乳酸菌发酵骨粉产品的研发及其补钙成骨作用的研究

发布时间：2018-07-07 10:55

本文选题：乳酸发酵 + 乳酸钙　；参考：《内蒙古大学》2017年硕士论文

【摘要】：响应自治区提出的绿色农畜产品输出基地建设与转型升级的需要,开发绿色无污染的高科技高端产品、增加畜产品附加值已成为当务之急。以牛羊鲜骨为原料通过发酵研发补钙又促成骨的纯天然高端产品,提高畜牧资源的附加值,为民生健康做出贡献。选用三种乳酸菌发酵牛羊鲜骨粉,使羟磷灰石的钙以乳酸钙形式释放。经牛羊鲜骨—粉碎—灭菌—发酵—总混—冻干—干法制粒—压片制成生物活性制剂(产品),并在细胞,组织和动物水平上验证了产品的功效。用湿灰法消化骨粉用EDTA滴定方法测定总钙含量,用EDTA滴定方法测定游离钙,以平板倾注法测定活菌数。培养人成骨细胞hFOB并用产品刺激12 h后提取RNA在以荧光定量PCR检测COLLAGEN,RANKL,OPG三种基因的表达。以切除卵巢的方法制作了骨质疏松模型动物,并用全自动生化仪测定血清指标,取股骨制作病理切片经HE染色观察大鼠骨质疏松程度。灌胃给药法研究个体水平上的功效,灌胃两个月后动脉取血测定血清生化指标,再处死大鼠后取股骨制作病理切片HE染色观察大鼠摄取不同钙产品后骨质疏松程度。目前我们的产品已将不能吸收的羟磷灰石的37.61%转化为可吸收的游离钙(乳酸钙)形式,即,游离钙的含量增加了近20倍。并在骨质疏松动物模型动物水平上证明了补钙又能促进骨形成。还在细胞水平也初步证明该产品具有增加骨保护素表达和成骨作用。因此,本研究对于钙补充、促进骨形成及预防骨质疏松症都具有重要意义。结论1、乳酸菌发酵有效游离出骨粉中以羟磷灰石形式存在的结合钙为乳酸钙,乳酸钙含量增加了近20倍;2、增加成骨细胞外钙离子浓度使OPG表达量增加,PANKL表达量增加,对GOLLLAGEN的表达没有影响,说明产品具有成骨作用;3、通过在细胞,组织和动物水平上实验证明产品对骨质疏松症的修复有显著性的效果;我们研发产品的性能测定结果显示,该产品具有产业化开发的前景。
[Abstract]:In response to the needs of the construction and transformation of the export base of green agricultural and livestock products proposed by the autonomous region, it is urgent to develop the green, non-polluting, high-tech and high-end products and to increase the added value of livestock products. Using fresh bone of cattle, sheep and sheep as raw material, calcium supplement and pure high-end product of bone can be developed through fermentation, which can improve the added value of animal husbandry resources and contribute to people's livelihood and health. The calcium of hydroxyapatite was released in the form of calcium lactate by using three kinds of lactic acid bacteria to ferment fresh bone powder of cattle and sheep. The bioactive preparation (product) was prepared by bovine and sheep fresh bone, crushing, sterilizing, fermenting, total mixing, freeze-drying, dry granulation and pressing tablets, and the efficacy of the product was verified at cell, tissue and animal levels. Total calcium content was determined by EDTA titration method, free calcium content was determined by EDTA titration method, and live bacteria number was determined by plate pour method. Human osteoblast hFOB was cultured and stimulated for 12 h, then RNA was extracted. The expression of three genes of COLLAGEN RANKL OPG was detected by fluorescence quantitative PCR. Osteoporosis model animal was made by ovariectomized method. Serum indexes were measured by automatic biochemical instrument. Pathological sections of femur were made by HE staining to observe the degree of osteoporosis in rats. The efficacy of intragastric administration was studied at individual level. The serum biochemical indexes were measured by arterial blood collection two months after gastric perfusion and then the femur was taken to make pathological sections by HE staining to observe the degree of osteoporosis after different calcium products were ingested in rats. At present, our products have converted 37.61% of unabsorbable hydroxyapatite into absorbable free calcium (calcium lactate), that is, the content of free calcium has increased nearly 20 times. At the animal level of osteoporosis animal model, calcium supplementation can promote bone formation. It was also proved that the product could increase osteoprotegerin expression and osteogenesis. Therefore, this study is important for calcium supplementation, bone formation and osteoporosis prevention. Conclusion 1. The calcium bound in hydroxyapatite was the calcium lactate in lactic acid bacteria. The content of calcium lactate increased by 20 times, and the concentration of extracellular calcium increased the expression of OPG and PANKL. There is no effect on the expression of GOLLLAGEN, which indicates that the product has osteogenic effect. It is proved by experiments on cell, tissue and animal level that the product has remarkable effect on the repair of osteoporosis. The product has the prospect of industrialization development.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS251.94;R580

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