Study On The Preparation And Calcium Bioavailability Of Ctenopharyngodon Idellus Bone Fermentation Liquid By Inoculating Leuconostoc Mesenteroides

With the rapid development of aquatic product processing in China, the byproducts of aquatic product processing haven’t been highly utilized today, about 50-60 percent of the fish waste is discarded, which results in a waste of protein source and the pollution of environment. Fish bones have rich calcium, and they also caused a waste of calcium source. Several studies have shown that lactic acid bacteria can transform calcium in fish bone to soluble calcium through fermentation, and for microorganisms, the nitrogen source is generally the most expensive resource in the medium, several studies have shown that fish waste can provide the nitrogen source for the growth of lactic acid bacteria. Therefore, the study on the preparation and bioavailability of fish bone fermented by lactic acid is a new way to study filling calcium products. Ctenophaiyngodon idellus bone was determined as the final material in this paper through screening raw material. Leuconostoc mesenteroides was determined as experimental Strains. The processing of softening, homogenizing, enzymatic hydrolysis on Ctenopharyngodon idellus bone and the condition of enzymatic hydrolysis were also determined. The fermentation processing was optimized by response surface methodology(RSM). A comprehensive calcium composition analysis of fermentation liquid was studied in this paper, the effects of fermentation liquid on calcium absorption and bone quality of rats were investigated, and the bioavailability of fish bone fermentation liquid in vivo was also analyzed.The main contents of this paper are as follows:1. The selection of experimental Strains and optimization of processing conditions on Ctenopharyngodon idellus boneAfter the effects of softening, enzymatic hydrolysis on the colony unit and soluble calcium content of fermentation liquid were investigated, softening, homogenizing, enzymatic hydrolysis were determined as the processing on Ctenopharyngodon idellus bone. After the effects of enzyme, hydrolysis time on the colony unit and soluble calcium content of fermentation liquid were tested, the best processing on Ctenopharyngodon idellus bone determined was:softening (121℃.20 min), homogenizing (medium speed,1 min), flavor protease enzymatic hydrolysis. temperature 50℃, pH 6.0,2.5:100 (enzyme:substrate, w/w, protein basis), enzymatic hydrolysis time 2.5 h. Under the optimized condition, the colony unit reached 3.27×1010, soluble calcium content was 85.28 mg/L, which have been significantly improved compared with the colony unit before optimization 1.04×109, soluble calcium content 20.61 mg/L, the result proved that the feasibility of Leuconostoc mesenteroides using the protein and calcium resource in Ctenopharyngodon idellus bone through fermentation.2. The optimization of fermentation processing by RSM, the calcium composition analysis of fermentation liquidThe soluble calcium content and calcium conversion rate were taken as the indexes respectively, the optimized fish bone concentration, carbon source, carbon source concentration, beginning pH, kinds of inorganic salt were determined by single factor experiment, based on the results on the single factor experiment, the three factors(fish bone concentration, carbon source concentration, beginning pH) were optimized by RSM. The optimized fermentation processing were:fish bone concentration 89.58 g/L, carbon source concentration 15.62 g/L, the fermentation beginning pH 5.25, and the calcium conversion rate is at 5.56%. The calcium composition was analyzed by high performance liquid chromatography(HPLC), solid phase micro extraction and gas chromatography mass spectrometry(GC-MS). The results showed that the soluble calcium(451.75 mg/L,11.29 mmol/L) in fermentation liquid existed in form of calcium lactate(3.89 mmol/L), calcium acetate (6.21 mmol/L), amino acid calcium, and small peptide calcium compounds together.3. The study on calcium bioavailability of Ctenopharyngodon idellus bone fermentation liquidThe calcium-deficiency model was established by three weeks’low calcium diet feeding. Then normal growth experiments and calcium metabolism experiment were established through different diet feeding of rats. The bioavailabiliy of fermentation liquid in rats were investigated by measuring the body weight gain of rats, calcium metabolism indexes, serum indexes and femur indexes. The results showed that the fermentation liquid could promote the growth of rats, the body weight gain of rats reached 41%, significantly higher than that of the low calcium group 31.88% (P <0.05); Many kinds of organic calcium in fermentation liquid got good absorption, the apparent absorption of rats reached 73.92%, which was significantly higher than other groups; Many kinds of organic calcium could also get good utilization and return the serum calcium, alkaline phosphatase(ALP) levels, femur weight, bone density and femur calcium content to normal levels in rats, and the fermentation liquid could maintain inner environment of organism and the stability of bone quality. The results showed that Ctenopharyngodon idellus bone fermentation liquid has good calcium bioavailability, which is expected to become a new filling calcium product.