Study On The Bio-characteristics Of Micro-smashed Meat And Bone Meal Fermented By Lactobacillus And Its Function Of Improving Bone Density

Calcium,which accounts for 1.5～2.0 percent of man's weight,is a kind of important metal element for people's health and longevity.It play a important role in physiological function, such as adjusting nerve or muscle stress,nerve impulse transmission,heart rhythm maintenance, blood solidification,cell adhesion and so on.So,short of calcium in body may induce many diseases.Of course,the absorptivity of calcium in people's body is influenced by many factors. Many overseas scholars also point out that people in whatever complexion are threatened by shorting of calcium.The recommended nutrients intake(RNI)of calcium from National Academic of Science is 1,000-1,200mg for an adult every day and that from Chinese nutrition society(1988)are 800mg for an adult and 1,200mg for the pregnant woman,the youth and the old every day.However,according the forth nutrition investigation(2002),the actual intake of calcium in our nation is generally low,that is half of RNI.So,developing calcium supplement is of significance.The bone,which accounts for 20～30 percent of the livestock's weight and consists of much protein,lipid and mineral,is a kind of valuable byproduct in meat industry.The calcium in the bone exist as hydroxyapatite crystalloid[Ca₁₀(PO₄)6(OH)₂]and unformed phosphate (CaHPO₄)which are wrapped by much collagen fibre.So,the bone need to be dealt with in order to destroy the organ and increase the absorptivity of calcium,when it is used as calcium source by man.When the microbe grow and propagate,the commutation of substance and energy take place between them and their surroundings.The metal ion acts as either its growth promoter or inhibitor.The quantity of metal ion absorbed by single cell of microbe could account for 0.5～1 percent of its dry weight,but the microbe has the characteristic of rapid growth speed and large propagation quantity.Moreover,the microbe could produce macromolecule substance that could combine metal.Originally,people take advantage of microbe to enrich heavy metals in the field of sewage disposal,and then the similar technique is used in food field to enrich many metals necessary for man,such as iron,zinc and selenium.According to many reported research,they have not only better absorptivity and availability,but also increase their security.Lactobacillus is a kind of beneficial bacteria,and it can prevent deleterious intestinal bacteria producing deleterious substance,stimulate phagocytosis of phagocyte,enhance the body's immunity and reduce lipid and cholesterin in blood.Furthermore,according to some research,Lactobacillus can enrich many metal iron,such as Fe²⁺.At present,there are more and more research about foods made from MBM abroad,but few about functional food.Furthermore,there is no report about using MBM fermented by Lactobacillus to produce the functional food which has the function of improving bone density. For the security problem,a large number of MBM are used in fodder and fuel industry in some courtries,therefore this wastes much protein,calcium and phosphorus in the bone that doesn't be taken good use of.In this paper,Our research,the functional food made from livestock's bone by the means of micro-smashing,Lactobacillus fermenting and enzymatic hydrolyzation are systematically studied.The are mainly three aspects of research:First,in order to establish the feasibility of fermenting MBM by Lactobacillus,the action of calcium enrichment with Lactobacillus and the work of screening out the good stain of Lactobacillus are investigated;Second,in order to provide theoretical and practical foundation of producing functional food with fermented MBM, the process of micro-smashed hog MBM and some of its characteristics,as well as the fermenting procedure and the function of fermented MBM,are studied;Third,the process and prescription of function food mainly made of fermented MBM,and its hygienic stability test, toxicological test and functional test are all studied,thus the security and the function are cleared about.The detailed research contents and results as followes:1.The study on the enriching action of calcium with LactobacillusWith studying the ability of growth,producing lactic acid and enriching calcium of six strains of Lactobacillus(Lactobacillus Bulgaria LB,Lactobacillus casei LC,Lactobacillus brevis Lb,Lactobacillus lactic LL,Lactobacillus acidophilus LA,Lactobacillus Plantarum LP),the result indicates Lactobacillus casei is better in all ability above.So,Lactobacillus casei is used as experimental strairt to study the calcium enriching action.When in MRS with calcium concentration of 1.2mg/mL(using calcium chloride as calcium source),the quantity of enriched calcium is 1.01μg/mg(based on wet microbe cell),and 78 percent of that is enriched in logarithmic phase;the high calcium concentration(12mg/mL)can retard the growth of Lactobacillus casei,and when calcium concentration ranges from 1mg/mL to 8mg/mL,the quantity of enriched calcium increases but increasing extent decreases,from 0.98μg/mg to 1.37μg/mg(based on wet microbe cell).When in MRS with calcium concentration of 3mg/mL(using calcium chloride,calcium phosphate and calcium carbonate as calcium source respectively),the quantity of enriched calcium is 1.2μg/mg with calcium source of calcium chloride,which is higher 0.3μg/mg and 0.22μg/mg than with calcium source of calcium phosphate and calcium carbonate respectively(based on wet microbe cell);When temperature is 42℃,the quantity of enriched calcium is higher than 37℃;The 59 percent of enriched calcium is in the cellular wall,and 41 percent of that in cytoplasm;When in simulant gastric juice,the Lactobacillus cell can release dissociative calcium with a postponing effect.2.The study on the process of micro—smashed hog MBM and some of its bio-characteristicsThe different process is investigated and the optimal process is gotten.The results indicate: When the micro—smashing machine goes along smoothly,the optimal process of fresh MBM is:the ratio of added water(-4℃)and hog bone is 1:1,and the method of adding is two times; the parameters of smashing machine and micro—smashing machine is two round and one round respectively;after first micro—smashing,the size of most bone particle is about 90μm, and after second micro—smashing,most of bone particle is about 55μm,so micro—smashing of two times is adopted;the fresh MBM contains 75%of water,12.3%of protein and 5.2%of lipid,and there are 783.5mg calcium in every 100g fresh MBM.The quantity of added water, temperature,added substance,pH,high-temperature-high-pressure process and enzymatic hydrolyzation can influence the viscosity of fresh MBM,and the latter two factors is more distinct in reducing it.After high-temperature-high-pressure process and hydrolyzed by papain, the amino acid nitrogen in MBM 52.65mg/100g to 123.4 mg/100g and the content of dissociative calcium is 1.3%,increasing about one times,but they can't change its pH value distinctly.3.The study on the fermenting process with Lactobacillus and the function of fermented MBMThe key points of fermentation are gotten from the study of the significant factors effecting on fermentation and the orthogonal experiment:4%of inoculation of two strains (Ll:Lp=1:1),5%of compound carbon source(glucose:sugar=3:7),15%of micro-smashed tomato juice,fermenting 16h in 42℃after 20h in 37℃.Especially,before sterilizing,the fresh MBM is hydrolyzed with 0.03%of papain 1h in 65℃,at pH 6.88,and removing most of lipid after cooling.During the fermentation,some of the physical and chemical characteristics are changed. The amino acid nitrogen in MBM decreases in the beginning and then increases a little(before fermentation:123.4 mg/100g,after 32h of fermentation:58.67 mg/100g,after 48h of fermentation:66.8 mg/100g).After fermentation,the size of bone particles in MBM is reduced distinctly.The results of analyzing the elementary calcium speciations with the 0.45μm film and LSA—10 adsorption colophony indicate that fermentation with Lactobacillus plays an important role in increasing the dissociative calcium content in the MBM and it increases from 12.6 mg/100g to 138.8 mg/100g,which accounts for 17.6%of the whole calcium in MBM. Especially,the organic calcium increases from 5.6 mg/100 to 113.1mg/100g,which accounts for 81.5%of the whole dissociative calcium.The functional test is carried out to investigate the function of improving bone density by feeding fresh MBM and fermented MBM to the male Wistar rats.The results indicates:the fermented MBM is significantly higher than the fresh MBM in improving the bone density of male Wistar rats' femur(P＜0.05).So,it provides the theoretic feasibility and necessity of producing calcium supplement food from the fermented MBM.4.The study on the functional food——biologic bone calcium capsuleThe functional food is prepared mainly with fermented MBM by spray granulating technique,and the technics and prescription is established.Spraying liquid is 2.8kg of fermented MBM adding with 0.6mg of vitamin D₃,and the mother granule is 1.4kg of calcium carbonate and 60g of CPP.After the mother granule has been pre-dried for 30min in the floating bed,the spraying liquid begins to spray into the floating bed at the pulse frequency of 60Hz,and the temperature of entering hot wind is 80℃. When preparing the capsule,the filling quantity and pressure is 0.25g per capsule and 0.6 MPa. The parameter of automatic bottle filling is:the filling scale is100 capsule per bottle,the filling speed is 50 bottle/min and the sealing temperature is 180℃.In the functional food product,30%of ingredient is bone powder which come from fermented MBM,and there are large amount of bone protein(＞15%),including amino acid and peptide.There are about 280mg calcium(12mg from fermented MBM),3μg vitamin D₃ and 30mg CPP in every gram product.The specification of the functional food recommends that people take it three times every day and two or three capsules once.5.The study on the hygienic stability of biologic bone calcium capsuleThe results of hygienic stability test are:there are 27.5g calcium,290μg vitamin D₃ and 17g protein in every 100g of biologic bone calcium capsule.The other results of testing index are:As＜0.1mg/kg,Hg＜0.1mg/kg,Pb＜1.5mg/kg,cfu＜180cfu/g,yeast＜5cfu/g,mold＜5cfu/g, MPN＜30 MPN/100g,pathogen not being detected(including salmonella,Shigella,Staph. aureus,hemolytic streptococcus).So,it indicates that the functional food——biologic bone calcium capsule,measure up according to the "The Management Method of Functional Food, China".The result of heat preservation test in 37℃and 75%of RH for 3 months,indicates that the product has a good hygienic stability. 6.The study on the toxicological test of biologic bone calcium capsuleThe toxicological test of biologic bone calcium capsule consists of acute toxicity test,three mutation tests(including Ames test,mice bone marrow polychromatic erythrocyte micronucleus test and mice sperm abnormality test)and 30 days feeding test.The results of acute toxicity test indicates:rats and mice(male and female)have a MTD(＞15000mg/kg.bw)for biologic bone calcium capsule,and the product is nontoxic;Three mutation tests and 30 days feeding test all reveal negative results.7.The study on the functional test of biologic bone calcium capsuleThe functional test is carried out to investigate the function of improving bone density by feeding biologic bone calcium capsule to the male Wistar rats.The results indicates:the product hasn't significantly difference in body weight between all groups of rats.But at feeding level of 375 mg/kg.bw and 1125 mg/kg.bw,biologic bone calcium capsule is significant higher in improving the bone density of male Wistar rats' femur(P＜0.05)compared to carbonate calcium of same dose.So,according to the "The Technical Criterion of Test and Evaluation about Functional Food,China,2003",biologic bone calcium capsule has a function of improving bone density.