Rearch On Screening The Bovine Bone-degraded Bacteria And Preparation Of Collagen-derived Polypeptides Chelated Calcium By Fermentation

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.