摘要
为综合开发利用畜禽骨骼资源,提高畜禽骨骼加工附加值,本文以牛骨为研究对象,从自然界筛选诱变高产胶原蛋白酶、降解骨骼的菌种,将其应用于发酵牛骨制备功能性的补钙产品—胶原多肽螯合钙(Bovine bone collagen-derived polypeptide chelated calcium, BBCP-Ca)的研究。主要的研究内容和结果如下：
从骨骼加工厂堆积骨骼处采集土样和水样,通过自行设计的筛选模式选出降解骨骼和产酶能力强的菌株MBL13(酶活为25.60±0.93 U/mL)。经鉴定该菌株为蜡样芽孢杆菌(Bacillus cereus)。对其进行亚硝基胍(NTG)处理和紫外线照射复合诱变,选育出一株遗传稳定性好,高产胶原蛋白酶的菌株B. cereus MBL13-U(酶活为36.80±1.39 U/mL),酶活较诱变前显著提高了43.75%(p<0.05)。
为进一步提高菌株产胶原蛋白酶和降解骨的能力,针对菌株发酵产酶条件进行了优化,以酶活为指标,确定了菌株产酶最佳的培养基组成为蔗糖10.0 g/L,骨明胶20.0 g/L,CaCl20.50 g/L, NaH2PO4·2H2O 0.50 g/L, K2HPO4·3H2O 2.50 g/L。通过响应面分析法确定了最佳发酵工艺条件为：接种量为3%,发酵温度为37℃、pH为7.0,发酵时间为36 h、装瓶量为25 mL/250 mL三角瓶,可获得到最大的酶活的实验值为48.24±0.71 U/mL,较优化前提高了31.09%。
对B. cereus MBL13-U菌株所产胶原蛋白酶(B. cereus collagenase, BCC)的分离纯化和酶学特性进行了研究。在产酶最佳发酵条件下,从发酵液中分离出分子量为38.0±1.5 kDa的纯化酶BCC。经酶学特性分析表明其富含天冬氨酸、赖氨酸和丝氨酸；最适的反应温度和pH分别为40℃和8.0；经金属离子、抑制剂和蛋白质底物对酶活影响实验,表明它是一种金属蛋白酶,对Ⅰ型胶原蛋白底物具有显著水解性。通过对比实验证实了其酶解牛骨胶原的能力显著优于标准的Ⅰ型胶原酶和常用于骨骼酶解的蛋白酶(p<0.05)；进一步证实了B. cereus MBL13-U为降解牛骨的优良菌株。
为了解菌株降解牛骨的机理,探讨了BCC对牛骨胶原蛋白的酶解动力学。实验确定了牛骨胶原蛋白最佳的提取工艺条件为：乙醚低温回流脱脂,0.48 mol/L盐酸脱钙,骨粒径为5 mm×10mm,提取介质为1%柠檬酸和1%胃蛋白酶复合液；经紫外(UV)光谱、傅里叶红外光谱(FT-IR)、差示扫描量热法(DSC)和SDS-PAGE凝胶电泳等对其结构表征,表明所提蛋白为保持完整三股螺旋结构的Ⅰ型胶原蛋白。然后以水解度(DH)为指标,实验确定了BCC酶解牛骨胶原蛋白的优化条件为底物浓度为30.0 g/mL,温度为45℃,0.35%(g/100 mL)的初始酶浓度,pH为8.0,酶解的时间为6 h；并拟合出了酶解动力学方程。
将单菌种B. cereus MBL13-U用于牛骨粉发酵制备牛骨胶原多肽(Bovine bone collagen-derived polypeptides, BBCP),研究结果表明发酵能显著提高牛骨粉溶液中游离氨基酸的含量；以水解度(DH)和胶原多肽含量为指标进行对比实验,确定预处理后的牛骨粉即骨素粉为发酵原料。经响应面分析法确定了最佳发酵条件为：接种量1%,pH值为6.0,骨素添加量为30.0 g/L,发酵温度为27℃,发酵时间为42 h,获得最大胶原多肽含量的实验值为5.54±0.09 mg/mL,骨胶原蛋白的转化率约为43%。
为进一步提高多肽得率,针对混合菌种(B. cereus MBL13-U+保加利亚乳杆菌Lactobacillus bulgaricu, Lb)发酵牛骨素粉制备BBCP进行了研究。以胶原多肽含量为指标,确定了最佳的菌种配比为MBL13-U:Lb=1:1;最佳的培养基组成为蔗糖含量10.00 g/L,牛骨素添加量30.00 g/L。在此基础上通过响应面分析法确定了最佳发酵工艺条件为：接种量2%,pH 7.5,发酵温度37°C、发酵时间48 h,获得最大胶原多肽含量的实验值为6.72±0.07 mg/mL,骨胶原蛋白的转化率约为52%。结果表明了混合菌种发酵效果优于单菌种发酵。为能指导生产,构建了混合菌种的发酵动力学模型。
为了获得螯合钙能力强的BBCP,对混合菌种发酵制备的BBCP进行了超滤膜(UF)分离研究。经过BBCP的氨基酸组成测定和UV光谱分析表明,发酵使骨中胶原蛋白降解为多肽、小分子量的短肽和氨基酸等。以膜透过速率(Tv)为指标,确定了四种超滤膜(截留分子量(MWCO)分别为10 kDa、6 kDa、4 kDa、2 kDa)在分离浓度为20 g/L的BBCP时,膜的最佳操作参数范围：压力为0.20 MPa-0.22 MPa,料液温度为30°C-35℃,料液的pH为6.0-7.0,运行周期为50 min-60 min。UF分离后发酵液中以分子量为2 kDa-4 kDa的BBCP为主,占了总肽量59.71%；对超滤各段经SDS-PAGE和Sephadex G-25凝胶层析分析分子量分布表明超滤能够较好的对胶原多肽进行分离纯化。
以超滤后的胶原多肽为基础,对BBCP-Ca的制备进行了研究,确定了钙源为牛骨钙,有机沉淀剂为无水乙醇,胶原多肽为2 kDa-4 kDa超滤片段,以螯合率和螯合物得率为指标,确定最佳螯合工艺参数为：多肽与骨钙的质量比为4:1,pH为7.0,多肽浓度为40.0 g/L,螯合温度和螯合时间分别为55℃和1.5 h,有机沉淀剂无水乙醇与水的体积比为8：1。在此条件下,得到钙的螯合率为47.85±2.24%,螯合物得率为77.89±3.32%。
对制备的BBCP-Ca理化性质和结构进行了研究。通过定性检测、成分分析、溶解度测定、持钙能力测定,确定BBCP-Ca是一种富含多肽和钙,且在水中有较好的溶解性和持钙能力较强的多肽螯合物。通过Tricine-SDS-PAGE、质谱(MS)、扫描电镜(SEM)、UV光谱、荧光光谱、IR、拉曼光谱(Raman)、园二色谱(CD)、X-射线能谱(EDS)、X-衍射(XRD)、核磁共振氢谱(HNMR)、HPLC对其结构进行测定,结果表明：螯合钙的胶原多肽大部分分子量在2000 Da-3326.4 Da之间,但仍含有部分胶原多肽的分子量<2000 Da；BBCP中氨基和羧基都参与了与Ca2+的配位结合,除此之外,还可能与骨钙之间有一定的吸附作用；BBCP-Ca的二级结构主要含有β-回折结构和无规则卷曲结构,几乎不含α-螺旋结构；且其富含钙和P元素,由结晶结构和无定型结构两部分组成,至少含有5个级分成分的一种新型螯合物。
In order to develop comprehensive utilization of livestock and poultry bone resources and enhance products added value, bovine bone was chosen as research object. The study was that the strain with high bone-degraded collagenase activity, being screened out and mutagensised from nature, was applied to ferment bovine bone powder for preparation of a novel functional calcium tonic of bovine bone collagen-derived polypeptides chelated calcium (BBCP-Ca). The main studies and results are as follows:
By self-designed screening model, strains with bone-degraded and collagense-produced ability were isolated from soil and water samples that were collected in the bone factories. The MBL13 strain with the highest bone-degraded ability was screened (enzyme activity was 25.60±0.93 U/mL). It was identified as a Bacillus cereus. B. cereus MBL13 was mutated by nitrosoguanidine (NTG) and ultraviolet (UV) complex mutagenesis and a mutant named MBL13-U with stable higher collagenase-productivity was obtained (collagenase activity was 36.80±1.39 U/mL). The enzyme activity significantly improved 43.75% compared with initial enzyme activity (p<0.05).
In order to increase the ability of the strain's producing collagenase and degrading bones, the optimal fermentation conditions for production of collagenase was investigated as guided by enzyme activity. The optimum fermentation medium was established as follows:sugar 10.0 g/L, bone gelatin 20.0 g/L, CaCl20.50 g/L, NaH2PO4-2H2O 0.50 g/L and K2HPO4-3H2O 2.50 g/L. The fermentation conditions were also optimized by response surface methodology (RSM) and the optimum conditions were inoculum 3%, fermentation temperature 37℃, pH7.0, fermentation time 36 h and medium volumes 25 mL in the 250 mL flask. The highest practice enzyme activity was 48.24±0.71 U/mL, increasing by 31.09% compared with initial fermented conditions.
From the culture supernatant under optimum conditions, B. cereus collagenase (BCC) with the molecular mass of 38.0±1.5 kDa was purified. And then the enzymatic properties were studied. After being hydrolyzed, it had high contents of amino acid of asparagine, lysine and serine. The optimum temperature and optimum pH for the enzyme activity were 40℃and pH=8.0, respectively. The results of the effects of some metal ions, inhibitors and protein substrates suggested that the purified collagenolytic protease was a member of the metalloproteases. The enzyme exhibited high hydrolytic activity for type I collagen. The comparative test indicated that BCC's hydrolysis capacity of bovine bone collagen was significantly higher than those of type I collagenase and other proteases which were commonly used in hydrolyzing bones (p<0.05). All these demonstrated that B. cereus MBL13-U was a fine strain to ferment bovine bone.
For research on the mechanism of strain degrading bones, the kinetics of BBC hydrolyzing bovine bone collagen was studied. The optimum process conditions of extracting collagen from bovine bones were defatted by reflowing ether at low-temperature,0.48% hydrochloric acid as decalcification solutions, bone particle size of 5 mm×10 mm and the compound of 1% citric acid and 1% pepsin as extraction medium. The structure of bone collagen was analyzed with UV spectrum, fourier transform infrared spectrum (FT-IR), differential scanning calorimetry (DSC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results confirmed that the extracted collagen was type I collagen, keeping three helical structure very well. The optimum conditions of BCC hydrolyzing bovine bone collagen were studied with the indexes degress of hydrolysis (DH%). The optimum conditions were 30 g/L bone collagen concentration, temperature of 45℃,0.35% (g/100 mL) the initial enzyme concentration, pH of 8.0 and hydrolysis time 6 h. Based on the experimental data, a kinetic model equation was obtained.
Bovine bone collagen-derived polypeptides (BBCP) were preparaed by B. cereus MBL13-U fermentation. The results showed that the fermentation could significantly improve the contents of free amino acid of bovine bone powder culture. Comparative experiment was performed choosing the degree of hydrolysis (DH) and concentration of polypeptides as the index. The result determined that bovine bone ossein powder was chosen as material for fermentation. The response surface showed that the optimum conditions for fermentation were inoculum 1%, pH 6.0, adding ossein amount 30.0 g/L, fermentation temperature 27℃and fermentation time 42 h. Under these fermentation conditions, the highest practice concentration of BBCP was 5.54±0.09 mg/mL, and conversion rate of bone collagen was about 43%.
BBCP were prepared by mixture strains fermentation in order to improve yield. Taking concentration of polypeptides as the index, the result indicated that the optimum conditions for preparation of BBCP were ratio of B. cereus MBL13-U to Lactobacillus bulgaricus (Lb) 1:1, sucrose concentration 10.0 g/L and ossein content 30.0 g/L. Based on these experiments, the response surface analysis indicated that the optimum fermentation conditions were inoculum 2%, pH7.5, fermentation temperature 37℃and fermentation time 48 h. Under these conditions, the highest practice concentration of BBCP was 6.72±0.07 mg/mL, and conversion rate of bone collagen was 52%-53%. The results showed that mixed fermentation was more effective than a single strain. The kinetics models of fermentation were established to guide production.
For the preparation of BBCP with the strong ability of chelated calcium, BBCP prepared by mixed strains fermentation were isolated through ultrafiltration (UF) membrane. Determination of the amino acid composition and UV spectrum analysis showed that the fermentation made the bone collagen degrade to polypeptides, small molecular mass peptides and amino acids. Taking the permeation rate as the index, the optimum separation parameters for different UF membranes (MWCO 10 kDa,6 kDa,4 kDa and 2 kDa) were that pressure 0.20 MPa-0.22 MPa, solution temperature 30℃-35℃, solution pH 6.0-7.0 and cycle time 50 min-60 min. The results of UF showed that polypeptides of MW 2 kDa-4 kDa were the main ingredient, about 59.71% of the total concentration of BBCP. The analysis of SDS-PAGE and sephadex G-25 gel chromatography on molecular weight distribution indicated that UF was an efficient method of separation and purification for BBCP.
Based on collagen peptides separated by UF, the preparation of the BBCP-Ca was studied. The result confirmed that calcium resource was bone calcium, organic precipitant was anhydrous ethanol, and molecular weight of collagen peptide ranged from 2 kDa to 4 kDa. Taking the chelation rate and the chelate yield as indexes, the optimum chelating parameters were that BBCP/bone calcium(w/w) was 4:1, pH was 7.0, concentration of BBCP was 40 g/L, temperature and time were 55℃and 1.5 h, organic precipitant/water (V/V) was 8:1. Under these conditions, chelation rate was 47.85±2.24%. chelate yield was 77.89±3.32%.
The physicochemical properties and structure characteristic of BBCP-Ca were researched. By qualitative detection, composition analysis, solubility test and ability of holding calcium determination, the results proved that BBCP-Ca was polypeptides chelate with high content of calcium and polypeptides, good solubility in water and high ability of holding calcium. The structure properties of BBCP-Ca products were characterized by Tricine-SDS-PAGE, MS, SEM, UV spectrum, Fluorescence, IR, Rman, CD, EDS, XRD, HNMR and HPLC measurements. The results indicated that the molecular weight of most chelated BBCP ranged from 2000 Da to 3326.4 Da, but also some collagen polypeptides MW<2 kDa. The amino group (-NH2) and the carboxyl group (-C=O) of BBCP participated in chelated calcium ion reaction. Except for these coordinate bonds, it may exist a certain adsorption effect between BBCP and bone calcium. The secondary structure of BBCP-Ca was mainlyβ-sheet and random coil, but the content of a-helix was very low. BBCP-Ca was a new type of chelate which were rich in mineral contents of Ca and P, consisted of crystalline structure and amorphous structure and contained at least five components.
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