| China is a large pig-breeding country with the production of live pig leaping into the front ranks of the world, and thus has abundant resource of pig bone. pig bone contains about 11% protein in which collagen protein accounts for 90% of the protein content. Collagen protein is a soluble protein with high nutritional quality and has the ability to improve the bioavailability of Ca in the body due to its adsorption for Ca2+. pig bone contains 19.3% calcium (Ca) and 9.39% phosphorus (P) with the Ca to P ratio approximately being 2:1 which is the most suitable ratio for absorption and utilization by human body. The saturated fatty acids (palmitic acid and stearic acid) to unsaturated fatty acids (oleic acid and linoleic acid) ratio is close to 1:1 that is the ideal composition ratio for absorption by human body. In addition, pig bone contains bioactive compounds such as composite phospholipids and neurotransmitter.For a long time, a vast number of pig bones have been going out of use or being processed into low value-added bone oil, bone glue and animal feed, thus resulting in huge environment pollution and resource waste. So it is of important significance to strengthen the development of deep processing of pig bones. bone extraction comprises nutrients extracted from fresh bones. It is an ideal alternative to monosodium glutamate and shows great market potential due to its higher safety, absorbability. This study was conducted to improve the existing processing technics of bone extraction and to develop new bone extraction product with high quality. The nutrient contents and bioactivities of the bone extraction product were also determined in this study. The research details and results are shown as follows:1. Study on the process conditions for boiling bones in the production of bone extraction Based on the investigations on single-factor experiment, a three-factor and three-level response surface design was applied to establish a quadratic regression mathematical model of response value vs. variables (factors) for predicting the theoretic extraction rate. The optimal parameters for extraction were thus determined using the model as follows: extraction pressure, 0.11 Mpa; extraction time, 226 min; liquid to bone ratio, 3:1. The bone extraction with high contents of proteins and free amino acids produced under these conditions could be used for the productions of hydrolyzed animal protein, seasoner and essence with meat flavor. The mathematical model developed in this study possesses good ability to predict factor effects on response variables and contributes to optimization of extraction parameters for the production ofbone extraction.2. Study on the enzymatic hydrolytic conditions in the production of bone extraction(1)Flavourzyme, trypsinase, Alcalase, papain, pepsin, AS.1398 neutrase, protamex and alkaline proteinase were individually used to hydrolyze bone protein. The AS.1398 neutrase was selected as the optimal enzyme for the hydrolysis of bone protein based on the degree of hydrolysis (DH), taste and clarity of hydrolysates.(2)Based on the single-factor experiment, a multiple linear regression orthogonal experimental design was employed to optimize the hydrolysis parameters of AS.1398 nuetrase. The optimal enzymatic hydrolytic parameters were as follows: hydrolysis temperature 55℃, pH value 8.0, enzyme addition 6000U/g,hydrolysis time 3h. The average DH obtained under the best experimental conditions mentioned above was 17.54%.(3) A uniform experimental design was applied to investigate the optimal conditions by step wise enzymatic hydrolysis with neutrase and Flavourzyme. The optimal enzymatic hydrolysis parameters by Flavourzyme were determined as enzyme addition 5000U/g, pH value7.6, hydrolysis temperature 55℃, hydrolysis time 120min, and the DH with 21.62% was obtained under these conditions. On the basis of investigations of single-factor experiment and L9(34) orthogonal experimental design, a quadratic orthogonal rotational combination design with three factors was implemented to optimize the experimental conditions by simultaneous enzymatic hydrolysis with neutrase and Flavourzyme, and the optimum enzymatic hydrolysis conditions derived via orthogonal rotational combination design were: substrate concentration 15%, neutrase dosage 6000 U/g, Flavourzyme dosage 5000 U/g, temperature 55℃, reaction time 4.5h, and pH 8.0. The DH obtained under the optimum conditions was 22.35%.(4)As judged by comparative experiments, the hydrolysis performance obtained by simultaneous enzymatic hydrolysis was better than by step wise enzymatic hydrolysis, in which the hydrolysis time could be shortened. The simultaneous enzymatic hydrolysis with neutrase and Flavourzyme was thus chosen based on the enzymatic hydrolytic performance and production time.3. Preparation of refined bone extraction powder by ultrasonic-assisted spray drying The composition and production rate were affected by entering air temperature, air flow speed and liquid flow speed. Based on the index of product composition (ash, lipid and protein), the range of variables of L9(34) orthogonal experimental design was determined by single-factor experiment, and then the optimum process parameters of spray drying were obtained as follows: entering air temperature 160℃, entering flow of substrate 6 ml/min, air flow 38m3/h. Three validation experiments were performed to produce refined bone extraction powder under the optimum process conditions. The resulted products contained 56.3% protein, 26.8% ash and14.5% lipid, and the average production rate was 32.7%. As compared to the results obtained by orthogonal experiment, the products contained higher protein and lower ash and lipid contents, and the production rate was relatively perfect, suggesting that the process parameters obtained were feasible. The products had uniform degree of fineness, uniform color and lustre, stable content, good solubility and low moisture-absorption ability, which shows good market potential and considerable economic value.4. Study on composition analysis and physical properties of refined bone extraction powder The protein, lipid, ash, moisture contents were 56.3%, 14.5%, 26.8% and 2.6% respectively, in the refined bone extraction powder. The indices of heavy metal, total arsenic, bacterial count, coliform in the product were in the range set in industry standards. The assay for Ca in the product had excellent reproducibility, precision and low relative standard deviation. The analysis result of Ca obtained under the assay conditions was reliable. The Ca content in product was measured as 249.1 mg/100g. The molecular weight distribution of the peptide in the products was determined in this study. The polypeptides with molecular weight above 1000 Da, ranging from 1000 to 544 Da, from 149 to 544 Da were 40.12%, 10.58% and 43.69%, respectively, indicating that the majority of the peptide composition is peptides with molecular weight under 1000 Da which account for 59.88%.5. Study on the supplemental effect of refined bone extraction powder on Ca status of rats (1) The bone extraction obtained by enzymatic hydrolysis of bone protein are rich in peptide-Ca, amino acid-Ca and other nutrients, and shows good solubility. The Ca metabolism status, change in weight and feed intake of rats were investigated after the rats were fed diets supplemented with bone extraction. Significant difference in apparent Ca absorptivity was observed in rats fed diets supplemented with high dosage of bone extraction compared with those in blank and control groups rats (p<0.01). No significant difference in body weight was observed between rats in blank, control, low dosage of bone extraction and medium dosage bone extraction groups (p>0.05). However, significant difference existed between high dosage group and the other four groups (p<0.05). The feed intake of rats in blank and control groups was significantly lower than in medium and high dosage groups (p<0.05).(2) No significant differences in serum Ca and cholesterol contents were observed between five groups (p>0.05). Serum Ca to P ratio in each group rats ranged from 0.898 to 1.005, which is in the normal range. Serum triglyceride content in rats fed diets supplemented with different dosage of bone extraction was significantly lower than in rats fed control diets (p<0.05). Significant difference in the activity of alkaline phosphatase in serum was observed between blank group rats and other four group rats (p<0.01). The activity of alkaline phosphatase in serum of rats fed bone extraction tended to be higher than that in control group rats (p>0.05) with the activity being the highest in high dosage group rats.(3) The thighbone of rats in bone extraction groups was very significantly longer than that of blank group rats (p<0.01) and significantly longer than that of control group rats (p<0.05). The thighbone diameter of rats inbone extraction groups tended to be bigger than that of blank and control groups rats but without significant difference (p>0.05). The wet weight of thighbone of rats inbone extraction groups was significantly higher than that of blank and control group rats (p<0.05 or p<0.01). The bone density of rats fed with bone extraction was higher than that of rats in blank group (p<0.05 or p<0.01), the bone density of rats fed medium dosage of bone extraction was significantly higher than that of control group rats (p<0.05), and the bone density of rats fed high dosage of bone extraction was very significantly higher than that of control group rats (p<0.01). Significant difference in bone Ca content was observed in rats fed bone extraction as compared to that of blank group rats (p<0.05 or p<0.01).(4) Micrographs of pathological tissue sections of shankbone metaphyseal showed that the growth and absorption of bone was active and osteoporosis was observed in rats fed diets with low Ca content. The supplemental efficiency of bone extraction on Ca status in rats was increased with the increase of bone extraction dosage.(5) According to Test Methods and Biological Functional Validation procedure for Nutraceuticals, all the results derived from animal experiment were positive, suggesting that the bone extraction could promote the bone development of rats, and the bioavailability of bone extraction is higher than that of calcium hydrogen phosphate. In a conclusion, the bone extractionis a Ca-strength product with high-quality, and can prevent osteoporosis. On the other hand, bone extraction possesses the function in decreasing triglyceride level of rats.
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