CN106755240A - A method for preparing black cucumber collagen oligopeptides by microwave/ultrasonic assistance and microbial protease hydrolysis - Google Patents

Abstract

The invention relates to a method for preparing black ginseng collagen oligopeptide by microwave/ultrasonic-assisted and microbial protease hydrolysis. The project adopts a microwave/ultrasonic wave auxiliary method, so that amino acid residues hidden in the collagen structure can be presented, and then the amino acid residues are hydrolyzed into smaller polypeptide under the action of microbial protease, and the yield of oligopeptide (with the molecular weight of less than 1000Da) can be obviously improved. The preparation method has the advantages of short production period, high yield, high product purity, obvious oxidation resistance, good solubility, high absorptivity and no peculiar smell or bitter taste; the sea cucumber is fully processed and utilized in a high-valued way.

Description

Preparation of collagen from black ginseng by microwave/ultrasonic assisted hydrolysis with microbial protease oligopeptide method

technical field

The invention relates to the preparation of collagen peptides, in particular to a method for preparing collagen oligopeptides from black ginseng with the aid of microwave/ultrasonic waves and hydrolysis of microbial proteases.

Background technique

Collagen polypeptide is a product between protein and amino acid obtained after collagen or gelatin is degraded by protease, etc. Generally, the linear peptide formed by 2 to 10 amino acids through peptide bonds is called oligopeptide or small peptide; A polymer of more than 10 amino acids bonded together is called a polypeptide. In recent years, many scholars have studied the digestion and absorption mechanism of peptides, and believe that peptides and even a considerable amount of proteins can be completely absorbed and utilized by the body in animals, such as oral vaccines and enzyme treatment of diseases. Peptide transport systems are faster than free amino acids. Numerous trials have shown that the addition of peptides can also increase the rate of amino acid absorption. In addition, peptides can also promote the rate of protein deposition, because peptides can be directly used in the synthesis of human proteins. Long-term consumption of foods added with collagen peptides can help improve skin elasticity, delay aging, and enhance immunity. It is also convenient to eat and is suitable for the general public.

my country's research on the utilization of Donghai black ginseng began in the early 1980s. Although there are products of Donghai black ginseng (Donghai ginseng) in Shanghai, Wenzhou and other places, it has not been industrialized so far. In recent years, with the rise of sea cucumber breeding in Bohai Bay, a sea cucumber industry with an annual output value of more than 10 billion has been generated, which has triggered many scientific research institutions to study the nutritional and medicinal components of sea cucumbers (including black cucumbers from the East China Sea). According to research by experts including Liao Yulin from the Institute of Oceanology, Chinese Academy of Sciences, Du Lijun from Tsinghua University’s postdoctoral mobile station, and Yi Yanghua from the Shanghai Second Army Marine Drug Research Institute, the black ginseng of the East China Sea is rich in nutrients and medicinal value. Among them, the total amount of amino acids, sea cucumber The content of saponin and sea cucumber polysaccharide is higher than that of topaz sea cucumber. However, it has also been suggested that the dirt layer on the surface of the black ginseng of the East China Sea contains a large amount of harmful heavy metals such as lead, cadmium, and mercury. The black ginseng of the East China Sea lives in the mud of the sea all the year round, and the body surface secretes a mucus rich in amino acids and collagen. This mucus is firmly combined with the harmful heavy metals in the mud of the sea, forming the dirt layer on the body surface of the black ginseng of the East China Sea. It is very difficult to remove this layer of dirt. It is difficult for Donghai black ginseng products to meet the national mandatory standard GB2762-2012 "National Standard for Food Safety/Pollution in Food" by conventional cleaning and cleaning with sodium hydroxide and hydrogen peroxide. Requirements of the standards of GB2733-2005 "Hygienic Standards for Fresh and Frozen Animal Aquatic Products" and NY/T1514-2007 "Green Food Sea Cucumber and Products" of the Ministry of Agriculture. According to the test results of Donghai black ginseng products collected in the market, the harmful heavy metals exceed the national standard by tens to hundreds of times, which is the fundamental reason why the rich resources of Donghai black ginseng have not been effectively utilized and industrialized.

Contents of the invention

The invention provides a method for preparing collagen oligopeptides of black ginseng by hydrolysis of microbial protease, which improves purity and extraction rate, removes bitter smell, improves mouthfeel, and facilitates human body absorption.

The present invention adopts the following technical solutions:

A method for preparing black cucumber collagen oligopeptides through microwave/ultrasonic assistance and microbial protease hydrolysis, comprising the following process steps:

a. Choose Donghai black ginseng rich in collagen as raw material, soak it in water to remove the salt, wash it and immerse it in the soaking solution for 50-60 minutes, remove and drain the water, use a beater for beating treatment, add 35-40 times the volume of After purifying water, use a colloid mill to grind and refine to obtain a slurry;

b. Heat the slurry obtained in step a until the central temperature reaches 130-145°C, keep it for 10 minutes, cool to 20-25°C, add protease for enzymolysis, turn on the computer microwave-ultrasonic combined catalytic synthesizer to carry out enzymatic hydrolysis Ultrasonic-microwave-assisted enzymatic hydrolysis, in which the insertion depth of the ultrasonic probe is fixed, the microwave power is 500W, the ultrasonic power is 100W, and the ultrasonic-microwave treatment temperature is 41°C. After 15 minutes of treatment, the enzymatic hydrolysis is continued to inactivate the enzyme; pass through 120 mesh filter screen to obtain collagen filtrate;

c. In the collagen filtrate obtained in step b, add glucose or sucrose by 0.5-3% by weight, adjust the pH to 6-7, and obtain the initial fermentation liquid;

d. Put the initial fermented liquid obtained in step c into an empty sterilization fermenter for actual sterilization. The conditions for empty sterilization and actual sterilization are both 121°C and 20min. After the temperature of the tank drops to 37°C, insert the fermented seeds. The inoculum size is The volume percentage of the initial fermentation liquid is 2-4.5%, and the inoculation mixture is sealed and fermented at 37°C and a stirring speed of 100-300rpm for 2-5 days, and the residue is filtered out of the fermentation liquid to obtain a fermentation filtrate;

e. Add 15-20% trehalose, 0.3-0.8% maltodextrin and 3-5% glycerol to the fermentation filtrate obtained in step d, pre-freeze at -50°C for 2-6h, then freeze at -70°C in a cold trap and Freeze-drying for 12-20 hours under a vacuum of <150Pa to directly obtain sea cucumber collagen peptides; or, use a tubular membrane with a pore size of 0.1-0.5 μm to remove macromolecules from the fermentation filtrate obtained in step d, and then use a membrane with a molecular weight cut-off of 3 kDa The ultrafiltration membrane cut-off classification, the retentate is concentrated and desalted by nanofiltration membrane, and then spray-dried under the conditions of 175-185°C inlet air temperature, 75-85°C outlet air temperature and 0.35-0.45MPa spray pressure to obtain sea cucumber collagen peptide.

As a preference, the soaking solution in step a consists of the following raw materials in parts by weight: 20-25 parts of hawthorn, 1-5 parts of wild chrysanthemum, 30-40 parts of patchouli, 15-18 parts of chitosan, 12-2 parts of sodium alginate 15 parts and 200-350 parts of water, the mass ratio of Donghai Wushen soaking liquid is 1:10.

Preferably, the fermented seeds in step d are in-situ composting Geobacillus, Bacillus licheniformis or Bacillus megaterium.

As a preference, the raw materials of the fermented seed culture medium are calculated in the following parts by weight: 3% soybean peptone, 5% selenium-enriched yeast, 6% asparagine, 5.5% methionine, 0.5% beef powder, 0.5% glucose, 0.1% chlorinated Sodium, 0.01% potassium dihydrogen phosphate, and the rest are distilled water, sterilized at 121°C for 20 minutes, and cooled for later use.

As a preference, inoculate the in-situ compost Geobacillus, Bacillus licheniformis or Bacillus megaterium into the fermented seed culture medium, shake the table at 180r/min, cultivate for 48h, and the Bacillus in the fermentation broth is >10 ⁸ cfu/mL.

The beneficial effects of the present invention are: the preparation method of the present invention has short production cycle, high yield, high product purity, remarkable oxidation resistance, good solubility, high absorption, no peculiar smell and bitter taste; Chemical processing and utilization.

detailed description

In order to enable those skilled in the art to better understand the technical solution of the present invention, these explanations do not limit the present invention in any form. Unless otherwise defined, all scientific and technical terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs and related fields.

The present invention will be described in further detail below in conjunction with specific examples, but it should be understood that the examples should not be construed as limiting the protection scope of the present invention.

Example 1

A method for preparing black cucumber collagen oligopeptides through microwave/ultrasonic assistance and microbial protease hydrolysis, comprising the following process steps:

a. Choose Donghai black ginseng rich in collagen as raw material, soak in water to remove salt, wash and immerse in the soaking solution for 50 minutes, remove and drain the water, use a beater for beating, add 35 times the volume of pure water and use The colloid mill is ground and refined to obtain a slurry; the soaking solution is composed of the following raw materials in parts by weight: 20 parts of hawthorn, 1 part of wild chrysanthemum, 30 parts of patchouli, 15 parts of chitosan, 12 parts of sodium alginate and 200 parts of water, the mass ratio of Donghai Wushen soaking liquid is 1:10;

b. Heat the slurry obtained in step a until the central temperature reaches 130°C, keep it for 10 minutes, cool to 20°C, add protease for enzymolysis, turn on the computer microwave-ultrasonic combined catalytic synthesizer to perform ultrasonic-microwave assistance on the enzymatic hydrolyzate Enzymolysis, in which the insertion depth of the ultrasonic probe is fixed, the microwave power is 500W, the ultrasonic power is 100W, and the ultrasonic-microwave treatment temperature is 41°C. After 15 minutes of treatment, the enzymatic hydrolysis is continued to inactivate the enzyme; through a 120-mesh filter, the obtained collagen filtrate;

c. In the collagen filtrate obtained in step b, add glucose at 0.5% by weight, adjust the pH to 6, and obtain the initial fermentation liquid;

d. Put the initial fermented liquid obtained in step c into an empty sterilization fermenter for actual sterilization. The conditions for empty sterilization and actual sterilization are both 121°C and 20min. After the temperature of the tank drops to 37°C, insert the fermented seeds. The inoculum size is 2% of the volume percentage of the initial fermentation broth, the inoculum mixture was sealed and fermented for 2 days under the conditions of 37°C and 100rpm stirring speed, the fermentation broth was filtered to remove the residue, and the fermentation filtrate was obtained; the fermented seeds were composted Geobacillus bacillus in situ; the fermented seeds were cultivated The raw materials of the base are calculated according to the following parts by weight: 3% soybean peptone, 5% selenium-enriched yeast, 6% asparagine, 5.5% methionine, 0.5% beef powder, 0.5% glucose, 0.1% sodium chloride, 0.01% diphosphate Potassium hydrogen, the rest is distilled water, sterilized at 121°C for 20 minutes, and cooled for later use;

Inoculate Geobacillus into the fermented seed medium for composting in situ, culture on a shaking table at 180r/min for 48h, and the Bacillus in the fermentation broth > 10 ⁸ cfu/mL;

e. Add 15% trehalose, 0.3% maltodextrin and 3% glycerol to the fermentation filtrate obtained in step d, pre-freeze at -50°C for 2 hours, and then freeze at -70°C cold trap temperature and <150Pa vacuum After drying for 12 hours, sea cucumber collagen peptides are obtained directly.

Example 2

A method for preparing black cucumber collagen oligopeptides through microwave/ultrasonic assistance and microbial protease hydrolysis, comprising the following process steps:

a. Choose Donghai black ginseng rich in collagen as raw material, soak in water to remove salt, wash and immerse in the soaking solution for 55 minutes, remove and drain the water, use a beater for beating, add 38 times the volume of pure water and use The colloid mill is ground and refined to obtain a slurry; the soaking liquid is composed of the following raw materials in parts by weight: 22 parts of hawthorn, 3 parts of wild chrysanthemum, 36 parts of patchouli, 17 parts of chitosan, 13 parts of sodium alginate and 250 parts of water, the mass ratio of Donghai Wushen soaking liquid is 1:10;

b. Heat the slurry obtained in step a until the central temperature reaches 135°C, keep it for 10 minutes, cool to 23°C, add protease for enzymolysis, turn on the computer microwave-ultrasonic combined catalytic synthesizer to perform ultrasonic-microwave assistance on the enzymatic hydrolyzate Enzymolysis, in which the insertion depth of the ultrasonic probe is fixed, the microwave power is 500W, the ultrasonic power is 100W, and the ultrasonic-microwave treatment temperature is 41°C. After 15 minutes of treatment, the enzymatic hydrolysis is continued to inactivate the enzyme; through a 120-mesh filter, the obtained collagen filtrate;

c. In the collagen filtrate obtained in step b, add sucrose by 2% by weight, adjust the pH to 6.5, and obtain the initial fermentation liquid;

d. Put the initial fermented liquid obtained in step c into an empty sterilization fermenter for actual sterilization. The conditions for empty sterilization and actual sterilization are both 121°C and 20min. After the temperature of the tank drops to 37°C, insert the fermented seeds. The inoculum size is 3.2% of the volume percentage of the initial fermentation broth, the inoculated mixed solution was sealed and fermented for 3 days under the conditions of 37°C and 200rpm stirring speed, the fermentation broth was filtered to remove the residue, and the fermentation filtrate was obtained; the fermented seed was Bacillus licheniformis; the raw material of the fermented seed culture medium According to the following parts by weight: 3% soybean peptone, 5% selenium-enriched yeast, 6% asparagine, 5.5% methionine, 0.5% beef powder, 0.5% glucose, 0.1% sodium chloride, 0.01% potassium dihydrogen phosphate, The rest is distilled water, sterilized at 121°C for 20 minutes, and cooled for later use;

Bacillus licheniformis was cultured in the fermented seed medium on a shaking table at 180r/min for 48 hours, and the Bacillus in the fermentation broth was >10 ⁸ cfu/mL;

e. The fermentation filtrate obtained in step d uses a tubular membrane with a pore size of 0.1-0.5 μm to remove macromolecules, and then adopts an ultrafiltration membrane with a molecular weight cut-off of 3kDa to cut off and grade, and the retentate is concentrated and desalted by a nanofiltration membrane, and then heated at 175°C The sea cucumber collagen peptide is obtained by spraying and drying under the conditions of an inlet air temperature, an outlet air temperature of 75° C. and a spray pressure of 0.35 MPa.

Example 3

A method for preparing black cucumber collagen oligopeptides through microwave/ultrasonic assistance and microbial protease hydrolysis, comprising the following process steps:

a. Choose Donghai black ginseng rich in collagen as raw material, soak in water to remove salt, wash and immerse in the soaking solution for 60 minutes, remove and drain the water, use a beater for beating, add 40 times the volume of pure water and use The colloid mill is ground and refined to obtain a slurry; the soaking solution is composed of the following raw materials in parts by weight: 25 parts of hawthorn, 5 parts of wild chrysanthemum, 40 parts of patchouli, 18 parts of chitosan, 15 parts of sodium alginate and 350 parts of water, the mass ratio of Donghai Wushen soaking liquid is 1:10;

b. Heat the slurry obtained in step a until the central temperature reaches 145°C, keep it for 10 minutes, cool to 25°C, add protease for enzymolysis, turn on the computer microwave-ultrasonic combined catalytic synthesizer to perform ultrasonic-microwave assistance on the enzymatic hydrolyzate Enzymolysis, in which the insertion depth of the ultrasonic probe is fixed, the microwave power is 500W, the ultrasonic power is 100W, and the ultrasonic-microwave treatment temperature is 41°C. After 15 minutes of treatment, the enzymatic hydrolysis is continued to inactivate the enzyme; through a 120-mesh filter, the obtained collagen filtrate;

c. In the collagen filtrate obtained in step b, add glucose at 3% by weight, adjust the pH to 7, and obtain the initial fermentation liquid;

d. Put the initial fermented liquid obtained in step c into an empty sterilization fermenter for actual sterilization. The conditions for empty sterilization and actual sterilization are both 121°C and 20min. After the temperature of the tank drops to 37°C, insert the fermented seeds. The inoculum size is 4.5% of the volume percentage of the initial fermentation broth, the inoculation mixture was sealed and fermented for 5 days under the conditions of 37°C and 300rpm stirring speed, the fermentation broth was filtered to remove the residue, and the fermentation filtrate was obtained; the fermented seeds were Bacillus megaterium; the raw material of the fermented seed culture medium According to the following parts by weight: 3% soybean peptone, 5% selenium-enriched yeast, 6% asparagine, 5.5% methionine, 0.5% beef powder, 0.5% glucose, 0.1% sodium chloride, 0.01% potassium dihydrogen phosphate, The rest is distilled water, sterilized at 121°C for 20 minutes, and cooled for later use;

Inoculate Bacillus megaterium in the fermented seed culture medium, shake the table at 180r/min, culture for 48h, and the Bacillus in the fermentation liquid>10 ⁸ cfu/mL;

e. Add 20% trehalose, 0.8% maltodextrin and 5% glycerin to the fermentation filtrate obtained in step d, pre-freeze at -50°C for 6 hours, and then freeze at -70°C cold trap temperature and <150Pa vacuum After drying for 20 hours, sea cucumber collagen peptides can be obtained directly.

Raw materials used in the present invention, equipment, if not specified, are commonly used raw materials, equipment in this area; Method used in the present invention, if not specified, are conventional methods in this area.

The above are only preferred embodiments of the present invention, and do not limit the present invention in any way. All simple modifications, changes and equivalent transformations made to the above embodiments according to the technical essence of the present invention still belong to the technical solution of the present invention. scope of protection.

Claims (5)

1. a kind of method that microwave/ultrasonic assistant, microprotein enzyme hydrolysis prepare crow ginseng collagen oligopeptide, its feature It is to comprise the technical steps that：

A. it is raw material to choose the black sea cucumbers from East China Sea rich in collagen, and clear water immersion removing salinity immerses soak 50- after cleaning 60min, is pulled out and drains away the water, and mashing treatment is carried out using beater, and colloid mill is used after adding the 35-40 times of pure water of volume Micronization processes are ground, slurry is obtained；

B. the slurry that step a is obtained is heated to central temperature and reaches 130-145 DEG C, keep 10min, be cooled to 20-25 DEG C, Protease is added to be digested, it is auxiliary that opening computer microwave-ultrasonic wave combined catalytic synthesizer carries out ultrasonic-microwave to zymolyte The probe insertion depth of enzymolysis, wherein ultrasonic wave is helped to immobilize, at microwave power 500W, ultrasonic power 100W, ultrasound-microwave After reason temperature under the conditions of 41 DEG C to process 15 minutes, continue to digest, go out enzyme；120 mesh filter screens are crossed, collagen filtrate is obtained；

C. in the collagen filtrate that step b is obtained, weight percent 0.5-3% adds glucose or sucrose, adjusts pH To 6-7, Preliminary fermentation liquid is obtained；

D. the Preliminary fermentation liquid for step c being obtained be put into sky disappear fermentation tank carry out it is real disappear, sky disappears and the real condition for disappearing is 121 DEG C, 20min, treat tank temperature drop to 37 DEG C, access fermentation seed, inoculum concentration is the 2-4.5% of Preliminary fermentation liquid percentage by volume, is connect Mixed liquor sealing and fermenting 2-5 days under conditions of 37 DEG C and 100-300rpm speeds of agitator are planted, zymotic fluid filters off residue, must ferment Filtrate；

E. 15-20% trehaloses, 0.3-0.8% maltodextrins and 3-5% glycerine are added in the ferment filtrate that step d is obtained ,- 50 DEG C of pre-freeze 2-6h, then the freeze-drying 12-20h under conditions of -70 DEG C of condenser temperatures and ＜ 150Pa vacuums, directly obtains To holothurian collagen peptide；Or, the ferment filtrate that step d is obtained uses aperture to divide for 0.1-0.5 μm of tubular membrane removing is big Son, then uses molecular cut off to retain classification for the milipore filter of 3kDa, and trapped fluid uses NF membrane concentrating and desalinating, Ran Hou It is spray-dried under conditions of 175-185 DEG C of EAT, 75-85 DEG C of leaving air temp and 0.35-0.45MPa atomisation pressures, is obtained Holothurian collagen peptide.

2. microwave/ultrasonic assistant according to claim 1, microprotein enzyme hydrolysis preparation crow ginseng collagen are oligomeric The method of peptide, it is characterised in that soak is made up of the raw material of following weight portion in step a：Hawthorn 20-25 parts, chrysanthemum indicum 1-5 Part, Pogostemon cablin 30-40 parts, shitosan 15-18 parts, sodium alginate 12-15 parts and water 200-350 parts, black sea cucumbers from East China Sea and soak Mass ratio be 1:10.

3. microwave/ultrasonic assistant according to claim 1, microprotein enzyme hydrolysis preparation crow ginseng collagen are oligomeric The method of peptide, it is characterised in that fermentation seed is compost ground bacillus, bacillus licheniformis or huge bud on the spot in step d Spore bacillus.

4. microwave/ultrasonic assistant according to claim 3, microprotein enzyme hydrolysis preparation crow ginseng collagen are oligomeric The method of peptide, it is characterised in that the raw material of fermentation seed culture medium is based on following parts by weight：3% soy peptone, 5% richness Selenium yeast, 6% asparagine, 5.5% methionine, 0.5% powdered beef, 0.5% glucose, 0.1% sodium chloride, 0.01% phosphoric acid Potassium dihydrogen, remaining is distilled water, 121 DEG C of high-temperature sterilization 20min, standby after cooling.

5. microwave/ultrasonic assistant according to claim 4, microprotein enzyme hydrolysis preparation crow ginseng collagen are oligomeric The method of peptide, it is characterised in that compost ground bacillus, bacillus licheniformis or bacillus megaterium will be inoculated in fermentation on the spot In seed culture medium, shaking table 180r/min cultivates 48h, bacillus ＞ 10 in zymotic fluid⁸cfu/mL。