CN108018326A - Novel microbial protease hydrolytic prepares collagen from black sea cucumbers from East China Sea peptide - Google Patents

Novel microbial protease hydrolytic prepares collagen from black sea cucumbers from East China Sea peptide Download PDF

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CN108018326A
CN108018326A CN201710859089.0A CN201710859089A CN108018326A CN 108018326 A CN108018326 A CN 108018326A CN 201710859089 A CN201710859089 A CN 201710859089A CN 108018326 A CN108018326 A CN 108018326A
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collagen
sea
east china
peptide
black
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金火喜
徐红萍
杨立业
欧阳小琨
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Zhejiang Ocean University ZJOU
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Abstract

The invention discloses a kind of novel microbial protease hydrolytic to prepare collagen from black sea cucumbers from East China Sea peptide, including：Pretreatment, extraction, fermentation, purifying, hydrolysis, purification.Have the beneficial effect that：2,3 dimethyl, 2 butanol and erythrose are added in enzymolysis process, coordinates bifrequency ultrasound to reduce E by reducing the energy barrier between ground state and activated stateaAnd KMValue, and then increase matrix and the affinity of enzyme, greatly improve enzyme's reaction speeding；The efficient enzymolysis vigor of bacillus protein enzyme can destroy three strands of superhelixes of Sea Cucumber collagen uniqueness, then the small-molecule substances such as the less polypeptide of relative molecular weight, small peptide, amino acid are hydrolyzed to, its absorption rate can improve much after hydrolysis, and the absorption of the other oroteins in food can be promoted, collagen peptide has a variety of medicinal health care functions, is a kind of nutraceutical agents for easily absorbing, having no toxic side effect.

Description

Novel microbial protease hydrolytic prepares collagen from black sea cucumbers from East China Sea peptide
Technical field
The present invention relates to the preparation field of collagen peptide, more particularly to novel microbial protease hydrolytic to prepare East Sea crow Join collagen peptide.
Technical background
Black sea cucumbers from East China Sea (scientific name：White anus Haiti melon Acaudina leucoprocta), it is that China's kind more than 20 can be edible One of sea cucumber, abounds with East Sea sea area, and in our province, particularly Ningbo, Zhoushan coast one is with widely distributed, only Xiangshan of Zhejiang Province one Band year amount of fishing just reaches tens thousand of tons of scales.Black sea cucumbers from East China Sea has high nutritive value and medical value, rich in protein, sea Join the components such as saponin(e, sea cucumber polysaccharide, collagen, vitamin, essential fatty acid and macro- and trace-element, and consolidate without courage Alcohol, long-term consumption can build up health, prevention disease, suppress tumour, slow down aging.However, for a long time, due to lacking industrialization Key technology, black sea cucumbers from East China Sea resource never obtain good development and utilization.In most cases, after black sea cucumbers from East China Sea fishing Abandoned as discarded object, cause also to pollute environment while the wasting of resources, therefore, exploitation black sea cucumbers from East China Sea resource is imperative.
Containing abundant collagen in black sea cucumbers from East China Sea body wall, there is good bioactivity and biological function, can participate in Migration, differentiation, the increment of cell, and due to its weaker antigenic and good biocompatibility, after specially treated, There is more application in terms of medicine, beauty, food.However, since its molecular weight is very big, it is unfavorable for the absorption of stomach and skin, Limit the performance of its bioactivity.Therefore, people have invested sight the collagen polypeptide of small molecule.With collagen phase Than collagen polypeptide not only has the nutritive peculiarity of collagen, but also with being easy to digest and assimilate, hypoallergenic, eats peace Congruent advantage.In addition, collagen polypeptide also has anti-oxidant, blood pressure lowering, improves immunity, improves memory and antitumor etc. Bioactivity.
Technology of preparing on collagen peptide has many methods, and the prior art is CN as authorized public platform The Chinese invention patent of 102251006 B, discloses a kind of manufacture method of collagen from black sea cucumbers from East China Sea, this method is in East Sea crow Add hydrolysising protease in ginseng slurries, compound protease, flavor protease are directly hydrolyzed, then through membrane filtration, concentration, do It is dry that collagen from black sea cucumbers from East China Sea product is prepared, have the characteristics that preparation method is simple, but this method process is difficult to control, by In three strands of superhelixes of collagen, easily hydrolysis is not thorough, and nutriment wastes, and is added in the preparation method Excessive protease, because enzyme digestion reaction speed has KMValue determines, rather than is determined by the amount of enzyme, therefore causes the wave of enzyme Take.
The content of the invention
The purpose of this method is that providing a kind of novel microbial protease hydrolytic prepares collagen from black sea cucumbers from East China Sea peptide Method, this method process is easily-controllable, and hydrolysis is thorough, and product Small Peptides and amino acid accounting are larger, are easy to be absorbed by organisms.
The ground bacillus of compost on the spot in the method for the present invention, bacillus licheniformis, bacillus megaterium are bought in State's Type Tissue Collection.
The present invention is directed to the problem of being mentioned in background technology, and the technical solution taken is：Novel microbial protease hydrolytic Collagen from black sea cucumbers from East China Sea peptide is prepared, including：Pretreatment, extraction, fermentation, purifying, hydrolysis, purification, specifically include following steps：
Pretreatment：Take the black sea cucumbers from East China Sea body wall of fresh clean, with 1-2% hydrochloric acid solutions immersion 36-48 it is small when, intermittent stirring, 12- 18 change a solution when small, with deionized water rinsing to neutrality after the completion of immersion；Then it is molten with 0.03-0.05% calcium hydroxides When liquid immersion 36-48 is small, intermittent stirring, changes a solution when 12-18 is small, with deionized water rinsing to neutrality after the completion of immersion； Sea Cucumber body wall is immersed in 5-6% sodium hydroxide solutions, is transparent and pulls out to wall of sea cucumber Stichopus japonicus, with deionized water rinsing into Property, then chopping homogenate；Pretreatment can remove calcareous, the non-Collagen material in wall of sea cucumber Stichopus japonicus, in order to further extract Collagen；
Extraction：Wall of sea cucumber Stichopus japonicus homogenate is placed in 0.05-0.08mol/L acetum systems, adds substrate weight 0.3- 0.5% pepsin limited enzymatic hydrolysis, at 5-10 DEG C, bifrequency ultrasonic wave added enzymolysis, continues under stirring at low speed control temperature When enzymolysis 48-72 is small, boiling water bath enzyme deactivation, by enzymolysis liquid in 10000-13000r/min high speed centrifugations, takes supernatant, precipitation repeats Enzymolysis operation 2-3 times, collects supernatant, is freeze-dried to obtain collagen from black sea cucumbers from East China Sea dry product；Restriction Endonuclease under weak acid system, Not only ensured while collagen consistency but also there is higher collagen recovery rate, recovery rate can reach more than 45%；
Fermentation：Fermentation culture is taken, under the conditions of 30-33 DEG C of temperature, shaking speed 230-250r/min, is inoculated with 4-6% gemma Bacillus bacterium solution, when fermentation 25-30 is small；Under this fermentation condition, fermentation of bacillus protease activity amount can reach maximum, favorably In the enzymolysis to collagen, technique is optimized, has saved cost；
Purifying：Thalline is removed after zymotic fluid is centrifuged 5-10 minutes at a temperature of 3-4 DEG C with 7000-8000r/min up to thick enzyme Liquid；Take crude enzyme liquid by it is a small amount of repeatedly in a manner of be slowly added to solid ammonium sulfate, it is molten to be configured to the ammonium sulfate that saturation degree is 70-72% Liquid, saturated solution is preserved overnight at 3-4 DEG C, is kept the temperature to centrifuge 10-15 minutes with 6000-6500r/min and is collected precipitation；Will It is precipitated and dissolved in water, enzyme must be purified by ultrafiltration membrance filter；Micro- life can be purified by ammonium sulfate precipitation and ultrafiltration membrance filter Thing protease, improves proteinase activity；
Hydrolysis：40-50 parts of lyophilized collagen from black sea cucumbers from East China Sea and 0.6-0.8 parts of bacillus protein enzymes are weighed, are added to Be completely dissolved in 100-150 parts of distilled water, add 0.002-0.006 parts 2,3- dimethyl -2- butanol, 0.002-0.003 parts it is red Moss sugar, adjusting pH is 7.5-8.0, is placed in 45-55 DEG C of thermostat water bath, bifrequency ultrasonic wave added enzymolysis, when hydrolysis 1.5-2 is small Afterwards with boiling water bath enzyme deactivation 15-20 minutes, enzymolysis liquid is cooled down to obtain；Three strands of superhelixes of collagen uniqueness, property are very steady Fixed, general processing temperature and short time heating cannot all make its decomposition, so that it is more difficult to cause it to digest and assimilate, be not easy by people Body makes full use of, and collagen from black sea cucumbers from East China Sea molecule primarily forms the less polypeptide of relative molecular weight, small peptide, amino after hydrolysis Acid, its absorption rate can improve much after hydrolysis, and can promote the absorption of the other oroteins in food；Micro 2, 3- dimethyl -2- butanol can improve the enzymolysis speed of bacillus protein enzyme, reduce hydrolysis time significantly, reduce the time Cost and cost of labor, and the rising removing when subsequent hydrolyzate boils, will not remain in collagen peptide product；
Purification：Hydrolyzate is purified with AB-8 macroreticular resins, its adsorption flow rate is 7-8mL/min, with the ethanol of 70-75% Solution elutes, elution flow rate 8.5-9.0mL/min, is concentrated under reduced pressure, is freeze-dried up to black sea cucumbers from East China Sea glue after collecting purification liquid Former protein peptides；After purification, amino acid, small peptide accounting in collagen peptide product improve, its molecular weight is between 180D-3000D Between, it is easy to be absorbed by organisms utilization, collagen peptide has a variety of medicinal health care functions, such as suppresses angiotensin converting enzyme Activity, its antioxidation activity having can eliminate free radical, can also reduce the Human Osteoarthritis such as knee joint or hip joint Pain, strengthens bone density, maintains bone metabolism balance etc., is a kind of nutraceutical agents for easily absorbing, having no toxic side effect.
Preferably, fermentation culture used is：Glycerine 35-40g/L, bean cake powder 30-35g/L, wheat bran 10-12g/L, Urea 5.0-8.0g/L, Na2HP044.0-5.0g/L、K2HP040.3-0.5g/L、 MgSO41.44-1.50g/L、CaCl21.1- 1.2g/L, NaCl5.0-6.0g/L and surplus distilled water, pH is naturally, 115-121 DEG C of autoclaving 20-35 minutes；Above-mentioned fermentation Nutrient solution can not only provide the environment that Institute of Micro-biology of the present invention needs, moreover it is possible to provide carbon source needed for growth breeding, nitrogen source and Growth factor, the reproduction speed of strain is fast, and can induce strain secretion and produce substantial amounts of protease, improves the production of protease Amount and yield.
Preferably, fermentation Bacillus used is compost ground bacillus or bacillus licheniformis or huge on the spot Bacterium anthracoides；The prolease activity of bacillus is higher, can be effectively small peptide and amino by collagen molecules enzymolysis The lower-molecular substances such as acid.
Preferably, it is 9-10 that erythrose used, which is weight ratio,:1 D- (-)-erythrose and D- (+)-erythrose Mixture；D- (-)-erythrose and the mixture of D- (+)-erythrose can produce synergistic effect with 2,3- dimethyl -2- butanol, The energy barrier between the ground state and excitation state of enzymatic hydrolysis system is reduced, and then increases the affinity of substrate and enzyme, greatly improves enzyme Promote reaction rate.
Preferably, the supersonic frequency of bifrequency ultrasonic wave added enzymolysis used is respectively 30-45kHz and 60-65kHz, The ultrasonic sound intensity is 0.4-0.5W/cm2；Bifrequency ultrasound can be by reducing enzyme digestion reaction EaAnd KMValue, increases enzyme-to-substrate Contact frequency and contact area, increase enzyme digestion reaction speed.
Compared with prior art, the advantage of the invention is that：1) by pretreated black sea cucumbers from East China Sea body wall in weak acid system Lower Restriction Endonuclease, while collagen consistency is not only ensured but also has a higher collagen recovery rate, and recovery rate can be with Reach more than 45%；2) D- (-)-erythrose of specific proportioning can be produced with D- (+)-erythrose with 2,3- dimethyl -2- butanol Raw synergistic effect, reduces the energy barrier between the ground state of enzymatic hydrolysis system and excitation state, coordinates bifrequency ultrasound is common to reduce enzyme Solution reaction EaAnd KMValue, and then increase matrix and the affinity of enzyme, greatly improve enzyme's reaction speeding；3) bacillus protein enzyme Efficient enzymolysis vigor can destroy three strands of superhelixes of Sea Cucumber collagen uniqueness, be then hydrolyzed to average molecular The small-molecule substances such as less polypeptide, small peptide, amino acid are measured, its absorption rate can improve much after hydrolysis, and can promote Into the absorption of the other oroteins in food, collagen peptide has a variety of medicinal health care functions, is a kind of easily absorption, nontoxic pair The nutraceutical agents of effect.
Brief description of the drawings
Fig. 1 is the graph of molecular weight distribution of black sea cucumbers from East China Sea collagen peptide in 1-3 of the embodiment of the present invention.
Embodiment
The present invention program is described further below by embodiment：
Embodiment 1：
Novel microbial protease hydrolytic prepares collagen from black sea cucumbers from East China Sea peptide, comprises the following steps：
1) wall of sea cucumber Stichopus japonicus homogenate is placed in 0.05mol/L acetum systems, adds the pepsin limit of substrate weight 0.3% Property enzymolysis processed, for control temperature at 5 DEG C, bifrequency ultrasonic wave added enzymolysis, supersonic frequency is respectively 30kHz and 60kHz, is continued low When the lower enzymolysis 48 of speed stirring is small, boiling water bath enzyme deactivation, by enzymolysis liquid in 10000r/min high speed centrifugations, takes supernatant, precipitation repeats Enzymolysis operation 2 times, collects supernatant, is freeze-dried to obtain collagen from black sea cucumbers from East China Sea dry product；
2) glycerine 35g, bean cake powder 30g, wheat bran 10g, urea 5.0g, Na are weighed2HP044g、K2HP040.3g/L、 MgSO41.44g、CaCl21.1g, NaCl5.0g and 910 parts of distilled water, pH trainings naturally, 115 DEG C of autoclavings must ferment for 20 minutes Nutrient solution, under the conditions of 30 DEG C of temperature, shaking speed 230r/min, is inoculated with 4% bacillus megaterium bacterium solution, when fermentation 25 is small； Under this fermentation condition, fermentation of bacillus protease activity amount can reach maximum, be conducive to the enzymolysis to collagen, optimize Technique, has saved cost；
3) zymotic fluid is removed into thalline up to crude enzyme liquid after five minutes at a temperature of 3 DEG C with 7000r/min centrifugations；Take crude enzyme liquid with A small amount of repeatedly mode is slowly added to solid ammonium sulfate, the ammonium sulfate that saturation degree is 70% is configured to, by saturated solution in 3 DEG C It is lower to preserve overnight, keep the temperature to centrifuge 10 minutes with 6000r/min and collect precipitation；It will be precipitated and dissolved in water, and pass through ultrafiltration membrance filter Enzyme must be purified；Microbial protease can be purified by ammonium sulfate precipitation and ultrafiltration membrance filter, improve proteinase activity；
4) 40 parts of lyophilized collagen from black sea cucumbers from East China Sea and 0.6 part of bacillus megaterium protease are weighed, are added to 100 parts of steamings It is completely dissolved in distilled water, it is 7.5 to adjust pH, is placed in 45 DEG C of thermostat water baths, bifrequency ultrasonic wave added enzymolysis, supersonic frequency point Not Wei 30kHz and 60kHz, the ultrasonic sound intensity is 0.4W/cm2, when hydrolysis 1.5 is small after with boiling water bath enzyme deactivation 15 minutes, cool down to obtain enzyme Solve liquid；Three strands of superhelixes of collagen uniqueness, property is sufficiently stable, and general processing temperature and short time heating are not It can make its decomposition, so that it is more difficult to cause it to digest and assimilate, be not easy to be made full use of by human body, collagen from black sea cucumbers from East China Sea molecule warp The less polypeptide of relative molecular weight, small peptide, amino acid are primarily formed after hydrolysis, its absorption rate can improve very after hydrolysis It is more, and the absorption of the other oroteins in food can be promoted；
5) hydrolyzate is purified with AB-8 macroreticular resins, its adsorption flow rate is 7mL/min, is washed with 70% ethanol solution De-, elution flow rate 8.5mL/min, is concentrated under reduced pressure after collecting purification liquid, is freeze-dried up to collagen from black sea cucumbers from East China Sea peptide.
Embodiment 2：
Novel microbial protease hydrolytic prepares collagen from black sea cucumbers from East China Sea peptide, comprises the following steps：
1) pre-process：The black sea cucumbers from East China Sea body wall of fresh clean is taken, successively with 2% hydrochloric acid solution and 0.05% calcium hydroxide solution point Not Jin Pao 48 it is small when, during intermittent stirring, 18 change a solution when small, with deionized water rinsing to neutrality after the completion of immersion； Sea Cucumber body wall is immersed in 6% sodium hydroxide solution, is transparent and pulls out to wall of sea cucumber Stichopus japonicus, with deionized water rinsing into Property, then chopping homogenate；Pretreatment can remove calcareous, the non-Collagen material in wall of sea cucumber Stichopus japonicus, in order to further extract Collagen；
2) extract：Wall of sea cucumber Stichopus japonicus homogenate is placed in 0.08mol/L acetum systems, adds the stomach egg of substrate weight 0.5% White enzyme limited enzymatic hydrolysis, control temperature is at 10 DEG C, when continuing that enzymolysis 72 is small under stirring at low speed, boiling water bath enzyme deactivation, by enzymolysis liquid in 13000r/min high speed centrifugations, take supernatant, and precipitation repeats enzymolysis operation 3 times, collects supernatant, be freeze-dried to obtain black sea cucumbers from East China Sea Collagen dry product；Restriction Endonuclease under weak acid system, while collagen consistency is not only ensured but also has higher collagen Protein extracting ratio, recovery rate can reach more than 45%；
3) ferment：Preparing fermentation culture used is：Glycerine 40g/L, bean cake powder 35g/L, wheat bran 12g/L, urea 8.0g/L, Na2HP045.0g/L、K2HP040.5g/L、MgSO41.50g/L、CaCl21.2g/L, NaCl6.0g/L and surplus distilled water, pH Naturally, 121 DEG C of autoclavings take fermentation culture in 35 minutes, and under the conditions of 33 DEG C of temperature, shaking speed 250r/min, inoculation 6% Bacillus licheniformis liquid, when fermentation 30 is small；Under this fermentation condition, the lichen bacillus ferments protease activity amount can be with Reach maximum, be conducive to the enzymolysis to collagen, optimize technique, saved cost；
4) purify：Zymotic fluid is removed into thalline up to crude enzyme liquid after ten minutes at a temperature of 4 DEG C with 8000r/min centrifugations；Take thick Enzyme liquid by it is a small amount of repeatedly in a manner of be slowly added to solid ammonium sulfate, be configured to the ammonium sulfate that saturation degree is 72%, saturation is molten Liquid preserves overnight at 4 DEG C, keeps the temperature to centrifuge 15 minutes with 6500r/min and collects precipitation；It will be precipitated and dissolved in water, and pass through ultrafiltration Membrane filtration must purify enzyme；Microbial protease can be purified by ammonium sulfate precipitation and ultrafiltration membrance filter, improve proteinase activity；
5) hydrolyze：50 parts of lyophilized collagen from black sea cucumbers from East China Sea and 0.8 part of bacillus licheniformis protease are weighed, are added to 150 It is completely dissolved in part distilled water, adds 0.006 part of 2,3- dimethyl -2- butanol, it is 8.0 to adjust pH, is placed in 55 DEG C of waters bath with thermostatic control In pot, when hydrolysis 2 is small after with boiling water bath enzyme deactivation 20 minutes, cool down to obtain enzymolysis liquid；2,3- dimethyl -2- butanol can effectively improve The enzymolysis speed of bacillus protein enzyme, reduces hydrolysis time significantly, reduces time cost and cost of labor, and with It is rising when hydrolyzate afterwards boils to remove, it will not remain in collagen peptide product；
6) purify：Hydrolyzate is purified with AB-8 macroreticular resins, its adsorption flow rate is 8mL/min, molten with 75% ethanol Liquid elutes, elution flow rate 9.0mL/min, is concentrated under reduced pressure, is freeze-dried up to collagen from black sea cucumbers from East China Sea after collecting purification liquid Peptide；After purification, amino acid, small peptide accounting in collagen peptide product improve, its molecular weight is between 180D-3000D, easily Being utilized in being absorbed by organisms, collagen peptide has a variety of medicinal health care functions, such as suppresses hypertensin conversion enzyme activity, its The antioxidation activity having can eliminate free radical, reduce the pain of the Human Osteoarthritis such as knee joint or hip joint, and enhancing bone is close Degree, maintains bone metabolism balance etc., is a kind of nutraceutical agents for easily absorbing, having no toxic side effect.
Embodiment 3：
The technical solution taken is：Novel microbial protease hydrolytic prepares collagen from black sea cucumbers from East China Sea peptide, including：Pretreatment, Extraction, fermentation, purifying, hydrolysis, purification, specifically include following steps：
Pretreatment：Take the black sea cucumbers from East China Sea body wall of fresh clean, with 2% hydrochloric acid solution immersion 45 it is small when, intermittent stirring, 15 change when small Solution, with deionized water rinsing to neutrality after the completion of immersion；Then with 0.04% calcium hydroxide solution immersion 45 it is small when, Have a rest stirring, 15 change a solution when small, with deionized water rinsing to neutrality after the completion of immersion；Sea Cucumber body wall is immersed in 6% hydrogen In sodium hydroxide solution, it is transparent and pulls out to wall of sea cucumber Stichopus japonicus, with deionized water rinsing to neutrality, then chopping homogenate；Pretreatment Calcareous, the non-Collagen material in wall of sea cucumber Stichopus japonicus can be removed, in order to further extract collagen；
Extraction：Wall of sea cucumber Stichopus japonicus homogenate is placed in 0.06mol/L acetum systems, adds the stomach cardia of substrate weight 0.4% Enzyme limited enzymatic hydrolysis, control temperature at 6 DEG C, continue stirring at low speed under enzymolysis 60 it is small when, boiling water bath enzyme deactivation, by enzymolysis liquid in 12000r/min high speed centrifugations, take supernatant, and precipitation repeats enzymolysis operation 3 times, collects supernatant, be freeze-dried to obtain black sea cucumbers from East China Sea Collagen dry product；Restriction Endonuclease under weak acid system, while collagen consistency is not only ensured but also has higher collagen Protein extracting ratio, recovery rate can reach more than 45%；
Fermentation：Preparing fermentation culture is：Glycerine 38g/L, bean cake powder 32g/L, wheat bran 10g/L, urea 6.5g/L, Na2HP044.5g/L、K2HP040.4g/L、MgSO41.45g/L、CaCl21.1g/L, NaCl5.5g/L and surplus distilled water, pH is certainly So, 120 DEG C of autoclavings take fermentation culture in 20 minutes, under the conditions of 32 DEG C of temperature, shaking speed 240r/min, inoculation 5% Compost ground bacillus bacterium solution on the spot, when fermentation 30 is small；Under this fermentation condition, compost ground bacillus fermentation protein enzyme on the spot Amount living can reach maximum, be conducive to the enzymolysis to collagen, optimize technique, saved cost；
Purifying：Thalline is removed after zymotic fluid is centrifuged 6 minutes at a temperature of 4 DEG C with 7500r/min up to crude enzyme liquid；Take crude enzyme liquid By it is a small amount of repeatedly in a manner of be slowly added to solid ammonium sulfate, the ammonium sulfate that saturation degree is 71% is configured to, by saturated solution in 4 Preserved overnight at DEG C, keep the temperature to centrifuge 12 minutes with 6400r/min and collect precipitation；It will be precipitated and dissolved in water, and pass through ultrafiltration membrane mistake Enzyme must be purified by filtering；Microbial protease can be purified by ammonium sulfate precipitation and ultrafiltration membrance filter, improve proteinase activity；
Hydrolysis：The compost ground bacillus protease on the spot of 50 parts of lyophilized collagen from black sea cucumbers from East China Sea and 0.7 part is weighed, is added to Be completely dissolved in 120 parts of distilled water, add 0.004 part of 2,3- dimethyl -2- butanol, 0.0018 part of D- (-)-erythrose, 0.0002 part of D- (+)-erythrose, it is 8.0 to adjust pH, is placed in 50 DEG C of thermostat water baths, bifrequency ultrasonic wave added enzymolysis, ultrasound Frequency is respectively 40kHz and 65kHz, and the ultrasonic sound intensity is 0.5W/cm2, when hydrolysis 2 is small after with boiling water bath enzyme deactivation 20 minutes, cooling Obtain enzymolysis liquid；The synergistic effect of 2,3- dimethyl -2- butanol and erythrose can improve the enzymolysis speed of bacillus protein enzyme Rate, reduces hydrolysis time significantly, reduces time cost and cost of labor, and transpiration removes when subsequent hydrolyzate boils Go, will not remain in collagen peptide product；Three strands of superhelixes of collagen uniqueness, property is sufficiently stable, generally Processing temperature and the short time heating cannot all make its decomposition so that it is more difficult to cause it to digest and assimilate, be not easy abundant by human body Utilize, collagen from black sea cucumbers from East China Sea molecule primarily forms the less polypeptide of relative molecular weight, small peptide, amino acid, water after hydrolysis Its absorption rate can improve much after solution, and can promote the absorption of the other oroteins in food；
Purification：Hydrolyzate is purified with AB-8 macroreticular resins, its adsorption flow rate is 8mL/min, with 72% ethanol solution Elution, elution flow rate 9.0mL/min, is concentrated under reduced pressure after collecting purification liquid, is freeze-dried up to collagen from black sea cucumbers from East China Sea peptide； After purification, amino acid, small peptide accounting in collagen peptide product improve, its molecular weight is easy between 180D-3000D Utilization is absorbed by organisms, collagen peptide has a variety of medicinal health care functions, such as suppresses hypertensin conversion enzyme activity, it has Some antioxidation activities can eliminate free radical, reduce the pain of the Human Osteoarthritis such as knee joint or hip joint, strengthen bone density, Bone metabolism balance etc. is maintained, is a kind of nutraceutical agents for easily absorbing, having no toxic side effect.
According to high performance gel filtration chromatography, (Zhang Yitao, Yu Wei, Jiang Guoyong, wait high performance gel filtration chromatographies to measure peptide Relative molecular weight distribution [J] feed herdings, 2012 (8):42-44), the black sea cucumbers from East China Sea glue in three embodiments is measured respectively The molecular weight distribution of former protein peptides, draws the distribution accounting between each molecular weight area according to regression equation and peak area, arranges number According to as shown in Figure 1.
As seen from Figure 1, novel microbial protease hydrolyzes the collagen from black sea cucumbers from East China Sea extracted through pepsin Into amino acid and small peptide, its Degree of Enzymatic Hydrolysis is more thorough, and enzymolysis efficiency is higher.It is seen also in fig. l that in embodiment 3, Extraction and enzymolysis process are optimized, is digested in extraction using bifrequency ultrasonic wave added, 2,3- diformazans are utilized while enzymolysis Base -2- butanol, D- (-)-erythrose, D- (+)-erythrose and bifrequency ultrasonic wave added, can greatly improve the progress of enzymolysis, make Molecular weight of product exceeded 75% between the accounting of the collagen peptide of 180-3000D, illustrate 2,3- dimethyl -2- butanol, D- (-)-erythrose, D- (+)-erythrose coordinate bifrequency ultrasound for improving enzyme digestion reaction efficiency, controlled enzymatic hydrolysis precision aspect Have the function that more excellent, the method for the present invention is that a kind of efficient, thorough, easily-controllable novel microbial protease hydrolytic prepares the East Sea The method of Sea Cucumber collagen peptide.
Routine operation in the operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme is described in detail in embodiment described above, it should be understood that the above is only For the specific embodiment of the present invention, it is not intended to limit the invention, all any modifications made in the spirit of the present invention, Supplement or similar fashion replacement etc., should all be included in the protection scope of the present invention.

Claims (10)

1. novel microbial protease hydrolytic prepares collagen from black sea cucumbers from East China Sea peptide, including：Pre-process, extract, fermenting, purifying, Hydrolysis, purification, it is characterised in that：The hydrolysing step is：Weigh lyophilized collagen from black sea cucumbers from East China Sea and bacillus egg White enzyme, is added in distilled water and is completely dissolved, and adds 2,3- dimethyl -2- butanol, erythrose, adjusting pH is 7.5-8.0, is placed in In 45-55 DEG C of thermostat water bath, bifrequency ultrasonic wave added enzymolysis, when hydrolysis 1.5-2 is small after with boiling water bath enzyme deactivation 15-20 minutes, Cool down to obtain enzymolysis liquid.
2. novel microbial protease hydrolytic according to claim 1 prepares collagen from black sea cucumbers from East China Sea peptide, its feature exists In：The pre-treatment step is：Take the black sea cucumbers from East China Sea body wall of fresh clean, with 1-2% hydrochloric acid solutions immersion 36-48 it is small when, Have a rest stirring, a solution is changed when 12-18 is small, with deionized water rinsing to neutrality after the completion of immersion；Then 0.03-0.05% hydrogen is used When calcium oxide solution immersion 36-48 is small, intermittent stirring, changes a solution, deionized water rinsing is used after the completion of immersion when 12-18 is small To neutrality；Sea Cucumber body wall is immersed in 5-6% sodium hydroxide solutions, is transparent and pulls out to wall of sea cucumber Stichopus japonicus, is rushed with deionized water Neutrality is washed till, then chopping homogenate.
3. novel microbial protease hydrolytic according to claim 1 prepares collagen from black sea cucumbers from East China Sea peptide, its feature exists In：The extraction step is：Wall of sea cucumber Stichopus japonicus homogenate is placed in 0.05-0.08mol/L acetum systems, adds substrate weight The pepsin limited enzymatic hydrolysis of 0.3-0.5% is measured, at 5-10 DEG C, bifrequency ultrasonic wave added enzymolysis, continues low speed and stir control temperature Mix lower enzymolysis 48-72 it is small when, by enzymolysis liquid in 10000-13000r/min high speed centrifugations, take supernatant, precipitation repeats enzymolysis behaviour Make 2-3 times, collect supernatant, be freeze-dried to obtain collagen from black sea cucumbers from East China Sea dry product.
4. novel microbial protease hydrolytic according to claim 3 prepares collagen from black sea cucumbers from East China Sea peptide, its feature exists In：The supersonic frequency of bifrequency ultrasonic wave added enzymolysis in the extraction step is respectively 30-45kHz and 60-65kHz, is surpassed Several is by force 0.4-0.5W/cm2。
5. novel microbial protease hydrolytic according to claim 1 prepares collagen from black sea cucumbers from East China Sea peptide, its feature exists In：The fermentation step is：Fermentation culture is taken, under the conditions of 30-33 DEG C of temperature, shaking speed 230-250r/min, is connect Kind 4-6% bacillus bacterium solutions, when fermentation 25-30 is small.
6. novel microbial protease hydrolytic according to claim 5 prepares collagen from black sea cucumbers from East China Sea peptide, its feature exists In：Bacillus in the fermentation step is compost ground bacillus or bacillus licheniformis or huge gemma bar on the spot Bacterium；Fermentation culture is：Glycerine 35-40g/L, bean cake powder 30-35g/L, wheat bran 10-12g/L, urea 5.0-8.0g/L, Na2HP044.0-5.0g/L、K2HP040.3-0.5g/L、MgSO41.44-1.50g/L、CaCl21.1-1.2g/L、NaCl5.0- 6.0g/L and surplus distilled water, pH is naturally, 115-121 DEG C of autoclaving 20-35 minutes.
7. novel microbial protease hydrolytic according to claim 1 prepares collagen from black sea cucumbers from East China Sea peptide, its feature exists In：The purification step is：Bacterium is removed after zymotic fluid is centrifuged 5-10 minutes at a temperature of 3-4 DEG C with 7000-8000r/min Body is up to crude enzyme liquid；Take crude enzyme liquid by it is a small amount of repeatedly in a manner of be slowly added to solid ammonium sulfate, it is 70-72%'s to be configured to saturation degree Ammonium sulfate, saturated solution is preserved overnight at 3-4 DEG C, is kept the temperature to centrifuge 10-15 minutes with 6000-6500r/min and is collected Precipitation；It will be precipitated and dissolved in water, enzyme must be purified by ultrafiltration membrance filter.
8. novel microbial protease hydrolytic according to claim 1 prepares collagen from black sea cucumbers from East China Sea peptide, its feature exists In：The hydrolysing step is：Weigh 40-50 parts of lyophilized collagen from black sea cucumbers from East China Sea and 0.6-0.8 parts of bacillus proteins Enzyme, is added in 100-150 parts of distilled water and is completely dissolved, and adds 0.002-0.006 parts 2,3- dimethyl -2- butanol, 0.002- 0.003 part of erythrose, adjusting pH are 7.5-8.0, are placed in 45-55 DEG C of thermostat water bath, bifrequency ultrasonic wave added enzymolysis, hydrolysis With boiling water bath enzyme deactivation 15-20 minutes after when 1.5-2 is small, enzymolysis liquid is cooled down to obtain.
9. novel microbial protease hydrolytic according to claim 8 prepares collagen from black sea cucumbers from East China Sea peptide, its feature exists In：Erythrose in the hydrolysing step is that weight ratio is 9-10:1 D-（-）- erythrose and D-（+）The mixing of-erythrose Thing.
10. novel microbial protease hydrolytic according to claim 1 prepares collagen from black sea cucumbers from East China Sea peptide, its feature exists In：The purification step is：Hydrolyzate is purified with AB-8 macroreticular resins, its adsorption flow rate is 7-8mL/min, with The ethanol solution elution of 70-75%, elution flow rate 8.5-9.0mL/min, is concentrated under reduced pressure, freeze-drying is after collecting purification liquid Obtain collagen from black sea cucumbers from East China Sea peptide.
CN201710859089.0A 2017-09-21 2017-09-21 Novel microbial protease hydrolytic prepares collagen from black sea cucumbers from East China Sea peptide Pending CN108018326A (en)

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