乳酸菌富集钙及发酵超微粉碎猪骨的研究
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摘要
微生物在生长繁殖过程中，不断进行着能量和物质的交换，其中金属离子既是生长所需的生长因子，也可能是生长干扰因素，尽管单个菌体吸收的金属量仅占菌体干物质的0.5%～1.0%左右，但是微生物繁殖量大，速度快，而且可以氧化还原金属的价态，通过自身代谢的高分子物质结合金属，利用这种特性，工业上首先在污水处理上出现了利用微生物降解重金属，进而在食品领域也出现了利用菌体富集人体所需的离子态金属元素，改变如铁、锌、硒等微量元素的化合态形式，据报道其更利于吸收和利用，而且食用安全性也提高了。
根据查新现有的利用食品微生物富集金属的研究，创新采用乳酸菌采研究微生物富集钙，了解金属元素与微生物相互作用中的因素影响机制，掌握金属元素是如何进入微生物和富集量的大小及分布，不仅为开发利用微生物富集人体所需金属元素生产营养强化食品提供一定的理论参考，也为开发利用过剩、低附加值的畜骨生产补钙新型营养食品提供科学的基础理论和数据；根据目前市场上现有的产品特点、中国人饮食习惯中缺乏吸收性好的钙源的现状和钙吸收利用的相关影响因素，设计研究含活性乳酸菌的发酵超微粉碎骨粉咀嚼片，作为保健食品提供高质量的补钙天然食品。
实验采用六种乳酸菌对微生物富集金属元素的机制和影响因素进行了实验，对富集钙量与菌株差异、生长培养时间、钙离子浓度的关系进行了研究，在本研究中创新根据生长曲线反映的个体增殖期(对数期)和物质代谢累积期(稳定期)来研究不同生长时段与钙离子富集量的关系研究方法，从而更深入地认识钙离子在整个菌体生长周期中富集的变化规律，推测菌体内外对钙离子的富集能力；通过对菌体破碎分离出细胞不溶成分和可溶成分，分别测定钙富集量，进一步验证乳酸菌与金属离子之间作用的关系；实验还对高浓度钙离子溶液是否影响乳酸菌生长代谢进行实验发现乳酸菌属中多数菌株之间的钙富集量无显著性差异；采用基础实验中获得的适宜菌株对超微粉碎骨粉发酵，对骨粉中钙和蛋白质的变化情况进行测定，优化发酵工艺参数；以生产含活菌体补钙食品为标准，研究真空干燥和制片工序的工艺，并进行了风味调试。最后对产品的理化特性和微生物指标进行了评定。
实验发现同属的乳酸菌除LB.1钙富集量明显较低以外，其他菌株的钙富集量无显著性差异；富集钙的高峰期出现在对数期，占整个生长周期富集量的80%，菌体最高富集钙量达1.4μg/mg；微生物富集钙量的增加幅度随钙离子浓度的增加逐渐减小；乳酸菌富集钙部位主要在细胞膜上。根据实验结果推测钙的富集与菌体细胞的协同转运吸收机制和膜成分存在相关性，是金属离子富集研究的重要方面。
Our study can be separated into two parts, one is the basic research on lactic acid bacteria' bio-accumulating Calcium, the other is the applicable study on Super-microsmashing Hogjxme Particles fermented by the lactobacillus. The first one can supply the fundamental data on the interaction of lactobacillus growing in the Calcium-adding culture medium to the last one, which is our design of producing the fermented bone food with the lactobacillus, which would be more efficient for persons to digest and absorb Calcium than non-fermented bone food or pure organic Calcium synthesized by chemical industry. The experiments in the basic research mainly concern about selecting species of lactic acid bacteria (LAB);analyzing the factors influencing the amount of Calcium absorbed (ACA) by the LAB, which include ACA difference due to species , culturing time arid Calcium concentration; detecting the Calcium distributed site in the cell and whether the lactobacillus is restrained by high Calcium concentration or not With the results got from the basic research ,we will choose the right species of lactobacillus to ferment the super-microsmashing hog-bone particles, through the experiments on fermentation, preferable techniques and recipes would be found ,which include the fermenting parameters which concern the fermentation time and temperature, the culture compositions, the ratio of bone particles to the culture, becasue the fermentation products designed by us should have not only abundant Calcium but lactobacillus, the experiments also concentrate on the techniques to protect the livability of lactobacillus after fermentation. The final work deals with the technology of producing the compound pill with bone Ca and active lactobacillus.
The results got from the basic research on factors influencing the amount of Calcium in the cells(ACC),determined by atom absorption spectrophotometer, indicate that: after 30hrs cultured with 1.2mgCa2+/ml MRS ,there is significant difference(p<0.05) in ACC between LB.l and other three species of LAB ,the highest ACC occurs in group LB.4(Ca 1.023 u g/mg Cell Weight); the main period for the LAB to accumulate the Calcium comes during logarithmic growth phase of microorganism, in which ACC adds to 80% of the whole period of culturing; the increase tendency of ACC by the increase of Ca2 Concentration doesn't appear linear ,and the slope decreasing till zero; the distribution of 41% ACC of cell binding Ca is in cell wall, 59% in the rest (cytoplasma etc.) , viewed by dry cell, the highest concentration of Ca occurs in cell wall;
Based on former study, we choose LB.4 to be the inoculated species of LBA to ferment superjnicrosmashing hog-bone(SMHB), during the fermentation, the diameter of SMHB and the ratio of amount of SMHB to culture volume significantly affect the Ca2 amount ionized from bone, the preferable fermentation parameter is: SMHB(<4u m) 45g/100ml, LB.4 2%, 36hrs at 42C. With automatic amino acid analysis eqiripment(BECKMAN), there is 63% protein hi SMHB hydrolyzed by fermentation, creating two functional amino acids: taurine and omitbine ; by viewing the microscopic SMHB with electron microscopy , the decomposition of SMHB is obvious and verifies the effect of fermentation to ionize Ca and hydrolyzes the macromolecule substrates. The final fermented product with 600mg weight comprising: Calcium 28mg, protein 6Qmg>amino acids 72mg, the active bacteria amount above 8.8 X106 cfu/g
根据查新现有的利用食品微生物富集金属的研究，创新采用乳酸菌采研究微生物富集钙，了解金属元素与微生物相互作用中的因素影响机制，掌握金属元素是如何进入微生物和富集量的大小及分布，不仅为开发利用微生物富集人体所需金属元素生产营养强化食品提供一定的理论参考，也为开发利用过剩、低附加值的畜骨生产补钙新型营养食品提供科学的基础理论和数据；根据目前市场上现有的产品特点、中国人饮食习惯中缺乏吸收性好的钙源的现状和钙吸收利用的相关影响因素，设计研究含活性乳酸菌的发酵超微粉碎骨粉咀嚼片，作为保健食品提供高质量的补钙天然食品。
实验采用六种乳酸菌对微生物富集金属元素的机制和影响因素进行了实验，对富集钙量与菌株差异、生长培养时间、钙离子浓度的关系进行了研究，在本研究中创新根据生长曲线反映的个体增殖期(对数期)和物质代谢累积期(稳定期)来研究不同生长时段与钙离子富集量的关系研究方法，从而更深入地认识钙离子在整个菌体生长周期中富集的变化规律，推测菌体内外对钙离子的富集能力；通过对菌体破碎分离出细胞不溶成分和可溶成分，分别测定钙富集量，进一步验证乳酸菌与金属离子之间作用的关系；实验还对高浓度钙离子溶液是否影响乳酸菌生长代谢进行实验发现乳酸菌属中多数菌株之间的钙富集量无显著性差异；采用基础实验中获得的适宜菌株对超微粉碎骨粉发酵，对骨粉中钙和蛋白质的变化情况进行测定，优化发酵工艺参数；以生产含活菌体补钙食品为标准，研究真空干燥和制片工序的工艺，并进行了风味调试。最后对产品的理化特性和微生物指标进行了评定。
实验发现同属的乳酸菌除LB.1钙富集量明显较低以外，其他菌株的钙富集量无显著性差异；富集钙的高峰期出现在对数期，占整个生长周期富集量的80%，菌体最高富集钙量达1.4μg/mg；微生物富集钙量的增加幅度随钙离子浓度的增加逐渐减小；乳酸菌富集钙部位主要在细胞膜上。根据实验结果推测钙的富集与菌体细胞的协同转运吸收机制和膜成分存在相关性，是金属离子富集研究的重要方面。
Our study can be separated into two parts, one is the basic research on lactic acid bacteria' bio-accumulating Calcium, the other is the applicable study on Super-microsmashing Hogjxme Particles fermented by the lactobacillus. The first one can supply the fundamental data on the interaction of lactobacillus growing in the Calcium-adding culture medium to the last one, which is our design of producing the fermented bone food with the lactobacillus, which would be more efficient for persons to digest and absorb Calcium than non-fermented bone food or pure organic Calcium synthesized by chemical industry. The experiments in the basic research mainly concern about selecting species of lactic acid bacteria (LAB);analyzing the factors influencing the amount of Calcium absorbed (ACA) by the LAB, which include ACA difference due to species , culturing time arid Calcium concentration; detecting the Calcium distributed site in the cell and whether the lactobacillus is restrained by high Calcium concentration or not With the results got from the basic research ,we will choose the right species of lactobacillus to ferment the super-microsmashing hog-bone particles, through the experiments on fermentation, preferable techniques and recipes would be found ,which include the fermenting parameters which concern the fermentation time and temperature, the culture compositions, the ratio of bone particles to the culture, becasue the fermentation products designed by us should have not only abundant Calcium but lactobacillus, the experiments also concentrate on the techniques to protect the livability of lactobacillus after fermentation. The final work deals with the technology of producing the compound pill with bone Ca and active lactobacillus.
The results got from the basic research on factors influencing the amount of Calcium in the cells(ACC),determined by atom absorption spectrophotometer, indicate that: after 30hrs cultured with 1.2mgCa2+/ml MRS ,there is significant difference(p<0.05) in ACC between LB.l and other three species of LAB ,the highest ACC occurs in group LB.4(Ca 1.023 u g/mg Cell Weight); the main period for the LAB to accumulate the Calcium comes during logarithmic growth phase of microorganism, in which ACC adds to 80% of the whole period of culturing; the increase tendency of ACC by the increase of Ca2 Concentration doesn't appear linear ,and the slope decreasing till zero; the distribution of 41% ACC of cell binding Ca is in cell wall, 59% in the rest (cytoplasma etc.) , viewed by dry cell, the highest concentration of Ca occurs in cell wall;
Based on former study, we choose LB.4 to be the inoculated species of LBA to ferment superjnicrosmashing hog-bone(SMHB), during the fermentation, the diameter of SMHB and the ratio of amount of SMHB to culture volume significantly affect the Ca2 amount ionized from bone, the preferable fermentation parameter is: SMHB(<4u m) 45g/100ml, LB.4 2%, 36hrs at 42C. With automatic amino acid analysis eqiripment(BECKMAN), there is 63% protein hi SMHB hydrolyzed by fermentation, creating two functional amino acids: taurine and omitbine ; by viewing the microscopic SMHB with electron microscopy , the decomposition of SMHB is obvious and verifies the effect of fermentation to ionize Ca and hydrolyzes the macromolecule substrates. The final fermented product with 600mg weight comprising: Calcium 28mg, protein 6Qmg>amino acids 72mg, the active bacteria amount above 8.8 X106 cfu/g
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