CN109907326A - One kind being based on the chelated calcium preparation method of wood frog bone and flesh collagen polypeptide - Google Patents
One kind being based on the chelated calcium preparation method of wood frog bone and flesh collagen polypeptide Download PDFInfo
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Abstract
The present invention relates to one kind to be based on the chelated calcium preparation method of wood frog bone and flesh collagen polypeptide, using a large amount of wood frog bone and flesh, carries out the chelated calcium research of wood frog bone and flesh, the problem of not only having dealt carefully with bone, the waste for avoiding resource also reduces the pressure of environment, increases economic efficiency and ecological benefits.Chelating including crude wood frog animal tankage, wood frog animal tankage enzymatic hydrolysis, the ultrafiltration of enzymolysis liquid and enzymolysis liquid and calcium chloride.It has the beneficial effect that and polypeptide is prepared using the method for trypsin digestion, small-molecular-weight polypeptide is prepared using the method for nanofiltration, to obtain the small collagen polypeptide liquid of molecular weight.High in the intracorporal absorptivity of people with the polypeptide chelating calcium obtained after this method and calcium ion chelating, advantages of good calcium supplying effect and easy to operate, not needing freeze-drying polypeptide powder can chelate, and high-efficient, easily-controllable property is strong, the collagen polypeptide chelating calcium chelation percent that obtains is higher.Wood frog bone and flesh polypeptide chelate calcium prepared by the present invention can be used for developing ordinary food, health care product or for field of medicaments.
Description
Technical field
The invention belongs to field of biotechnology, are related to a kind of preparation method of collagen polypeptide chelating calcium, more particularly to one kind
Based on the chelated calcium preparation method of wood frog bone and flesh collagen polypeptide.
Background technique
Dongbei forest-frog is the pure wild animal for being grown in Heilungkiang, Jilin and Liaoning San Sheng, it collects beauty, medicinal, edible
It is integrated, is the famous economic frog kind in China.According to incompletely statistics, it is about more than 1,000,000,000 or so that the Northeast, which produces wood frog amount per year,
Most purposes of wood frog are to extract oviductus ranae, but oviductus ranae only accounts for 15% or so of the whole frog, and can only be in the female frog
It extracts, other positions such as brain, skin, meat and bone etc. cause the serious wasting of resources essentially as feed or offal treatment.Woods
Contain collagen quite abundant in the bone and flesh of the frog, all fails to be used appropriately.Therefore, it is taken a hint by the research of forefathers,
It proposes that one kind efficiently prepares chelated calcium method with calcium chelating using the collagen polypeptide in wood frog bone and flesh, promotion China wood frog is produced
The development of industry has important impetus.
Naturally occurring active peptides due in organism content it is low, and extract difficult, it is difficult to realize mass production
Needed for supply, chemical artificial synthetic polypeptide is expensive.It is obtained so sight is more invested exploitation protein hydrolysate by people
It obtains to come in this approach of active peptides.
Bone mineral increase in everyone growth period is one of the principal element of peak bone mass after the determination date, with old age in the future
The development of phase osteoporosis also has certain relationship.Many factors, such as heredity, gender, endocrine and trophic factors will affect
The increase of peak bone mass.If peak bone mass will be made to reduce after skeletal maturation, and improve old age in puberty calcium insufficiency of intake
The risk that phase osteoporotic fracture occurs.Therefore, it is necessary to additionally replenish the calcium.The calcium supplement about 200 of health care currently on the market
More than, food calcium additive be about hundreds of, more than more than agriculture calcareous fertilisers about more than 30, livestock feed calcium additive 30, but it is few
See the product of polypeptide chelate calcium class.Polypeptide chelate calcium is prepared, exactly adds a kind of method of calcium source, it not only improves microelement
It is completely transported absorption with the situation of chelate, and effectively calcium ion can be released.Since it is easily absorbed, biology
The characteristics such as potency height become the Main way of Recent study calcium preparation.
Summary of the invention
The purpose of the present invention is to provide a kind of comprehensive utilization wood frog bone and flesh collagen polypeptides, and replenish the calcium for presently commercially available
Product has intense irritation to stomach, and bioavailability is low, and the low problem of the preferable product calcium content of dissolubility provides Rana sylvatica Le conte bone
The chelated calcium preparation method of meat collagen polypeptide.
To solve above-mentioned technical problem, the technical solution adopted by the present invention is that:
(1) preparation of wood frog animal tankage: the female frog bone and flesh for removing oviductus ranae or male frog bone and flesh are first cleaned with clear water, dried
55-65 DEG C of temperature drying, time 2-4h cross 40-60 mesh sieve, obtain wood frog to be crushed to tiny residue with multifunctional crusher
Bone and flesh fine powder；
(2) it is digested using wood frog animal tankage, solid-liquid ratio (mass volume ratio g/ml) is 1:5-1:20；Enzyme concentration is
11000-40000U/mg, enzymolysis time 3h-5h, hydrolysis temperature are 40-60 DEG C, ph 6.5-8.5；90- after the completion of enzymatic hydrolysis
100 DEG C of enzyme deactivation 20min；Supercentrifuge 10000r/min is centrifuged 30min, takes supernatant, i.e. acquisition wood frog bone and flesh digests collagen
Polypeptide liquid.
(3) it obtains wood frog bone and flesh and digests collagen polypeptide liquid, molecular cut off is used to carry out ultrafiltration for the ultrafiltration membrane of 3000Da,
Filtrate is collected to get molecular weight < 3000Da wood frog bone and flesh polypeptide liquid below is arrived.
(4) the wood frog bone and flesh enzymolysis polypeptide liquid and anhydrous Cacl small using molecular weight2Carry out chelation reaction, polypeptide liquid with
Cacl2Mass ratio be 10:2-10:0.5ml/g, chelating temperature be 20-40 DEG C, the chelating time be 30-50min, chelation reaction
After, the dehydrated alcohol that 8-10 times of volume is added into solution precipitates chelating object, and precipitation system settles 1- under the conditions of 4 DEG C
3h, 4000-5000r/min are centrifuged 10-20min, take precipitating, wood frog bone and flesh collagen polypeptide chelating to obtain the final product after vacuum freeze drying
Calcium.
Preferably, clean wood frog bone and flesh is placed in the baking oven that temperature is 60 DEG C and is dried in the step (1)
2.5h, dried wood frog animal tankage is broken, and mesh number is 60 mesh.
Preferably, in the step (2) solid-liquid ratio be 1:10g/ml, enzyme concentration 38000U/mg,
Enzymolysis time is 4h, and hydrolysis temperature is 45 DEG C, and ph when enzymatic hydrolysis is 8.2.
Preferably, using molecular cut off for the super of 3000Da wood frog bone and flesh enzymolysis polypeptide liquid in the step (3)
Filter membrane carries out ultrafiltration, collects filtrate to get molecular weight < 3000Da wood frog bone and flesh polypeptide liquid below is arrived.
Preferably, chelates temperature is 40 DEG C in the step (4), the chelating time is 40min, and peptide calcium ratio is 10:1ml/
G, the addition multiple of dehydrated alcohol are 9, and setting revolving speed is 4000r/min when centrifugation, are centrifuged 15min.
Application of the wood frog bone and flesh polypeptide chelate calcium in ordinary food.
Application of the wood frog bone and flesh polypeptide chelate calcium in health care product.
Application of the wood frog bone and flesh polypeptide chelate calcium in field of medicaments.
Compared with the prior art, the advantages of the present invention are as follows:
(1) after enzymatic hydrolysis is completed to obtain collagen polypeptide liquid, ultrafiltration is carried out, having obtained molecular cut off is less than 3000Da's
Polypeptide liquid is to get the polypeptide liquid for having arrived small-molecular-weight, this is for human body to the bio-absorbable of calcium constituent in chelate using very
Favorably, there is higher dissolubility in water, and within the scope of 30 DEG C -40 DEG C close to body temperature, solubility is maximum.
(2) present invention is chelated using small-molecular-weight polypeptide liquid with calcium ion, than carrying out chela at powder by freeze-drying
It closes, the required time is short, energy consumption is small, chelation percent is high, and biological value is high.
(3) a large amount of wood frog bone and flesh is utilized, the chelated calcium research of wood frog bone and flesh is carried out, has not only dealt carefully with asking for bone
Topic, avoids the waste of resource, also reduces the pressure of environment, accomplish to turn waste into wealth, and increases economic efficiency and ecological benefits.
Specific embodiment
The present invention program is described further below by embodiment:
Embodiment 1
(1) the wood frog bone and flesh that 100g is fresh is cleaned, and is placed in 60 DEG C of air dry oven dry 2.5h, is dried postposition
It is crushed in high speed disintegrator, crosses 50 meshes, obtain wood frog animal tankage 86g；
(2) by smashed wood frog animal tankage, it is that distilled water is added in 1:10g/ml with solid-liquid ratio, is digested, digest item
Part setting are as follows: the enzyme concentration of trypsase is 38000U/mg, and enzymolysis time 4h, hydrolysis temperature is 45 DEG C, ph 8；It has digested
In 90 DEG C of enzyme deactivation 20min after；Polypeptide liquid after enzyme deactivation is placed in supercentrifuge 10000r/min centrifugation 30min, that is, is obtained
Wood frog bone and flesh digests collagen polypeptide liquid 832mL and residue 14.6g.
(3) it obtains wood frog bone and flesh and digests collagen polypeptide liquid, molecular cut off is used to carry out ultrafiltration for the filter membrane of 3000Da, receive
Collect filtrate to get 465mL and molecular weight < 3000Da collagen polypeptide liquid below is arrived.
(4) the wood frog bone and flesh enzymatic hydrolysis collagen polypeptide liquid and the anhydrous Cacl of 46.5g that the 465mL obtained has been concentrated2Carry out chelating
Instead, chelating temperature is 40 DEG C, and the chelating time is 30min, and after chelation reaction, the anhydrous second of 8 times of volumes is added into solution
Alcohol precipitates chelating object, and precipitation system settles 2h under the conditions of 4 DEG C, and 4000r/min is centrifuged 20min, takes precipitating, place it in freezing
It dries in drying machine to get wood frog bone and flesh collagen polypeptide chelating calcium 302.7g.
Embodiment 2
(1) the wood frog bone and flesh that 150g is fresh is cleaned, and is placed in 55 DEG C of air dry oven dry 2.5h, is dried postposition
It is crushed in high speed disintegrator, crosses 40 meshes, obtain wood frog animal tankage 122g；
(2) by smashed wood frog animal tankage, it is that distilled water is added in 1:12g/ml with solid-liquid ratio, is digested, digest item
Part setting are as follows: the enzyme concentration of trypsase is 30000U/mg, and enzymolysis time 4h, hydrolysis temperature is 45 DEG C, ph 8.2；Enzymatic hydrolysis
After the completion in 95 DEG C of enzyme deactivation 15min；Polypeptide liquid after enzyme deactivation is placed in supercentrifuge 10000r/min centrifugation 30min, that is, is obtained
It obtains wood frog bone and flesh and digests collagen polypeptide liquid 1372mL and residue 22.6g.
(3) it obtains wood frog bone and flesh and digests collagen polypeptide liquid, molecular cut off is used to carry out ultrafiltration for the filter membrane of 3000Da, receive
Collect filtrate to get 755mL and molecular weight < 3000Da collagen polypeptide liquid below is arrived.
(4) the wood frog bone and flesh enzymatic hydrolysis collagen polypeptide liquid and the anhydrous Cacl of 75.5g that the 755mL obtained has been concentrated2Carry out chelating
Reaction, chelating temperature are 40 DEG C, and the chelating time is 40min, and after chelation reaction, the anhydrous of 9 times of volumes is added into solution
Ethanol precipitation chelating object, precipitation system settle 3h under the conditions of 4 DEG C, and 4000r/min is centrifuged 15min, takes precipitating, place it in cold
It dries in lyophilizer to get wood frog bone and flesh collagen polypeptide chelating calcium 482.4g.
Embodiment 3
(1) the wood frog bone and flesh that 200g is fresh is cleaned, and dry 2h is placed in 65 DEG C of air dry oven, and drying is placed on
It is crushed in high speed disintegrator, crosses 50 meshes, obtain wood frog animal tankage 133g；
(2) by smashed wood frog animal tankage, it is that distilled water is added in 1:15g/ml with solid-liquid ratio, is digested, digest item
Part setting are as follows: the enzyme concentration of trypsase is 15000U/mg, and enzymolysis time 4h, hydrolysis temperature is 45 DEG C, ph 7.5；Enzymatic hydrolysis
After the completion in 90 DEG C of enzyme deactivation 20min；Polypeptide liquid after enzyme deactivation is placed in supercentrifuge 10000r/min centrifugation 25min, that is, is obtained
It obtains wood frog bone and flesh and digests collagen polypeptide liquid 1447mL and residue 37.6g.
(3) it obtains wood frog bone and flesh and digests collagen polypeptide liquid, molecular cut off is used to carry out ultrafiltration for the filter membrane of 3000Da, receive
Collect filtrate to get 698mL and molecular weight < 3000Da collagen polypeptide liquid below is arrived.
(4) the wood frog bone and flesh enzymatic hydrolysis collagen polypeptide liquid and the anhydrous Cacl of 69.8g that the 698mL obtained has been concentrated2Carry out chelating
Reaction, chelating temperature are 40 DEG C, and the chelating time is 30min, and after chelation reaction, the anhydrous of 10 times of volumes is added into solution
Ethanol precipitation chelating object, precipitation system settle 2.5h under the conditions of 4 DEG C, and 4000r/min is centrifuged 20min, takes precipitating, place it in
It dries in freeze drier to get wood frog bone and flesh collagen polypeptide chelating calcium 322.7g.
Embodiment 4
(1) the wood frog bone and flesh that 250g is fresh is cleaned, and is placed in 65 DEG C of air dry oven dry 1.0h, is dried postposition
It is crushed in high speed disintegrator, crosses 60 meshes, obtain wood frog animal tankage 156g；
(2) by smashed wood frog animal tankage, it is that distilled water is added in 1:20g/ml with solid-liquid ratio, is digested, digest item
Part setting are as follows: the enzyme concentration of trypsase is 32000U/mg, and enzymolysis time 4.5h, hydrolysis temperature is 45 DEG C, ph 7；Enzymatic hydrolysis
After the completion in 100 DEG C of enzyme deactivation 15min；Polypeptide liquid after enzyme deactivation is placed in supercentrifuge 10000r/min centrifugation 30min, that is, is obtained
It obtains wood frog bone and flesh and digests collagen polypeptide liquid 2789mL and residue 30.3g.
(3) it obtains wood frog bone and flesh and digests collagen polypeptide liquid, molecular cut off is used to carry out ultrafiltration for the filter membrane of 300Da, receive
Collect filtrate to get 1788mL and molecular weight < 3000Da collagen polypeptide liquid below is arrived.
(4) the wood frog bone and flesh enzymatic hydrolysis collagen polypeptide liquid and the anhydrous Cacl of 178.8g that the 1788mL obtained has been concentrated2Carry out huge legendary turtle
Reaction is closed, chelating temperature is 30 DEG C, and the chelating time is 30min, and after chelation reaction, the nothing of 10 times of volumes is added into solution
Water-ethanol precipitates chelating object, and precipitation system settles 1h under the conditions of 4 DEG C, and 4500r/min is centrifuged 15min, takes precipitating, place it in
It dries in freeze drier to get wood frog bone and flesh collagen polypeptide chelating calcium 821g.
Embodiment 5
(1) the wood frog bone and flesh that 300g is fresh is cleaned, and dry 4h is placed in 45 DEG C of air dry oven, and drying is placed on
It is crushed in high speed disintegrator, crosses 40 meshes, obtain wood frog animal tankage 277g；
(2) by smashed wood frog animal tankage, it is that distilled water is added in 1:10g/ml with solid-liquid ratio, is digested, digest item
Part setting are as follows: the enzyme concentration of trypsase is 38000U/mg, and enzymolysis time 4h, hydrolysis temperature is 45 DEG C, ph 8；It has digested
In 95 DEG C of enzyme deactivation 15min after；Polypeptide liquid after enzyme deactivation is placed in supercentrifuge 12000r/min centrifugation 20min, that is, is obtained
Wood frog bone and flesh digests collagen polypeptide liquid 2674mL and residue 43.2g.
(3) it obtains wood frog bone and flesh and digests collagen polypeptide liquid, molecular cut off is used to carry out ultrafiltration for the filter membrane of 3000Da, receive
Collect filtrate to get 1277mL and molecular weight < 3000Da collagen polypeptide liquid below is arrived.
(4) the wood frog bone and flesh enzymatic hydrolysis collagen polypeptide liquid and the anhydrous Cacl of 127.7g that the 1277mL obtained has been concentrated2Carry out huge legendary turtle
Reaction is closed, chelating temperature is 40 DEG C, and the chelating time is 40min, and after chelation reaction, the nothing of 9 times of volumes is added into solution
Water-ethanol precipitates chelating object, and precipitation system settles 2h under the conditions of 4 DEG C, and 5000r/min is centrifuged 20min, takes precipitating, place it in
It dries in freeze drier to get wood frog bone and flesh collagen polypeptide chelating calcium 644.3g.
Embodiment 6
(1) the wood frog bone and flesh that 350g is fresh is cleaned, and is placed in 50 DEG C of air dry oven dry 2.5h, is dried postposition
It is crushed in high speed disintegrator, crosses 60 meshes, obtain wood frog animal tankage 296g；
(2) by smashed wood frog animal tankage, it is that distilled water is added in 1:5g/ml with solid-liquid ratio, is digested, enzymatic hydrolysis condition
Setting are as follows: the enzyme concentration of trypsase is 25000U/mg, and enzymolysis time 4h, hydrolysis temperature is 45 DEG C, ph 6.5；It has digested
In 100 DEG C of enzyme deactivation 10min after；Polypeptide liquid after enzyme deactivation is placed in supercentrifuge 10000r/min centrifugation 30min, that is, is obtained
Wood frog bone and flesh digests collagen polypeptide liquid 1307mL and residue 77.4g.
(3) it obtains wood frog bone and flesh and digests collagen polypeptide liquid, molecular cut off is used to carry out ultrafiltration for the filter membrane of 3000Da, receive
Collect filtrate to get 607mL and molecular weight < 3000Da collagen polypeptide liquid below is arrived.
(4) the wood frog bone and flesh enzymatic hydrolysis collagen polypeptide liquid and the anhydrous Cacl of 60.7g that the 607mL obtained has been concentrated2Carry out chelating
Reaction, chelating temperature are 20 DEG C, and the chelating time is 50min, and after chelation reaction, the anhydrous of 8 times of volumes is added into solution
Ethanol precipitation chelating object, precipitation system settle 1.5h under the conditions of 4 DEG C, and 4500r/min is centrifuged 20min, takes precipitating, place it in
It dries in freeze drier to get wood frog bone and flesh collagen polypeptide chelating calcium 203.9g.
Claims (8)
1. one kind is based on the chelated calcium preparation method of wood frog bone and flesh collagen polypeptide, which comprises the following steps:
Step 1: the preparation of wood frog animal tankage
Wood frog bone and flesh is cleaned, 45-65 DEG C of drying, time 2-4h is placed in pulverizer and is crushed to 40-60 mesh, obtains thinner
Wood frog animal tankage.
Step 2: enzymatic hydrolysis
After crushing, trypsase is added and is digested, solid-liquid ratio (mass volume ratio g/ml) is 1:5-1:20；Enzyme concentration is
11000-40000U/mg, enzymolysis time 3h-5h, hydrolysis temperature are 40-60 DEG C, ph 6.5-8.5；90- after the completion of enzymatic hydrolysis
100 DEG C of enzyme deactivation 20min；Supercentrifuge 10000r/min is centrifuged 30min, takes supernatant, i.e. acquisition wood frog bone and flesh digests collagen
Polypeptide liquid.
Step 3: ultrafiltration freeze-drying
Gained filtrate in step 2 is concentrated by ultrafiltration with ultrafilter by 3000Da ultrafiltration membrane, light yellow clarification Rana sylvatica Le conte bone is obtained
Meat collagen polypeptide liquid obtains the wood frog bone and flesh polypeptide liquid that molecular weight is less than 3000Da.
Step 4: chelating
The wood frog bone and flesh enzymolysis polypeptide liquid of the small-molecular-weight used and anhydrous Cacl2Carry out chelation reaction, polypeptide liquid and Cacl2's
Mass ratio is 10:2-10:0.5ml/g, and chelating temperature is 20-40 DEG C, and the chelating time is 30-50min, after chelation reaction,
The dehydrated alcohol that 8-10 times of volume is added into solution precipitates chelating object, and precipitation system settles 1-3h, 4000- under the conditions of 4 DEG C
5000r/min is centrifuged 10-20min, takes precipitating, wood frog bone and flesh collagen polypeptide chelating calcium to obtain the final product after vacuum freeze drying.
2. according to claim 1 a kind of based on the chelated calcium preparation method of wood frog bone and flesh collagen polypeptide, which is characterized in that
60 DEG C of drying temperature in the step 1, time 2.5h, crushing number are 60 mesh.
3. according to claim 1 a kind of based on the chelated calcium preparation method of wood frog bone and flesh collagen polypeptide, which is characterized in that
Solid-liquid ratio is 1:10g/ml in the step 2, and the enzyme concentration in enzymolysis process is 38000U/mg, enzymolysis time 4h, enzymatic hydrolysis
Temperature is 45 DEG C, and ph when enzymatic hydrolysis is 8.2.
4. according to claim 1 a kind of based on the chelated calcium preparation method of wood frog bone and flesh collagen polypeptide, it is characterised in that
The enzymolysis liquid obtained after enzymatic hydrolysis is collected into the filtrate for being less than 3000Da component by the ultrafiltration membrane of 3000Da in the step 3.
5. according to claim 1 a kind of based on the chelated calcium preparation method of wood frog bone and flesh collagen polypeptide, it is characterised in that
Polypeptide liquid and Cacl in the step 42Mass ratio be 10:1ml/g, chelates temperature be 40 DEG C, the chelating time be 40min, nothing
The addition multiple of water-ethanol is 9, and 2h is settled under the conditions of 4 DEG C, and revolving speed is set as 4000r/min, is centrifuged 15min.
6. application of the wood frog bone and flesh polypeptide chelate calcium prepared according to claim 1 in ordinary food.
7. application of the wood frog bone and flesh polypeptide chelate calcium prepared according to claim 1 in health care product.
8. application of the wood frog bone and flesh polypeptide chelate calcium prepared according to claim 1 in field of medicaments.
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| CN111254178A (en) * | 2020-03-23 | 2020-06-09 | 吉林大学 | Preparation method of wood frog skin small-molecular peptide chelated iron |
| CN111334544A (en) * | 2020-03-26 | 2020-06-26 | 吉林大学 | Preparation method of wood frog bone and meat oligopeptide with immunocompetence |
| CN114601171A (en) * | 2022-03-28 | 2022-06-10 | 吉林大学 | Preparation method of rana japonica bone meat polypeptide chelated zinc microcapsule |
| CN116616425A (en) * | 2023-05-25 | 2023-08-22 | 青岛瑞可莱餐饮配料有限公司 | Flavoring and preparation method thereof |
| CN117986354A (en) * | 2024-03-25 | 2024-05-07 | 通化康元生物科技有限公司 | Wood frog bone collagen polypeptide chelated calcium and preparation method thereof |
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| CN117986354A (en) * | 2024-03-25 | 2024-05-07 | 通化康元生物科技有限公司 | Wood frog bone collagen polypeptide chelated calcium and preparation method thereof |
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