CN102871122B - Method for extracting bone calcium through defatting fermentation and bone gla protein - Google Patents
Method for extracting bone calcium through defatting fermentation and bone gla protein Download PDFInfo
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Abstract
The invention discloses a method for extracting bone calcium through defatting fermentation and bone gla protein prepared by means of the method. The method comprises the following steps of three-time defatting, two-time centrifugation, fermentation and protease hydrolysis. Fat in bone meal prepared by adopting a defatting process is completely removed basically, so that the fat content in calcium obtained by means of enzymolysis is lower than 0.1%, and the intracellular free calcium content is increased. The bone gla protein is safe and healthy to consumers with cardiovascular diseases, and economical burden to the consumers is not increased.
Description
Technical field
The present invention relates to bone calcium extraction process, belong to food processing field.
Background technology
Along with social aging, in western countries, the illness rate of osteoporosis occupies first of metabolic bone disease.The misery that osteoporosis illness brings patient and family is self-evident.According to statistics, China approximately has the patients with osteoporosis that exceedes 100,000,000 at present, expects the year two thousand fifty will be increased to 200,000,000 1 thousand ten thousand people.Cause the factor of increasing people's calcium deficiency: live and work rhythm is accelerated, operating pressure is large and live irregular, the structure that is not careful in one's diet reasonably combined, calcium was taken in and was less than 600 milligrams of persons and can thinks calcium insufficiency of intake every day, easily caused the shortage of calcium in body; Some a middle-aged person's work strains, lack self health consciousness, think little of outdoor activity, and proper interior synthetic vitamin D is reduced, and affect absorption and the utilization of calcium; Unbalanced or the necessary nutrient of shortage human body is taken in nutrition.
The calcium agent of existing market mostly is the synthetic calcium of industry.Industrial synthetic calcium agent nutritional labeling is single, is difficult to meet many-sided nutritional need in bone growth metabolic process, and effect of supplemented calcium is undesirable.Animal Bone is rich in the several kinds of mineral elements such as calcium, phosphorus, iron, magnesium, and with people's bone photo seemingly, skeleton cell has stronger affinity to homologue's cell, thereby utilization rate is higher.
Chinese patent CN1087793A and U.S. Pat 6342252B1 disclose a kind of technique of utilizing enzyme solution to extract calcium element from ox bone.Enzymolysis bone calcium has suitable, nutritious, the calcareous absorption rate high of calcium phosphorus ration.From market sale situation, enzymolysis bone calcium is subject to liking of consumers in general deeply, takes the osteoporotic successful of rear improvement, has obtained significant economic benefit.
But in the bone calcium that above-mentioned technique makes, degreasing method is mainly to implement in boiling mode, is difficult to remove the fat of high-load in sclerotin, cause final product fat content higher.For the consumer with angiocardiopathy, too high fat intake obviously can raise morbidity hazards.Therefore, from animal skeleton, extract in the process of calcium element, remove excess fat, reduce the onset risk of angiocardiopathy, become technical staff's urgent problem.
Summary of the invention
The extraction process that the object of the present invention is to provide a kind of new enzymolysis bone calcium, described technique can better be removed the fat in sclerotin compared with prior art.
Another object of the present invention is to provide a kind of more healthy calcium supplementing product production technology, and the calcium supplementing product that described technique makes has lower fat content.
Another object of the present invention is to provide a kind of calcium supplementing product with the market competitiveness, and described product is not because the design of degreasing process increases production cost.
For achieving the above object, the present invention adopts following technical proposal to realize.
The present invention adopts three degreasing modes to remove animal oil, is 60～80 ℃ for the first time, 45～75min; 80～90 ℃ for the second time, 30min; Lipase enzymolysis for the third time.Three degreasings, except having the fatty effect of removing in sclerotin, also have effect of sterilization simultaneously.For strengthening degreasing effect, the present invention is preferably broken to bone meal 5～40mm particle, the too small production cost that increases of particle, and the excessive degrease effect of particle is poor.Inventor, through test of many times and experience, finally determines that 5～40mm granulometric range reaches the best cost performance of bone fat and cost.
After alcohol steep degreasing, the present invention also adopts hot-water soak rinse method to remove the fat existing in aggregate; After rinsing, again with 4000～8000r/min, centrifugal 30min, can remove most fat in bone meal like this.
For further removing remaining fat, process choice of the present invention again adds alkaline lipase to carry out fat remaining in enzymolysis aggregate after centrifugal.Alkaline lipase A, B, by 1.5～3: 1 compound lipases mixing, account for aggregate than 0.1～0.4%, and 20～30 ℃, hydrolysis 40～60min.After enzymolysis, secondary centrifuging, further removes remaining trace fat.
After above-mentioned defatting step, the aggregate of the method that the present invention adopts fermentation after to degreasing carries out pretreatment, and then carries out enzymolysis and extraction calcium element.
For strengthening ferment effect, the present invention is preferably broken to bone meal 5～40mm particle, the too small production cost that increases of particle, and the excessive effect of particle is poor.Inventor, through test of many times and experience, finally determines that 5～40mm granulometric range reaches the best cost performance of the extraction of calcium element and cost.
Enzymolysis primary fermentation of the present invention adopts lactic acid bacteria and aggregate by certain weight proportion, and 35～50 ℃, fermentation 12h.Weight proportion, concentration, fermentation temperature and the time of lactic acid bacteria in zymotic fluid of lactic acid bacteria and aggregate have important impact to the product Free Calcium content finally making, and the weight proportion of lactic acid bacteria and aggregate and the lactic acid bacteria concentration in zymotic fluid having the greatest impact to free calcium.Inventor, according to oneself working experience for many years, determines after repetition test that it is 0.01～0.04% that lactic acid bacteria accounts for aggregate weight ratio, under the condition of aggregate and water w/v 1: 3～5, can reach the best cost performance of input and output.
Inventor provides great many of experiments, it is unexpected that discovery various lactobacillus ferment according to certain proportioning combination, final product Free Calcium content improves more than 8% with certain lactobacillus-fermented more separately, and it is better to adopt bifidobacterium lactis (B.lactis), lactobacillus bulgaricus (L.bulgaricus) and three kinds of lactic acid bacterias of Lactobacillus helveticus (L.helviticus) to carry out ferment effect; Adopt these three kinds of lactic acid bacteria proportioning after fermentation effects best, in the time that three kinds of lactic acid bacterias combine with weight ratio at 1: 3: 2, after the more single lactobacillus-fermented of enzymolysis liquid Free Calcium content, improve 10%～30%.
Enzymolysis of the present invention adopts secondary enzymolysis method, and after screening, final discovery is carried out enzymolysis with pepsin and papain and had better effect compared with other protease.Certainly, select other protease to carry out enzymolysis and also can realize object of the present invention.Enzymolysis scheme best in extraction process of the present invention is pH value 1～3, adds pepsin, and 35～40 ℃ of hydrolysis temperatures stir 1～2h, and pH value 6～7, adds papain, 40～60 ℃, stirs 1～2h.
It is 0.04～0.08% for good that hydrolysis bone meal two kinds of protease used respectively account for aggregate ratio.
The alkaline lipase that the Penicillium expansion PF868 that alkaline lipase A is developed by Biological Engineering College of Fujian Normal University produces, by Shenzhen, Lv Weikang bioengineering Co., Ltd provides; Alkaline lipase B adopts biotechnology to be refined by fungi fermentation and the alkaline lipase that produces, and by Haining, Jin Chao Industrial Co., Ltd. provides.
The various formulations that in composition of the present invention and any or more than one pharmacies, auxiliary material is mixed as starch, dextrin, lactose, microcrystalline cellulose, HPMC, polyethylene glycol, dolomol, superfine silica gel powder, xylitol, lactitol, glucose, glycine, sweet mellow wine, glycine etc., for example, can be made into tablet, sustained release tablets, dripping pill, granule, capsule, fine granule.Preferred dosage form is tablet or granule.
Adopt in the bone meal that degreasing process of the present invention makes, fat is completely removed substantially, makes in calcium agent that enzymolysis obtains fat content lower than 0.1%.Product safety and health more concerning having the consumer of angiocardiopathy, but financial burden therefore do not increased again.
Test example
Further illustrate beneficial effect of the present invention by some concrete experimental datas below, all embodiment of the present invention all can make close therewith experiment effect, below data just illustrate.
1, materials and methods
1.1 ox bones: be purchased from Hebei Fu Chengwufeng food limited company
1.2 samples 1: the sample that uses ox bone to obtain according to Chinese patent CN1087793A; Sample 2: the sample that uses ox bone to obtain according to U.S. Pat 6342252B1; Sample 3: according to " variation of ultramicro grinding yak bone paste free calcium and amino-acid nitrogen after fermentation and enzymolysis " (Chen Dan, Zhang Chuanlin etc., total the 178th phase of " China brewages " the 1st phase in 2008) in the sample of " 1.5 lactobacillus-fermenteds are processed yak bone pastes ... the concentration that ox bone mud is 10% with distilled water diluting ... ferment 36 hours " use ox bone acquisition; Sample 4: the sample that uses ox bone to obtain according to the embodiment of the present invention 4.
1.3 methods: adopt atomic absorption spectroscopy determination free calcium content.
2, result
The free calcium content of 2.1 samples 1 is 170.05mg/100g, and the free calcium content of sample 2 is 341.11mg/100g, and the free calcium content of sample 3 is 1934.83mg/100g, and the free calcium content of sample 4 is 2302.67mg/100g.
2.2 through Data Comparison analysis, and the free calcium content of the prepared product of the present invention, compared with sample 1, sample 2, has the raising of highly significant; Compared with sample 3, tool increases significantly, and increase rate reaches 19.01%.
The specific embodiment
Preparation culture medium: MRS culture medium: peptone 1%, beef extract 1%, yeast extract 0.5%, glucose 2%, dipotassium hydrogen phosphate 0.2%, sodium acetate 0.5%, magnesium sulfate 0.02%, manganese sulfate 0.005%, Tween-80 0.1%, Triammonium citrate 0.2%, PH5.5～6.0,115 ℃, sterilizing 30min.
Embodiment 1
Get animal skeleton and be crushed to 40mm particle, add ethanol, 80 ℃, lixiviate 75min, abandons supernatant; 90 ℃ of hot-water soak rinsing 30min, by aggregate 8000r/min, centrifugal 30min, abandons supernatant; Aggregate after centrifugal is added water, and aggregate and water w/v 1: 5, adjust pH value 10, adds alkaline lipase A, B by the compound lipases of mixing in 1.5: 1, accounts for aggregate than 0.4%, 30 ℃, and hydrolysis 60min is centrifugal, removes supernatant; Bifidobacterium lactis (B.lactis), lactobacillus bulgaricus (L.bulgaricus) and Lactobacillus helveticus (L.helviticus), three kinds of lactic acid bacteria weight ratios 1: 3: 2; Aggregate and water w/v 1: 5, add 5% sucrose of gross weight, and it is 0.04%, 50 ℃ that lactic acid bacteria accounts for aggregate weight ratio, fermentation 12h, 120 ℃ of sterilizing 30min; Add hydrochloric acid adjust pH 3, add pepsin, accounting for aggregate ratio is 0.08%, and 40 ℃ of hydrolysis temperatures stir 2h, adds alkali and regulates pH value to 7, adds papain, and accounting for aggregate ratio is 0.08%, 60 ℃, stirs 2h, heats the enzyme that goes out; Add alkali neutralization, dry, to obtain final product.
Embodiment 2
Get animal skeleton and be crushed to 5mm particle, add ethanol, 60 ℃, lixiviate 45min, abandons supernatant; 80 ℃ of hot-water soak rinsing 30min, by aggregate 4000r/min, centrifugal 30min, abandons supernatant; Aggregate after centrifugal is added water, and aggregate and water w/v 1: 3, adjust pH value 8, adds alkaline lipase A, B by the compound lipases of mixing in 3: 1, accounts for aggregate than 0.1%, 20 ℃, and hydrolysis 40min is centrifugal, removes supernatant; Bifidobacterium lactis (B.lactis), lactobacillus bulgaricus (L.bulgaricus) and Lactobacillus helveticus (L.helviticus), three kinds of lactic acid bacteria weight ratios 1: 3: 2; Aggregate and water w/v 1: 3, add 5% sucrose of gross weight, and it is 0.01%, 35 ℃ that lactic acid bacteria accounts for aggregate weight ratio, fermentation 12h, 120 ℃ of sterilizing 30min; Add hydrochloric acid adjust pH 1, add pepsin, accounting for aggregate ratio is 0.04%, and 35 ℃ of hydrolysis temperatures stir 1h, adds alkali and regulates pH value to 6, adds papain, and accounting for aggregate ratio is 0.04%, 40 ℃, stirs 1h, heats the enzyme that goes out; Add alkali neutralization, dry, to obtain final product.
Embodiment 3
Get animal skeleton and be crushed to 20mm particle, add ethanol, 70 ℃, lixiviate 60min, abandons supernatant; 85 ℃ of hot-water soak rinsing 30min, by aggregate 8000r/min, centrifugal 30min, abandons supernatant; Aggregate after centrifugal is added water, and aggregate and water w/v 1: 4, adjust pH value 9, adds alkaline lipase A, B by the compound lipases of mixing in 2: 1, accounts for aggregate than 0.3%, 25 ℃, and hydrolysis 50min is centrifugal, removes supernatant; Bifidobacterium lactis (B.lactis), lactobacillus bulgaricus (L.bulgaricus) and Lactobacillus helveticus (L.helviticus), three kinds of lactic acid bacteria weight ratios 1: 3: 2; Aggregate and water w/v 1: 4, add 5% sucrose of gross weight,, it is 0.03%, 40 ℃ that lactic acid bacteria accounts for aggregate weight ratio, fermentation 12h, 120 ℃ of sterilizing 30min; Add hydrochloric acid adjust pH 2, add pepsin, accounting for aggregate ratio is 0.06%, and 37 ℃ of hydrolysis temperatures stir 1.5h, adds alkali and regulates pH value to 6.5, adds papain, and accounting for aggregate ratio is 0.06%, 50 ℃, stirs 1.5h, heats the enzyme that goes out; Add alkali neutralization, dry, to obtain final product.
Embodiment 4
Get animal skeleton and be crushed to 10mm particle, add ethanol, 80 ℃, lixiviate 75min, abandons supernatant; 89 ℃ of hot-water soak rinsing 30min, by aggregate 6000r/min, centrifugal 30min, abandons supernatant; Aggregate after centrifugal is added water, and aggregate and water w/v 1: 3.5, adjust pH value 8.5, adds alkaline lipase A, B by the compound lipases of mixing in 2: 1, accounts for aggregate than 0.2%, 30 ℃, and hydrolysis 60min is centrifugal, removes supernatant; Bifidobacterium lactis (B.lactis), lactobacillus bulgaricus (L.bulgaricus) and Lactobacillus helveticus (L.helviticus), three kinds of lactic acid bacteria weight ratios 1: 3: 2; Aggregate and water w/v 1: 5, add 5% sucrose of gross weight, and it is 0.04%, 50 ℃ that lactic acid bacteria accounts for aggregate weight ratio, fermentation 12h, 120 ℃ of sterilizing 30min; Add hydrochloric acid adjust pH 1, add pepsin, accounting for aggregate ratio is 0.08%, and 37 ℃ of hydrolysis temperatures stir 1.2h, adds alkali and regulates pH value to 6.7, adds papain, and accounting for aggregate ratio is 0.06%, 45 ℃, stirs 1h, heats the enzyme that goes out; Add alkali neutralization, dry, to obtain final product.
Embodiment 5
Get animal skeleton and be crushed to 25mm particle, add ethanol, 75 ℃, lixiviate 65min, abandons supernatant; 88 ℃ of hot-water soak rinsing 30min, by aggregate 8000r/min, centrifugal 30min, abandons supernatant; Aggregate after centrifugal is added water, and aggregate and water w/v 1: 4, adjust pH value 9.5, adds alkaline lipase A, B by the compound lipases of mixing in 2.8: 1, accounts for aggregate than 0.3%, 30 ℃, and hydrolysis 50min is centrifugal, removes supernatant; Bifidobacterium lactis (B.lactis), lactobacillus bulgaricus (L.bulgaricus) and Lactobacillus helveticus (L.helviticus), three kinds of lactic acid bacteria weight ratios 1: 3: 2; Aggregate and water w/v 1: 5, add 5% sucrose of gross weight,, it is 0.04%, 40 ℃ that lactic acid bacteria accounts for aggregate weight ratio, fermentation 12h, 120 ℃ of sterilizing 30min; Add hydrochloric acid adjust pH 3, add pepsin, accounting for aggregate ratio is 0.08%, and 40 ℃ of hydrolysis temperatures stir 2h, adds alkali and regulates pH value to 7, adds papain, and accounting for aggregate ratio is 0.08%, 60 ℃, stirs 1h, heats the enzyme that goes out; Add alkali neutralization, dry, to obtain final product.
Embodiment 6
Get animal skeleton and be crushed to 35mm particle, add ethanol, 78 ℃, lixiviate 70min, abandons supernatant; 87 ℃ of hot-water soak rinsing 30min, by aggregate 7000r/min, centrifugal 30min, abandons supernatant; Aggregate after centrifugal is added water, and aggregate and water w/v 1: 5, adjust pH value 8, adds alkaline lipase A, B by the compound lipases of mixing in 1.8: 1, accounts for aggregate than 0.25%, 20 ℃, and hydrolysis 50min is centrifugal, removes supernatant; Get bifidobacterium lactis (B.lactis), lactobacillus bulgaricus (L.bulgaricus) and Lactobacillus helveticus (L.helviticus), three kinds of lactic acid bacteria weight ratios 1: 3: 2; Aggregate and water w/v 1: 5, add 5% sucrose of gross weight, and it is 0.04%, 45 ℃ that lactic acid bacteria accounts for aggregate weight ratio, fermentation 12h, 120 ℃ of sterilizing 30min; Add hydrochloric acid adjust pH 1, add pepsin, accounting for aggregate ratio is 0.08%, and 36 ℃ of hydrolysis temperatures stir 2h, adds alkali and regulates pH value to 7, adds papain, and accounting for aggregate ratio is 0.08%, 55 ℃, stirs 2h, heats the enzyme that goes out; Add alkali neutralization, dry, to obtain final product.
Embodiment 7
Get animal skeleton and be crushed to 10mm particle, add ethanol, 60 ℃, lixiviate 75min, abandons supernatant; 86 ℃ of hot-water soak rinsing 30min, by aggregate 5000r/min, centrifugal 30min, abandons supernatant; Aggregate after centrifugal is added water, and aggregate and water w/v 1: 4, adjust pH value 10, adds alkaline lipase A, B by the compound lipases of mixing in 3: 1, accounts for aggregate than 0.35%, 24 ℃, and hydrolysis 45min is centrifugal, removes supernatant; Bifidobacterium lactis (B.lactis), lactobacillus bulgaricus (L.bulgaricus) and Lactobacillus helveticus (L.helviticus), three kinds of lactic acid bacteria weight ratios 1: 3: 2; Aggregate and water w/v 1: 5, add 5% sucrose of gross weight, and it is 0.03%, 50 ℃ that lactic acid bacteria accounts for aggregate weight ratio, fermentation 12h, 120 ℃ of sterilizing 30min; Add hydrochloric acid adjust pH 1.3, add pepsin, accounting for aggregate ratio is 0.08%, and 35 ℃ of hydrolysis temperatures stir 2h, adds alkali and regulates pH value to 7, adds papain, and accounting for aggregate ratio is 0.08%, 45 ℃, stirs 2h, heats the enzyme that goes out; Add alkali neutralization, dry, to obtain final product.
Embodiment 8
Get animal skeleton and be crushed to 15mm particle, add ethanol, 80 ℃, lixiviate 75min, abandons supernatant; 84 ℃ of hot-water soak rinsing 30min, by aggregate 8000r/min, centrifugal 30min, abandons supernatant; Aggregate after centrifugal is added water, and aggregate and water w/v 1: 5, adjust pH value 8.5, adds alkaline lipase A, B by the compound lipases of mixing in 2.6: 1, accounts for aggregate than 0.14%, 30 ℃, and hydrolysis 60min is centrifugal, removes supernatant; Bifidobacterium lactis (B.lactis), lactobacillus bulgaricus (L.bulgaricus) and Lactobacillus helveticus (L.helviticus), three kinds of lactic acid bacteria weight ratios 1: 3: 2; Aggregate and water w/v 1: 5, add 5% sucrose of gross weight, and it is 0.03%, 37 ℃ that lactic acid bacteria accounts for aggregate weight ratio, fermentation 12h, 120 ℃ of sterilizing 30min; Add hydrochloric acid adjust pH 2, add pepsin, accounting for aggregate ratio is 0.07%, and 38 ℃ of hydrolysis temperatures stir 1.5h, adds alkali and regulates pH value to 6, adds papain, and accounting for aggregate ratio is 0.08%, 40 ℃, stirs 2h, heats the enzyme that goes out; Add alkali neutralization, dry, to obtain final product.
Embodiment 9
Get animal skeleton and be crushed to 30mm particle, add ethanol, 75 ℃, lixiviate 55min, abandons supernatant; 83 ℃ of hot-water soak rinsing 30min, by aggregate 8000r/min, centrifugal 30min, abandons supernatant; Aggregate after centrifugal is added water, and aggregate and water w/v 1: 5, adjust pH value 8, adds alkaline lipase A, B by the compound lipases of mixing in 3: 1, accounts for aggregate than 0.35%, 28 ℃, and hydrolysis 60min is centrifugal, removes supernatant; Get bifidobacterium lactis (B.lactis), lactobacillus bulgaricus (L.bulgaricus) and Lactobacillus helveticus (L.helviticus), three kinds of lactic acid bacteria weight ratios 1: 3: 2; It is 0.04% that lactic acid bacteria accounts for aggregate weight ratio, and aggregate and water w/v 1: 3 add 5% sucrose of gross weight, 50 ℃, and fermentation 12h, 120 ℃ of sterilizing 30min; Add hydrochloric acid adjust pH 3, add pepsin, accounting for aggregate ratio is 0.08%, and 39 ℃ of hydrolysis temperatures stir 2h, adds alkali and regulates pH value to 7, adds papain, and accounting for aggregate ratio is 0.08%, 50 ℃, stirs 2h, heats the enzyme that goes out; Add alkali neutralization, dry, to obtain final product.
Embodiment 10
Get animal skeleton and be crushed to 20mm particle, add ethanol, 80 ℃, lixiviate 60min, abandons supernatant; 82 ℃ of hot-water soak rinsing 30min, by aggregate 6000r/min, centrifugal 30min, abandons supernatant; Aggregate after centrifugal is added water, and aggregate and water w/v 1: 5, adjust pH value 10, adds alkaline lipase A, B by the compound lipases of mixing in 3: 1, accounts for aggregate than 0.3%, 30 ℃, and hydrolysis 60min is centrifugal, removes supernatant; Get bifidobacterium lactis (B.lactis), lactobacillus bulgaricus (L.bulgaricus) and Lactobacillus helveticus (L.helviticus), three kinds of lactic acid bacteria weight ratios 1: 3: 2; It is 0.04% that lactic acid bacteria accounts for aggregate weight ratio, and aggregate and water w/v 1: 3 add 5% sucrose of gross weight, 40 ℃, and fermentation 12h, 120 ℃ of sterilizing 30min; Add hydrochloric acid adjust pH 2, add pepsin, accounting for aggregate ratio is 0.05%, and 38 ℃ of hydrolysis temperatures stir 2h, adds alkali and regulates pH value to 6, adds papain, and accounting for aggregate ratio is 0.07%, 55 ℃, stirs 1h, heats the enzyme that goes out; Add alkali neutralization, dry, to obtain final product.
Embodiment 11
Bone is crushed to 40mm particle, adds ethanol, 80 ℃, lixiviate 75min, abandons supernatant; 81 ℃ of hot-water soak rinsing 30min, by aggregate 8000r/min, centrifugal 30min, abandons supernatant; Aggregate after centrifugal is added water, and aggregate and water w/v 1: 5, adjust pH value 10, adds alkaline lipase A, B by the compound lipases of mixing in 3: 1, accounts for aggregate than 0.1%, 30 ℃, and hydrolysis 60min is centrifugal, removes supernatant; Extracting lactic acid bacterium adds in aggregate, and aggregate and water w/v 1: 5 add 5% sucrose of gross weight,, 42 ℃, fermentation 12h, 120 ℃ of sterilizing 30min; Add hydrochloric acid adjust pH 2, add pepsin, accounting for aggregate ratio is 0.08%, 38 ℃ of hydrolysis temperatures, and hydrolysis 2h, adjusts pH value 7, then adds papain, and accounting for aggregate ratio is 0.06%, 55 ℃, stirs 2h, heats the enzyme that goes out; Add alkali neutralization, dry, to obtain final product.
Embodiment 12
Bone is crushed to 5mm particle, adds ethanol, 60 ℃, lixiviate 45min, abandons supernatant; 90 ℃ of hot-water soak rinsing 30min, by aggregate 4000r/min, centrifugal 30min, abandons supernatant; Aggregate after centrifugal is added water, and aggregate and water w/v 1: 3, adjust pH value 8, adds compound lipases, accounts for aggregate than 0.1%, 20 ℃, and hydrolysis 60min is centrifugal, removes supernatant; Bifidobacterium lactis (B.lactis), bulgarian milk bar mattress (L.bulgaricus) and Lactobacillus helveticus (L.helviticus), three kinds of lactic acid bacteria weight ratios 1: 3: 2; Aggregate and water w/v 1: 4, add 5% sucrose of gross weight, and it is 0.04%, 45 ℃ that lactic acid bacteria accounts for aggregate weight ratio, fermentation 12h, 120 ℃ of sterilizing 30min; Add hydrochloric acid adjust pH, add protease, accounting for aggregate ratio is 0.08%, and hydrolysis 2h, heats the enzyme that goes out; Add alkali neutralization, dry, to obtain final product.
Embodiment 13
Bone is crushed to 30mm particle, adds ethanol, 75 ℃, lixiviate 60min, abandons supernatant; 90 ℃ of hot-water soak rinsing 30min, by aggregate 8000r/min, centrifugal 30min, abandons supernatant; Aggregate after centrifugal is added water, and aggregate and water w/v 1: 4, adjust pH value 9, adds compound lipases, accounts for aggregate than 0.4%, 30 ℃, and hydrolysis 60min is centrifugal, removes supernatant; Bifidobacterium lactis (B.lactis), bulgarian milk bar mattress (L.bulgaricus) and Lactobacillus helveticus (L.helviticus), three kinds of lactic acid bacteria weight ratios 1: 3: 2; Aggregate and water w/v 1: 3.5, add 5% sucrose of gross weight, and it is 0.04%, 50 ℃ that lactic acid bacteria accounts for aggregate weight ratio, fermentation 12h, 120 ℃ of sterilizing 30min; Add hydrochloric acid adjust pH 2, add pepsin, accounting for aggregate ratio is 0.06%, and 40 ℃ of hydrolysis temperatures stir 2h, adjust pH value 7, again add papain, and accounting for aggregate ratio is 0.08%, 60 ℃, stirs 2h, heats the enzyme that goes out; Add alkali neutralization, dry, to obtain final product.
Embodiment 14
Bone is crushed to 10mm particle, adds ethanol, 80 ℃, lixiviate 75min, abandons supernatant; 88 ℃ of hot-water soak rinsing 30min, by aggregate 8000r/min, centrifugal 30min, abandons supernatant; Aggregate after centrifugal is added water, and aggregate and water w/v 1: 5, adjust pH value 10, and the compound lipases that adds alkaline lipase A, B to mix, accounts for aggregate than 0.4%, 30 ℃, and hydrolysis 60min is centrifugal, removes supernatant; Bifidobacterium lactis (B.lactis), bulgarian milk bar mattress (L.bulgaricus) and Lactobacillus helveticus (L.helviticus), three kinds of lactic acid bacteria weight ratios 1: 3: 2; Aggregate and water w/v 1: 5, add 5% sucrose of gross weight, and it is 0.04%, 50 ℃ that lactic acid bacteria accounts for aggregate weight ratio, fermentation 12h, 120 ℃ of sterilizing 30min; Add hydrochloric acid adjust pH 1.5, add pepsin, 37 ℃ of hydrolysis temperatures, stir 2h, add alkali and regulate pH value to 6.7, add neutral proteinase, and accounting for aggregate ratio is 0.08%, 50 ℃, stirs 2h, heats the enzyme that goes out; Add alkali neutralization, dry, to obtain final product.
Embodiment 15
Bone is crushed to 25mm particle, adds ethanol, 80 ℃, lixiviate 65min, abandons supernatant; 90 ℃ of hot-water soak rinsing 30min, by aggregate 6000r/min, centrifugal 30min, abandons supernatant; Aggregate after centrifugal is added water, and aggregate and water w/v 1: 4, adjust pH value 8.5, adds compound lipases, accounts for aggregate than 0.3%, 30 ℃, and hydrolysis 60min is centrifugal, removes supernatant; Extracting lactic acid bacterium adds in aggregate, and it is 0.02% that lactic acid bacteria accounts for aggregate weight ratio, and aggregate and water w/v 1: 4 add 5% sucrose of gross weight, and 50 ℃, fermentation 12h, 120 ℃ of sterilizing 30min; Add hydrochloric acid adjust pH 3, add pepsin, accounting for aggregate ratio is 0.08%, and 40 ℃ of hydrolysis temperatures stir 1h, adds alkali and regulates pH value to 7, adds papain, and accounting for aggregate ratio is 0.08%, 45 ℃, stirs 2h, heats the enzyme that goes out; Add alkali neutralization, dry, to obtain final product.
Claims (5)
1. a method for bone calcium is extracted in degreasing fermentation, comprises the steps:
(1) bone is pulverized, the lixiviate oil of boning, abandons supernatant;
(2) aggregate after lixiviate is soaked to rinsing, centrifugal, abandon supernatant;
(3) aggregate after centrifugal is added water, adjust pH value, add alkaline fat enzyme hydrolysis, centrifugal, remove supernatant;
(4) aggregate after centrifugal in step (3) is added to lactobacillus-fermented;
(5) zymotic fluid in step (4) adds the hydrolysis of protease secondary again, adds alkali neutralization, is drying to obtain;
Step (1) particles of aggregates 5～40mm adds ethanol, and 60～80 ℃, 45～75min;
In step (2) by the rinsing of particles of aggregates hot water, then 4000～8000r/min, centrifugal 30min;
Aggregate and water w/v 1: 3～5 in step (3), adjust pH value 8～10, the compound lipases that adds alkaline lipase A, B to mix;
Fermentation described in step (4) is bifidobacterium lactis (B.lactis), lactobacillus bulgaricus (L.bulgaricus) and Lactobacillus helveticus (L.helviticus), three kinds of lactic acid bacteria weight ratios 1: 3: 2; Aggregate and water w/v 1: 3～5, add 5% sucrose of gross weight, and it is 0.01～0.04%, 35～50 ℃ that lactic acid bacteria accounts for aggregate weight ratio, fermentation 12h, 120 ℃ of sterilizing 30min;
Hydrolysis described in step (5) is to add hydrochloric acid adjust pH 1～3, adds pepsin, and 35～40 ℃ of hydrolysis temperatures stir 1～2h, adds alkali and regulates pH value to 6～7, adds papain, 40～60 ℃, stir 1～2h, and heat the enzyme that goes out.
2. bone calcium extracting method according to claim 1, is characterized in that, described compound lipases neutral and alkali lipase A, B weight ratio are 1.5～3: 1.
3. bone calcium extracting method according to claim 1 and 2, is characterized in that, described compound lipases accounts for aggregate than 0.1～0.4%.
4. bone calcium extracting method according to claim 1, is characterized in that, it is 0.04～0.08% for good that hydrolysis bone meal two kinds of protease used respectively account for aggregate ratio.
5. a BGP that is extracted preparation by animal skeleton, is characterized in that, adopts following methods preparation: bone is crushed to 5～40mm particle, adds ethanol, 60～80 ℃, lixiviate 45～75min, abandons supernatant; Aggregate hot-water soak rinsing, water temperature 80-90 ℃, soaks 30 minutes, and by aggregate 4000～8000r/min, centrifugal 30min, abandons supernatant; Aggregate after centrifugal is added water, and aggregate and water w/v 1: 3～5, adjust pH value 8～10, add alkaline lipase A, B by 1.5～3: 1 compound lipases mixing, accounts for aggregate than 0.1～0.4%, 20～30 ℃, hydrolysis 40～60min, centrifugal, remove supernatant; Bifidobacterium lactis (B.lactis), lactobacillus bulgaricus (L.bulgaricus) and Lactobacillus helveticus (L.helviticus), three kinds of lactic acid bacteria weight ratios 1: 3: 2; Aggregate and water w/v 1: 3～5, add 5% sucrose of gross weight, and it is 0.01～0.04%, 35～50 ℃ that lactic acid bacteria accounts for aggregate weight ratio, fermentation 12h, 120 ℃ of sterilizing 30min; Add hydrochloric acid adjust pH 1～3, add pepsin, accounting for aggregate weight ratio is 0.04～0.08%, 35～40 ℃ of hydrolysis temperatures, stir 1～2h, add alkali and regulate pH value to 6～7, add papain, accounting for aggregate weight ratio is 0.04～0.08%, 40～60 ℃, stir 1～2h, heat the enzyme that goes out; Add alkali neutralization, dry, to obtain final product.
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