乙酸乙酯,在温度70℃下热回流萃取1.5h；微波水解法皂化精糠蜡的最优工艺条件为：微波功率700w,以料碱比1：2(m/V)的NaOH溶液,微波皂化精糠蜡12min;高真空蒸馏工艺在蒸馏温度220～240℃,真空度11.3Pa的条件下可获得含量为53.72%的二十八烷醇。同时,建立了毛细管柱气相色谱法对二十八烷醇进行定性定量分析的测定方法。
将含二十八烷醇的受试物应用于饲料中进行了急性毒性、遗传毒性和30d喂养试验研究,旨在为二十八烷醇在饲料中的应用提供安全性依据。采用霍恩氏法,对40只(20±2)g雌雄各半清洁级ICR小白鼠和40只(200±20)g雌雄各半清洁级Wistar大鼠进行急性经口毒性试验；选用TA97、TA98、TA100、TA102四种经生物特性鉴定合格的菌株,进行加与不加大鼠肝S9的标准平皿掺入法Ames试验;选用50只体重25～30g雌雄各半的清洁级ICR小白鼠进行小鼠骨髓细胞微核试验；选用25只体重25-35g清洁级雄性ICR小白鼠进行小鼠精子畸变试验；采用饲料掺入法,选用80只刚断乳的体重(85±10)g,雌雄各半的Wistar大白鼠,进行30d喂养试验。结果表明：含二十八烷醇的受试物对ICR小白鼠、Wistar大鼠的急性经口LD50＞10.0g/(kg·BW),属实际无毒物质；遗传毒性试验表明受试物无致突变作用；大鼠30d喂养试验中未见动物生长、健康状况、生化、血液学指标和器官组织形态的异常变化,推测二十八烷醇用于动物体的最大无作用剂量大于40mg/kg。
为了探索二十八烷醇对畜禽动物的饲用有效性,本文对二十八烷醇在断奶仔猪和蛋鸡上的应用效果进行了系统的研究。选取105头21日龄、初始体重为(5.70±1.41)kg的健康“杜×长×大”断奶仔猪,随机分为对照组、泰妙菌素组(泰妙菌素33g/kg)和二十八烷醇组(二十八烷醇8mg/kg),每组7个重复,每个重复5头猪,进行饲养试验,试验期42d,研究二十八烷醇对仔猪生长性能、养分消化率、腹泻情况、血液指标和激素分泌量及机体GLUT-4、GS和AMPK基因的表达量的影响。结果表明,整个试验期内,二十八烷醇组显著提高仔猪的日增重和饲料转化率(P<0.05),对仔猪的腹泻程度有明显改善(P<0.05),对血液生化和抗氧化指标也有显著影响(P<0.05),并可提高日粮中干物质和粗蛋白质的表观消化率(P>0.05)；与泰妙菌素组相比,二十八烷醇组仔猪的血液指标显著优于泰妙菌素组(P<0.05),日增重、饲料转化率、营养成分消化率和腹泻程度指标与泰妙菌素组效果无显著差异(P>0.05)。试验前后,断奶仔猪血液中生长激素、胰高血糖素、肾上腺素三种激素含量的增加量比对照组和泰妙菌素组均有显著提高(P<0.05),肝脏和肌肉中组织中GLUT-4、GS、AMPK三种基因表达量也比对照组和泰妙菌素组有明显增加(P<0.05)。研究结果表明,二十八烷醇对断奶仔猪的生产性能有显著地促进作用,可减少饲料中抗生素的添加,通过促进机体GLUT-4、GS和AMPK基因的表达量的增加,促进体内多个有关能量代谢的酶类物质的合成,调节机体能量代谢。
研究了日粮中添加不同比例的二十八烷醇对蛋鸡生产性能、蛋品质、血液指标和粪便菌群的影响。选用采食正常、产蛋率和体重相近的健康的46周龄、海兰褐产蛋鸡384只,随机分成4组,每组8个重复,每个重复12只鸡。对照组饲喂基础日粮,3个试验组OCTO1、OCTO2、OCT03组饲粮分别在对照组日粮基础上添加8、16、24mg/kg二十八烷醇。试验预饲期1周,正饲期6周。结果表明：1)与对照组相比,OCT02和OCT03组显著提高了蛋鸡的产蛋率(P<0.05)和蛋重；2)试验结束时,3个试验组均显著提高了鸡蛋的蛋白高度和哈夫单位(P<0.05),各试验组间无显著差异(P＞0.05)；OCT02和OCT03组还可促进蛋壳强度的提升(P<0.05)；二十八烷醇对鸡蛋的蛋黄直径、蛋黄颜色、蛋壳厚度影响不明显(P>0.05);3)OCT02和OCT03组显著降低了蛋鸡血清中总胆固醇(TC)、甘油三酯(TG)和低密度脂蛋白(LDL-C)的含量(P<0.05),不影响高密度脂蛋白(HDL-C)的含量(P>0.05);4)OCT02和OCT03组显著降低蛋鸡粪便中大肠杆菌数量(P<0.05),提高乳酸杆菌数量(P＜0.05);5)OCT02与OCT03组对各指标的影响无显著差异,从成本角度综合考虑,蛋鸡日粮中二十八烷醇的添加量以16mg/kg为宜。研究结果表明,二十八烷醇可提高蛋鸡产蛋率、改善鸡蛋品质、降低血脂、调节肠道菌群平衡,对蛋鸡生产性能的提高具有显著地促进作用。
综上所述,本研究为二十八烷醇在饲料领域的应用提供了科学基础和理论依据,对倡导绿色养殖具有重要的实用价值,同时,对促进农副产品的高效利用有积极的推动作用。
In this paper, extraction process in rice bran wax of octacosanol was optimized and toxicology and application effect of octacosanol were studied as a new feed addictive.
The extraction process in rice bran wax of octacosanol was studied, which may obtained high-purity octacosanol. Purified bran wax was obtained from rough bran wax by solvent extraction method and four factors of the variety of solvent, extraction time and extraction temperature on the yield and purity of bran wax were studied. Then the mixture of senior fatty alcohols in purified bran wax was isolated by microwave hydrolysis method and the effect of microwave power, saponification time, alkali liquor types and the ratio of material to alkali on saponification yield were studied. High vacuum distillation process was used to extract and purify of octacosano. At last, the process was optimized by single factor test and orthogonal test. The results showed the optimal process conditions of purifing the rough bran wax was using acetic acid ethyl ester in solvent ratio1:8(m/V) under the temperature70℃and extraction1.5h. The optimal process conditions of microwave hydrolysis method of saponification purified bran wax was under microwave power700w, with alkali ratio1:2(m/V) of NaOH solution, microwave saponification12min. High vacuum distillation process was in the distillation temperature220～240℃and under the condition of vacuum11.3Pa. Thus octacosanol content of53.72%can be obtained. In addition, the determination method of octacosanol was established;
In order to confirm the security of octacosanol in feed the safety toxicology evaluate was proceeded which for providing a theoretical basis of security for the application of octacosanol in feed. The HuoEnShi method was adopted to test the acute oral toxicity which was with40ICR mice of clean level whose weight were (20±2)g and40Wistar rats of clean level whose weight were (200±2)g,and ICR mice and Wistar rats were all male and female half. TA97, TA98, TA100, TA102strains passed biological characteristics identification were choosed in Ames test and the integrated method of the studard AGAR was conducted with add or not the rat liver S9. In bone marrow cell micronucleus test50ICR mice of clean level weight were25-30g and male and female were half were tested. And in mice sperm malformation test25male ICR mice of clean level weight were25-35g were choosed. The feed mixing method was adopted in30days feeding test of Wistar rats.80weanling Wistar rats, weight(85±10)g and male and female half, were divided into four groups averagely and used for high, medium and low dose test groups and control group. The results showed that the feed addictive containing octacosanol was nonpoisonous in reality with oral LD50>10.0g/(kg·BW) to ICR rats and Wistar mice. The results of Ames test, mice bone marrow micronucleus test and mice sperm malformation test were all negative active, which mean no inducing effect of pathologocal changes and mutation. After30days feeding experiment to Wistar mice, there were no abnormal changes in the terms of health status, biological chemical indexes, hematology indexes, as well as organ and tissue morphology. It was included that feed additive containing octacosanol can be used in animal feed safely.
In order to explore the effectiveness of octacosanol to livestock and poultry, the application effect of octacosanol to weanling piglets and laying hens were researched systematically. The effect of octacosanol on growth performance, nutrient digestibility, diarrhea, blood index and hormone secretion content of weanling piglets were stuied. Selected a total of10521-day-old piglets were randomly divided into three groups:Control group; tiamulin group (tiamulin33mg/kg) and octacosanol group (octacosanol8mg/kg). Each group set up7repetition with5pigs and feeding period was42days. The results showed that in the whole experiment period octacosanol group had significant enhanced on ADG and G/F (P<0.05) and improved on diarrhea degree (P<0.05); the blood biochemical and antioxidant index also had a significant differences (P<0.05) and the digestibility of dry matter and crude protein had a significant improved (P <0.05) with control group. Compared with tiamulin group, Octacosanol group showed greater superiority in blood parameters (P<0.05). Although there is no significant difference in ADG, G/F, nutrient digestibility and diarrhea degree (P>0.05), the effect is better than that of tiamulin group. The content of Growth hormone, Adrenaline and Glucagon had significant improved compared with control group and tiamulin group (P＜0.05). In the liver and muscle tissues the gene expression quantity of GLUT-4, GS and AMPK were significantly increased in octacosanol group (P<0.05) which indicated octacosanol could regulate the body's energy metabolism. So this research proved that octacosanol could improve growth performance, immunity and anti-stress ability of weanling piglet through increasing the gene expression quantity of GLUT-4, GS and AMPK which involved in energy metabolism.
The effect of different proportion octacosanol in laying hens feed to hens on laying performance, egg quality, serum parameters and fecal microorganism was conducted. Fourty six-week old laying hens (n=384) were fed with a diet containing0,8,16,24mg/kg octacosanol fors6weeks. We observed that comparing with control feeding octacosanol to laying hens significantly increased the rate of laying eggs and egg weight (P<0.05) and the egg albumen height and haugh unit had significantly increased because octacosanol (P＜0.05). OCT02and OCT03group also could promote the ascension of eggshell intensity (P<0.05). There was no observed adverse effects of the octacosanol on egg yolk diameter, egg yolk color and shell thickness(P>0.05). The levels of TC, TG and LDL-C in serum significantly reduced (P<0.05) after feeding octacosanol were found and the content of HDL-C was not affected by octacosanol (P>0.05). Compared with control group, OCT02and OCT03group could significantly reduce the number of e. coli in laying hens manure (P<0.05) and increase the number of lactobacillus (P<0.05). The results found OCT02and OCT03group of no significant difference (P<0.05). So it could be confirmed that adding16mg/kg octacosanol into laying hens feed was asvisable.
To sum up, this research for the application of octacosanol in the field of feed provides a scientific basis and theoretical basis and has important practical value to advocate green farming. At the same time, it may promote the efficient utilization of subsidiary agricultural products.
引文
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