羊胎素及其免疫调节功能的研究
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摘要
羊胎素就是指羊胚胎或胎盘(统称为羊胎)中的活性物质。通过查阅大量的文献资料得知，羊胎中的活性物质主要是小分子多肽类物质。此外，羊胎中也含有丰富的氨基酸，包括人体所必需的8种氨基酸。本文以羊胚胎及胎盘为研究对象，利用超滤法提取其中的主要生物活性成分，对活性成分提取后的剩余蛋白质进行了酶水解，选择了适用于羊胎水解的最适酶种类及最适水解条件。对酶水解液进行了乳酸发酵，筛选出最适发酵菌种及最适发酵条件。并对水提液、水提液+水解液、水提液+发酵液进行了免疫调节功能评价和对比，对水提液(小分子多肽)的加工稳定性进行了研究，并对羊胎素口服液进行了安全性毒理学评价。结果表明：
采用目前较为成熟的超滤法提取羊胎中的活性物质，所得小分子肽为无色或微黄色透明液体，pH6.8～7.2，采用聚丙酰胺凝胶电泳测定其分子量介于3800～10 000Dal之间。紫外扫描分析，最大紫外吸收波长在256nm附近，紫外吸收光密度E_(260)/E_(280)≥2.0，表明该物质为核苷酸和肽类物质，多肽含量为797.22±107.07ug/ml。
以水解度和氮收率为指标，通过中性蛋白酶、胃蛋白酶、胰酶和木瓜蛋白酶4种蛋白酶对羊胎蛋白质水解效率的比较，选择胰酶为水解羊胎的最适酶。通过均匀设计法选得羊胎水解的最适条件为酶用量2.5％(100g原料加胰酶2.5g)，底物浓度5.0％(100ml水中加原料5.0g)，反应温度40℃，反应时间3h，此时水解度为39.28％。羊胎水解液中含有19种组成蛋白质的氨基酸，包括人体所必需的8种氨基酸，其游离氨基酸总量为45.37mg/ml。
乳酸发酵可脱除羊胎水解液中的苦味和腥味，改善羊胎水解液的感官品质。适用于羊胎水解液发酵的微生物菌种组合为嗜酸乳杆菌1.1854与嗜热链球菌1.1855。通过发酵参数的单因子试验及正交试验得出最优发酵条件为：1.1854与1.1855配比4：1、接种量5％、发酵温度39℃、发酵时间20h、最适糖种类及用量为乳糖2％、发酵起始pH6.5。发酵后水解液中部分与苦味有关的疏水性氨基酸的含量增加，从而使水解液苦味降低。
动物试验表明，低剂量羊胎水提液可抑制SRBC所致的迟发性变态反应，提高小鼠血清溶血素水平，增强小鼠特异性免疫功能；可极显著增强单核-巨噬细胞吞噬功能，增强小鼠非特异性免疫功能，因此具有免疫调节作用。
羊胎蛋白质水解液＋水提液或水解液＋A酵液对小鼠的非特异性免疫功能较
水提液有显著提高（P功．05），也即水解液或发酵液对小鼠的免疫功能具有提
高作用。水解液经发酵后与水解液的功能相比无显著提高（P＞0．05），但测定
值有一定的提高。
温度、。pH、胃蛋白酶对小分子肽的免疫活性有一定的影响．随着温度的升
高，小分子肽的免疫活性降低。在 pH3－10的范围内，小分子肽相对稳定，
免疫活性变化很小。胃蛋白酶可以将小分子肽水解为游离氨基酸，从而使其活
性降低。
大、，1、鼠经口急性毒性试验，羊胎素口服液 LDw均＞10．00wg体重，属
实际无毒．30天喂养试验表明，较长期受试羊胎素口服液对大鼠生长、发育、
血液、生化、病理等指标未见异常，30天喂养最大无作用剂量＞5 00（）mgh
体重。
In this paper, we extracted the bioactive composition of goat embryos with the ultrafiltration and studied its physical and chemical characters; hydrolyzed the protein of the goat embryos with different proteases; selected the optimal hydrolyzed condition, screened the optimal strains and condition of lactic acid ferment; evaluated the function of the extract with water, extract with water and hydrolyzed products of protein, extract with water and fermented products of hydrolyzed products. The results of research are summed up as the followings:
The extract with water of dark goat embryos is colorless or light yellow; its molecular weight is between 3800 ~ 10 OOODal. The maximum absorb wavelength is around 256nm; E260/E2go > 2.0 shows its nature is polypeptide; the content of polypeptide is about 797.22 ?107.07ug/ml.
According to the DH% and recieval of nitrogen degree, we selected the pancreatin as the optimal enzyme from the neutral protease, pepsin, papain and pancreatin. The best hydrolyzed condition is 2.5g pancreatin/lOOgmaterial, 5.0g substrate/100ml water, reacting in 40 癈 for three hours. The hydrolyzed products includes nineteen kinds of amino acids; the total amount of free amino acids is 45.37mg/ml
Fermented with Lactic acid bacteria, the sense of the hydrolyzed products is improved greatly. The optimal ferment condition is 1.1854:1.1855=4:1,inoculated amount 5%, 39癈 for 20 hours with 2% lactose, initial pH 6.5.
The extract of goat embryos can effectively enhance the cellar immunity and
humoral immunity, specific and nonspecific immunity of mouse. But the hydrolyzed products + extract and fermented products + extract have no distinguished difference with each other.
Temperature, pH and pepsin all can influence the activity of the extract. With the Temperature advancing, the activity of the extract cuts down. Within pH3 ~ 10, the activity of the extract is relatively stable. Pepsin can hydrolyze the peptide to low its activity.
The LD50 of Juice of goat embryos by the rat and mice is more than 10.00g/kg. The result of experiment of 30 days feeding shows the extract has no bad effect on the growth, development, blood, pathology and the maximum dose without action is more than 5 000mg/kg.
采用目前较为成熟的超滤法提取羊胎中的活性物质，所得小分子肽为无色或微黄色透明液体，pH6.8～7.2，采用聚丙酰胺凝胶电泳测定其分子量介于3800～10 000Dal之间。紫外扫描分析，最大紫外吸收波长在256nm附近，紫外吸收光密度E_(260)/E_(280)≥2.0，表明该物质为核苷酸和肽类物质，多肽含量为797.22±107.07ug/ml。
以水解度和氮收率为指标，通过中性蛋白酶、胃蛋白酶、胰酶和木瓜蛋白酶4种蛋白酶对羊胎蛋白质水解效率的比较，选择胰酶为水解羊胎的最适酶。通过均匀设计法选得羊胎水解的最适条件为酶用量2.5％(100g原料加胰酶2.5g)，底物浓度5.0％(100ml水中加原料5.0g)，反应温度40℃，反应时间3h，此时水解度为39.28％。羊胎水解液中含有19种组成蛋白质的氨基酸，包括人体所必需的8种氨基酸，其游离氨基酸总量为45.37mg/ml。
乳酸发酵可脱除羊胎水解液中的苦味和腥味，改善羊胎水解液的感官品质。适用于羊胎水解液发酵的微生物菌种组合为嗜酸乳杆菌1.1854与嗜热链球菌1.1855。通过发酵参数的单因子试验及正交试验得出最优发酵条件为：1.1854与1.1855配比4：1、接种量5％、发酵温度39℃、发酵时间20h、最适糖种类及用量为乳糖2％、发酵起始pH6.5。发酵后水解液中部分与苦味有关的疏水性氨基酸的含量增加，从而使水解液苦味降低。
动物试验表明，低剂量羊胎水提液可抑制SRBC所致的迟发性变态反应，提高小鼠血清溶血素水平，增强小鼠特异性免疫功能；可极显著增强单核-巨噬细胞吞噬功能，增强小鼠非特异性免疫功能，因此具有免疫调节作用。
羊胎蛋白质水解液＋水提液或水解液＋A酵液对小鼠的非特异性免疫功能较
水提液有显著提高（P功．05），也即水解液或发酵液对小鼠的免疫功能具有提
高作用。水解液经发酵后与水解液的功能相比无显著提高（P＞0．05），但测定
值有一定的提高。
温度、。pH、胃蛋白酶对小分子肽的免疫活性有一定的影响．随着温度的升
高，小分子肽的免疫活性降低。在 pH3－10的范围内，小分子肽相对稳定，
免疫活性变化很小。胃蛋白酶可以将小分子肽水解为游离氨基酸，从而使其活
性降低。
大、，1、鼠经口急性毒性试验，羊胎素口服液 LDw均＞10．00wg体重，属
实际无毒．30天喂养试验表明，较长期受试羊胎素口服液对大鼠生长、发育、
血液、生化、病理等指标未见异常，30天喂养最大无作用剂量＞5 00（）mgh
体重。
In this paper, we extracted the bioactive composition of goat embryos with the ultrafiltration and studied its physical and chemical characters; hydrolyzed the protein of the goat embryos with different proteases; selected the optimal hydrolyzed condition, screened the optimal strains and condition of lactic acid ferment; evaluated the function of the extract with water, extract with water and hydrolyzed products of protein, extract with water and fermented products of hydrolyzed products. The results of research are summed up as the followings:
The extract with water of dark goat embryos is colorless or light yellow; its molecular weight is between 3800 ~ 10 OOODal. The maximum absorb wavelength is around 256nm; E260/E2go > 2.0 shows its nature is polypeptide; the content of polypeptide is about 797.22 ?107.07ug/ml.
According to the DH% and recieval of nitrogen degree, we selected the pancreatin as the optimal enzyme from the neutral protease, pepsin, papain and pancreatin. The best hydrolyzed condition is 2.5g pancreatin/lOOgmaterial, 5.0g substrate/100ml water, reacting in 40 癈 for three hours. The hydrolyzed products includes nineteen kinds of amino acids; the total amount of free amino acids is 45.37mg/ml
Fermented with Lactic acid bacteria, the sense of the hydrolyzed products is improved greatly. The optimal ferment condition is 1.1854:1.1855=4:1,inoculated amount 5%, 39癈 for 20 hours with 2% lactose, initial pH 6.5.
The extract of goat embryos can effectively enhance the cellar immunity and
humoral immunity, specific and nonspecific immunity of mouse. But the hydrolyzed products + extract and fermented products + extract have no distinguished difference with each other.
Temperature, pH and pepsin all can influence the activity of the extract. With the Temperature advancing, the activity of the extract cuts down. Within pH3 ~ 10, the activity of the extract is relatively stable. Pepsin can hydrolyze the peptide to low its activity.
The LD50 of Juice of goat embryos by the rat and mice is more than 10.00g/kg. The result of experiment of 30 days feeding shows the extract has no bad effect on the growth, development, blood, pathology and the maximum dose without action is more than 5 000mg/kg.
引文
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71．熊燕飞，王惠平，陈怀新，等．酶法水解蚕蛹蛋白制备多肽的工艺研究．湖北大学学报，1999，21(3)：18～20．
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73．毛建伟等．肉类发酵混合培养菌种的选择．食品科学，1991，12(1)：21～25
74．乔发东．超滤技术制备浓缩乳酸菌发酵剂的研究．中国畜产与食品．1998，5(2)：112～113
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79．卫生部卫生监督司《保健食品功能学评价程序和检验方法》
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74．乔发东．超滤技术制备浓缩乳酸菌发酵剂的研究．中国畜产与食品．1998，5(2)：112～113
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78．武建晔．羊肉加工利用现状．肉类工业，1996，(9)：21～23
79．卫生部卫生监督司《保健食品功能学评价程序和检验方法》
80．李衡，王季，区明勋．食品感官鉴定方法及实践．上海科学技术出版杜，1990
81．朱纪．食品感官分析入门．中国轻工出版社，1990
82．中华人民共和国国家标准，乳酸菌饮料中乳酸菌的微生物学检验GB/T 16347---1996
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