Microbial agent for increasing sea cucumber peptide content and application of microbial agent in sea cucumber peptide craft beer

Abstract

The invention discloses a microbial agent for increasing the content of sea cucumber peptides and application of the microbial agent in sea cucumber peptide craft beer, and relates to the technical field of food processing. The microbial agent which can take the sea cucumber protein as a sole nitrogen source is firstly provided, the microbial agent can efficiently degrade the sea cucumber protein, and in the prepared sea cucumber peptide, the content of soluble protein is greatly increased; the contents of water-soluble proteins obtained by adopting the leuconostoc mesenteroides subsp. Mesenteroides bacterial suspension and the lactobacillus plantarum bacterial suspension can reach 55g / L and 48g / L respectively; the content of protein in the prepared beer is also greatly improved, and the content of protein in the beer prepared from the complex microbial inoculant F-05 is 2.13 (g / 100g).

Description

Technical Field

[0001] The present invention relates to a microbial agent for increasing the content of sea cucumber peptides and its application in sea cucumber peptide craft beer, and relates to the technical field of food processing. Background Art

[0002] Sea cucumbers are rich in protein, low in fat and cholesterol, and are rich in minerals and vitamins. They also contain nearly 20 kinds of amino acids such as glycine, glutamic acid, and aspartic acid, and most of them are essential amino acids for the human body. Collagen accounts for about 70% of the total protein in the sea cucumber body wall. Collagen constitutes the skeleton of the sea cucumber body wall and wraps other nutrients. The effective utilization rate of sea cucumber protein is low and it is difficult to be utilized. Enzymatic hydrolysis of sea cucumbers can improve the biological utilization rate of sea cucumbers. When sea cucumber protein is degraded into sea cucumber polypeptides, it has better solubility and stability, is more easily digested and absorbed, and has good antioxidant activity;

[0003] Sea cucumber protein is difficult to digest, and the salt content of the processed sea cucumber body wall is high. The salt tolerance of current commercial strains and enzyme preparations is not high. If the sea cucumber protein is directly degraded, the activity of biodegradation will be inhibited due to the high salt concentration. Since fresh sea cucumbers are difficult to store, about 90% of sea cucumbers are salted to extend their storage period. However, the salt tolerance of current general commercial enzymes and microorganisms is low, so the sea cucumbers need to be desalted before degradation. Relevant research has found that the desalting process of sea cucumbers will cause losses of protein, polysaccharides and characteristic flavor substances, and will also damage their tissue morphology; and while reducing the molecular weight of peptide segments by enzymatic hydrolysis, a large amount of ketones, aldehydes, alkanes and other fishy and bitter substances are also produced. Coupled with the fishy smell of sea cucumbers themselves, it seriously affects their flavor and taste. In the prior art, salt-tolerant strains are screened for the degradation of sea cucumber protein. However, most of them are non-edible strains, posing potential food safety hazards; therefore, a microbial preparation that can improve the soluble protein content and is safe without the enzymatic hydrolysis conditions of enzyme preparations is needed.

[0004] Beer is a low-alcohol beverage containing carbon dioxide brewed by the fermentation of yeast using grains, water, and hops as the main raw materials. The grains are heated and soaked in water to convert the starch in the grains into sugar; after completing the previous process, yeast is added for fermentation after cooling, and the sugar is converted into alcohol. Each process can endow the beer with different flavors; the grains have a sweet taste, the fragrance of grass, and the burnt fragrance after baking; hops not only have a bitter taste, but also have a floral fragrance and a citrus fragrance. Yeast in some beers can provide an earthy smell and the taste of toasted bread; and alcohol can give a stimulating feeling to the mouth.

[0005] In recent years, with the in-depth understanding of the concept of healthy living among people, the scientific consumption of sea cucumber peptides, a nutritious and healthy food, has received more and more attention. How to integrate sea cucumber peptides and beer has not appeared before. Summary of the Invention

[0006] The present invention provides a microbial inoculum that can increase the content of sea cucumber polypeptide. The microbial inoculum is prepared according to the following steps:

[0007] (1) Prepare a solid sea cucumber culture medium: Add agar and glucose to the sea cucumber cell wall lysate. The sea cucumber cell wall lysate is prepared as follows: Take fresh sea cucumbers, wash them, remove the internal organs, add water to the sea cucumbers and boil them, break the cells with a cell disruptor, and after sieving, obtain the sea cucumber cell wall lysate;

[0008] (2) Inoculate Leuconostoc mesenteroides subsp. mesenteroides and Lactiplantibacillus plantarum into the solid sea cucumber culture medium respectively. After culturing, obtain single colonies. Inoculate the obtained single colonies into a new solid sea cucumber culture medium, repeat the above steps, and after culturing for 30 to 60 generations, obtain the single colonies of Leuconostoc mesenteroides subsp. mesenteroides and Lactiplantibacillus plantarum after subculture;

[0009] (3) Inoculate the single colonies obtained in step (2) after subculture into a liquid sea cucumber culture medium for culturing to obtain a fermentation broth; Centrifuge the prepared fermentation broth and collect the bacterial cells;

[0010] (4) Add sterile water to the bacterial cells obtained in step (3) to prepare a bacterial suspension, and then obtain the microbial inoculum.

[0011] In one embodiment of the present invention, Leuconostoc mesenteroides subsp. mesenteroides and Lactiplantibacillus plantarum are purchased from CICC, and the numbers are CICC 22184 and CICC 21810 respectively.

[0012] In one embodiment of the present invention, step (1) is to add 1.5 - 2% (w / w) agar and 5 - 8% (w / w) glucose to the sea cucumber cell wall lysate, add water to the sea cucumbers and steam them. The addition amount of water is added according to a weight ratio of 1:5 - 6. After boiling for 30 - 50 min, cool and let stand for 1 - 1.5 h, then pour the whole system into a cell disruptor, break the cells with the cell disruptor, and after sieving through a 200 - 300 mesh sieve, obtain the sea cucumber cell wall lysate;

[0013] In an embodiment of the present invention, step (2) is as follows: the cultivation method is to inoculate Leuconostoc mesenteroides subsp. mesenteroides and / or Lactobacillus plantarum into a solid sea cucumber medium respectively, and cultivate in a constant temperature incubator at 35-37 °C for 10-12 h. Select single colonies with a strain diameter greater than 1.5 mm and inoculate them into a new solid sea cucumber medium. Repeat the above steps. After culturing for 30-60 generations, single colonies of Leuconostoc mesenteroides subsp. mesenteroides and Lactobacillus plantarum after subculture are obtained respectively.

[0014] In an embodiment of the present invention, in step (3), the cultivation is carried out at 35-37 °C under the condition of a rotation speed of 150-200 r / min for 16-18 h to prepare a seed liquid; the prepared seed liquid is cultured at 35-37 °C under the condition of a rotation speed of 150-200 r / min for 20-22 h with an inoculation amount of 5-10%, and the pH is controlled at 6.5-7.0; a fermentation broth is obtained; after centrifuging the prepared fermentation broth, the thalli are collected.

[0015] In an embodiment of the present invention, in step (4), the microbial inoculum is a Leuconostoc mesenteroides subsp. mesenteroides bacterial suspension, a Lactobacillus plantarum bacterial suspension, or a mixture prepared by mixing the prepared Leuconostoc mesenteroides subsp. mesenteroides bacterial suspension and Lactobacillus plantarum bacterial suspension according to a volume ratio of 1-5:1-5.

[0016] In an embodiment of the present invention, in the bacterial suspension, the total bacterial concentration is: 1-5×10 6 CFU / ml; preferably, the bacterial concentration is 1×10 6 CFU / ml.

[0017] The present invention also provides a preparation method of sea cucumber peptides, and the method includes the following steps:

[0018] (1) Add water to the sea cucumber and cook it. The addition amount of water is added according to a weight ratio of 1:5-6. After cooking for 30-50 min, cool and let it stand for 1-1.5 h, then pour the whole system into a wall breaker, break the wall with the wall breaker, and pass through a 200-300 mesh sieve to obtain a sea cucumber wall-breaking liquid.

[0019] (2) Inoculate the above microbial inoculum into the sea cucumber wall-breaking liquid obtained in step (1) according to a ratio of 3-5% (v / v) respectively, and cultivate at 35-37 °C under the condition of a rotation speed of 150-200 r / min for at least 20 h, and control the pH at 6.5-7.0; sea cucumber peptides are prepared.

[0020] In an embodiment of the present invention, the microbial inoculum is a mixture prepared by mixing the prepared Leuconostoc mesenteroides subsp. mesenteroides bacterial suspension and Lactobacillus plantarum bacterial suspension according to a volume ratio of 1-5:1-5.

[0021] In one embodiment of the present invention, the microbial inoculum is prepared by mixing the prepared Leuconostoc mesenteroides subsp. mesenteroides bacterial suspension and Lactobacillus plantarum bacterial suspension in a volume ratio of 1:1 or 2:1.

[0022] The present invention also provides a sea cucumber peptide, which is prepared by the following method:

[0023] (1) Add 1.5 - 2% (w / w) agar and 5 - 8% (w / w) glucose to the sea cucumber broken wall liquid, add water to the sea cucumber and cook it. The addition amount of water is added according to a weight ratio of 1:5 - 6. After cooking for 30 - 50 min, cool and let stand for 1 - 1.5 h, then pour the whole system into a wall breaker, break the wall with the wall breaker, and pass through a 200 - 300 mesh sieve to obtain the sea cucumber broken wall liquid;

[0024] (2) Inoculate the above microbial inoculum into the sea cucumber broken wall liquid obtained in step (1) according to a ratio of 3 - 5% (v / v), and culture at 35 - 37 °C and a rotation speed of 150 - 200 r / min for at least 20 h, and control the pH at 6.5 - 7.0; thus obtaining the sea cucumber peptide.

[0025] In one embodiment of the present invention, the microbial inoculum is prepared by mixing the prepared Leuconostoc mesenteroides subsp. mesenteroides bacterial suspension and Lactobacillus plantarum bacterial suspension in a volume ratio of 1 - 5:1 - 5;

[0026] In one embodiment of the present invention, the microbial inoculum is prepared by mixing the prepared Leuconostoc mesenteroides subsp. mesenteroides bacterial suspension and Lactobacillus plantarum bacterial suspension in a volume ratio of 1:1 or 2:1.

[0027] The present invention also provides the application of the above method, or the sea cucumber peptide prepared by the above method, or the above sea cucumber peptide in the preparation of craft beer.

[0028] The present invention also provides a method for preparing a sea cucumber peptide craft beer, and the method includes the following steps:

[0029] (1) Add oatmeal with a mass fraction of 0.5 - 1.0%, 20 - 40 mL of α - high - temperature amylase, and water to a mashing pot, and raise the temperature step - by - step at a rate of 1 - 5 °C / min to 98 °C to obtain mash;

[0030] (2) Add 10 - 15 parts of Canadian barley malt, 40 - 45 parts of Australian barley malt, and 35 - 40 parts of wheat malt according to the mass fraction, add them to a grinder for grinding to obtain a ground mixture;

[0031] Add water to the saccharification pot. When the temperature rises to 40 - 45 °C, add the above-mentioned crushed and mixed materials. At the same time, add citric acid with a mass fraction of 0.08 - 0.1%, lactic acid with a mass fraction of 0.007 - 0.01%, calcium sulfate with a mass fraction of 0.09 - 0.1%, xylanase with an activity of 0.15 - 0.2 IU / g, and β-glucan complex enzyme with an activity of 1.5 - 2.0 IU / g; keep warm for 10 - 15 min, then raise the temperature to 50 - 55 °C. After keeping warm for 15 - 20 minutes, transfer the mash from the gelatinization pot to the saccharification pot, stir, and raise the temperature to 68 - 72 °C, keep warm for 25 - 30 minutes; after passing the iodine test, raise the temperature to 70 - 80 °C and then end the saccharification; filter the saccharified liquid after saccharification is completed and collect the original juice; after washing the grains, combine the collected wort. After washing the grains, the residual sugar in the spent grains is above 1.5 °P; combine the original juice and the wort after washing the grains;

[0032] (3) Add calcium sulfate and calcium chloride to the wort collected in step (2), stir evenly. After heating the clarified wort to boiling, add hops in three portions. After boiling for 10 - 15 min, add 0.1% - 0.25% of SAAZ hops based on the mass of the wort. After boiling for 20 - 25 min, add 0.5% - 0.75% of hops based on the mass of the wort again. Boil for 20 - 25 min, and finally add 0.1% - 0.3% of SAAZ hops based on the mass of the wort, continue to boil for 5 - 10 min, and then cool down;

[0033] (4) After precipitation and filtration, aerate the wort, with the aeration amount being 9 - 10 mg / L, and the addition amount of brewer's yeast at full tank being 10 - 11 million / mL;

[0034] (5) Primary fermentation: Transfer the wort added with brewer's yeast to the fermentation tank, seal the tank for fermentation (control the temperature at 18 - 20 °C). When the diacetyl content is 0.09 mg / L and the fermentation degree is 63 - 65%, the primary fermentation ends;

[0035] (6) After the primary fermentation ends, add the sea cucumber peptide prepared by the above method or the said sea cucumber peptide to the fermentation tank, with the addition amount being 1 - 2%; after addition, cool down and store cold. After cold storage for 45 - 60 days, centrifuge using a centrifuge, and the number of yeast cells in the filtered wine is 8 - 10 million / mL;

[0036] After cold storage for 45 - 60 days, centrifuge using a centrifuge and then package the finished product.

[0037] In an embodiment of the present invention, the hops are SAAZ hops; the enzyme activity of the α-thermostable amylase is 20,000 U / ml.

[0038] The present invention also provides a sea cucumber peptide craft beer prepared by the above preparation method.

[0039] The present invention also provides a sea cucumber peptide, which is: WEPPPTGPGSP peptide, GPIDGLP peptide or RPDIPGPR peptide.

[0040] The present invention also provides the application of WEPPPTGPGSP peptide, GPIDGLP peptide or RPDIPGPR peptide in preparing a tyrosinase inhibitor or in preparing a whitening cosmetic.

[0041] The present invention also provides the application of a WEPPPTGPGSP peptide, GPIDGLP peptide or RPDIPGPR peptide in inhibiting the activity of tyrosinase.

[0042] Beneficial effects

[0043] (1) The present invention firstly provides a microbial inoculant capable of using sea cucumber protein as the sole nitrogen source. The microbial inoculant can efficiently degrade sea cucumber protein. In the prepared sea cucumber peptide, the content of soluble protein is greatly increased. The water-soluble protein contents obtained by using the Leuconostoc mesenteroides subsp. mesenteroides bacterial suspension and Lactobacillus plantarum bacterial suspension can reach up to 55 g / L and 48 g / L respectively.

[0044] (2) By using the method of the present invention, a compound inoculant F-05 is obtained by mixing the Leuconostoc mesenteroides subsp. mesenteroides bacterial suspension and Lactobacillus plantarum bacterial suspension in a volume ratio of 2:1. The water-soluble protein content in the prepared sea cucumber peptide is the highest, achieving a synergistic effect, and the water-soluble protein content can reach up to 110 g / L respectively.

[0045] (3) By using the method of the present invention, the protein content in the prepared beer is also greatly increased. Among them, a compound inoculant F-05 is obtained by mixing the Leuconostoc mesenteroides subsp. mesenteroides bacterial suspension and Lactobacillus plantarum bacterial suspension in a volume ratio of 2:1, and the protein content in the prepared beer is 2.13 (g / 100 g).

[0046] (4) The process is scientific, reasonable, safe and reliable, and simple to manufacture. It is suitable for both home-made and large-scale industrial production. Specific embodiments

[0047] In the following examples, Leuconostoc mesenteroides subsp. mesenteroides, Lactiplantibacillus plantarum, Bacillus subtilis, and Bacillus amyloliquefaciens were purchased from CICC, and their numbers were: CICC 22184, CICC 21810, CICC 23093, and CICC 24519, respectively.

[0048] The culture media involved in the following examples are as follows:

[0049] MRS liquid medium (1 L): Tryptone (trypsin digested) 10.0 g, Beef extract powder 10.0 g, Yeast extract powder 5.0 g, Glucose 20.0 g, Tween 80 1.0 g, Sodium acetate 5.0 g, Ammonium citrate tribasic 2.0 g, K2HPO4 2.0 g, MgSO4·7H2O

[0050] 0.2 g, MnSO4·H2O 0.2 g, pH 6.2 - 6.5. The above components were sterilized at 121°C for 15 min.

[0051] MRS solid medium: Add 15 g of agar to the MRS liquid medium.

[0052] Nutrient broth medium (1 L): Peptone 5.0 g, Beef extract powder 3.0 g, NaCl 5.0 g, 5 mg MnSO4·H2O, pH 7.0; The above components were sterilized at 121°C for 15 min.

[0053] Nutrient broth solid medium: Add 15 g of agar to the nutrient broth medium.

[0054] The detection methods involved in the following examples

[0055] Determination of water-soluble protein content: Use the Lowry protein concentration assay kit to determine the water-soluble protein content as required

[0056] Protein content of beer

[0057] Determined by the Kjeldahl method GB5009 2010.

[0058] Example 1: Preparation of microbial inoculant

[0059] 1. Preparation of single-strain inoculant

[0060] The specific method is as follows:

[0061] (1) Take the fresh sea cucumbers purchased from the market, wash them, remove the internal organs, add water to the sea cucumbers (add water according to the weight ratio of 1:6), boil for 40 min, then cool and let stand for 1 h. Pour the whole system (sea cucumbers and water) into a blender, break the cells with the blender, and pass through a 200-mesh sieve to obtain the broken sea cucumber liquid.

[0062] Prepare the solid sea cucumber medium: Add 1.5 - 2% (w / w) agar and glucose (the addition amount is 5% of the total mass of the broken sea cucumber liquid) to the broken sea cucumber liquid.

[0063] Liquid sea cucumber medium: Add glucose (the addition amount is 5% of the total mass of the broken sea cucumber liquid) to the broken sea cucumber liquid.

[0064] (2) Inoculate Leuconostoc mesenteroides subsp. mesenteroides, Lactiplantibacillus plantarum, Bacillus subtilis, and Bacillus amyloliquefaciens into the solid sea cucumber medium for seed pre-culture respectively. Culture in a 37°C constant temperature incubator for 12 h. Select single colonies with a strain diameter greater than 1.5 mm and inoculate them into a new solid sea cucumber medium. Repeat the above steps. After culturing for 50 generations, obtain the single colonies of Leuconostoc mesenteroides subsp. mesenteroides, Lactiplantibacillus plantarum, Bacillus subtilis, and Bacillus amyloliquefaciens after subculture respectively.

[0065] (3) Inoculate the single colonies of each strain obtained in step (2), namely: the single colonies of Leuconostoc mesenteroides subsp. mesenteroides, Lactiplantibacillus plantarum, Bacillus subtilis, and Bacillus amyloliquefaciens, into the liquid sea cucumber medium respectively. Culture at 37°C and a rotation speed of 150 - 200 r / min for 16 h to prepare the seed liquid.

[0066] (4) Inoculate the activated seed liquid into the liquid sea cucumber medium respectively, with an inoculation amount of 5%. Culture at 37°C and a rotation speed of 150 - 200 r / min for 20 h, and control the pH at 6.5 - 7.0 to obtain the fermentation broth. Centrifuge the prepared fermentation broth at a rotation speed of 4000 r / min for 30 min to obtain the bacterial cells, and collect the bacterial cells.

[0067] (5) Prepare the bacterial suspension: Mix the bacterial cells with sterile water respectively to prepare the bacterial suspensions of Leuconostoc mesenteroides subsp. mesenteroides, Lactiplantibacillus plantarum, Bacillus subtilis, and Bacillus amyloliquefaciens respectively. Adjust the concentration of the bacterial suspensions to: 1×10 6 CFU / ml.

[0068] 2. Preparation of compound microbial agents

[0069] The cell suspensions of Leuconostoc mesenteroides subsp. mesenteroides and Lactobacillus plantarum were mixed at a volume ratio of 1:1 to obtain the compound microbial agent F-01. In the compound microbial agent, the cell concentration of the cell suspension was: 1×10 6 CFU / ml.

[0070] The cell suspensions of Leuconostoc mesenteroides subsp. mesenteroides and Lactobacillus plantarum were mixed at a volume ratio of 1:2 to obtain the compound microbial agent F-02. In the compound microbial agent, the cell concentration of the cell suspension was: 1×10 6 CFU / ml.

[0071] The cell suspensions of Leuconostoc mesenteroides subsp. mesenteroides and Lactobacillus plantarum were mixed at a volume ratio of 1:3 to obtain the compound microbial agent F-03. In the compound microbial agent, the cell concentration of the cell suspension was: 1×10 6 CFU / ml.

[0072] The cell suspensions of Leuconostoc mesenteroides subsp. mesenteroides and Lactobacillus plantarum were mixed at a volume ratio of 1:4 to obtain the compound microbial agent F-04. In the compound microbial agent, the cell concentration of the cell suspension was: 1×10 6 CFU / ml.

[0073] The cell suspensions of Leuconostoc mesenteroides subsp. mesenteroides and Lactobacillus plantarum were mixed at a volume ratio of 2:1 to obtain the compound microbial agent F-05. In the compound microbial agent, the cell concentration of the cell suspension was: 1×10 6 CFU / ml.

[0074] The cell suspensions of Leuconostoc mesenteroides subsp. mesenteroides and Lactobacillus plantarum were mixed at a volume ratio of 3:1 to obtain the compound microbial agent F-06. In the compound microbial agent, the cell concentration of the cell suspension was: 1×10 6 CFU / ml.

[0075] The cell suspensions of Leuconostoc mesenteroides subsp. mesenteroides and Lactobacillus plantarum were mixed at a volume ratio of 4:1 to obtain the compound microbial agent F-07. In the compound microbial agent, the cell concentration of the cell suspension was: 1×10 6 CFU / ml.

[0076] 3. Preparation of control microbial agents

[0077] According to the method in steps (3) to (5) of step 1, prepare the bacterial suspension, with the difference that steps (1) to (2) are omitted. At the same time, adjust the culture media for Leuconostoc mesenteroides subsp. mesenteroides and Lactiplantibacillus plantarum to: MRS culture medium, and adjust the culture media for Bacillus subtilis and Bacillus amyloliquefaciens to: nutrient broth culture medium.

[0078] Respectively prepare Leuconostoc mesenteroides subsp. mesenteroides bacterial suspension - W1, Lactiplantibacillus plantarum bacterial suspension - W1, Bacillus subtilis bacterial suspension - W1, and Bacillus amyloliquefaciens bacterial suspension - W1; adjust the concentration of the bacterial suspensions to: 1×10 6 CFU / ml.

[0079] Example 2: Preparation of sea cucumber peptides

[0080] 1. Preparation of sea cucumber cell wall - broken liquid

[0081] Take the fresh sea cucumbers purchased from the market, wash them and remove the internal organs. Add water to the sea cucumbers (add water according to a weight ratio of 1:6), boil for 40 minutes, then cool and let stand for 1 hour. Pour the whole system (sea cucumbers and water) into a wall - breaker, break the cells with the wall - breaker, and then pass through a 200 - mesh sieve to obtain the sea cucumber cell wall - broken liquid;

[0082] 2. Preparation of sea cucumber peptides

[0083] Respectively inoculate the bacterial suspensions prepared in Example 1 into the sea cucumber cell wall - broken liquid at a ratio of 3% (v / v), culture at 37°C under the condition of a rotation speed of 150 - 200 r / min for 20 h, and control the pH at 6.5 - 7.0; after 20 h of treatment, detect the content of water - soluble protein in the fermentation broth.

[0084] The results of single - strain agents are shown in Table 1:

[0085] Table 1: Effects of different bacterial suspensions on sea cucumber peptides

[0086] Bacterial suspension Water-soluble protein content (g / L) Blank group (without adding bacterial suspension) 3.0 Leuconostoc mesenteroides subsp. mesenteroides bacterial suspension 55 Leuconostoc mesenteroides subsp. mesenteroides bacterial suspension - W1 37 Lactiplantibacillus plantarum bacterial suspension 48 Lactiplantibacillus plantarum bacterial suspension - W1 32 Bacillus subtilis bacterial suspension 39 Bacillus subtilis bacterial suspension - W1 25 Bacillus amyloliquefaciens bacterial suspension 36 Bacillus amyloliquefaciens bacterial suspension - W1 27

[0087] The results of compound bacterial agents are shown in Table 2

[0088] Table 2: Effects of different compound bacterial agents on sea cucumber peptides

[0089]

[0090]

[0091] In summary, mix the Leuconostoc mesenteroides subsp. mesenteroides bacterial suspension and the Lactiplantibacillus plantarum bacterial suspension in a volume ratio of 2:1 to obtain the compound bacterial agent F - 05; in the compound bacterial agent, the bacterial concentration of the bacterial suspension is: 1×10 6CFU / ml; The water-soluble protein content in sea cucumber peptides prepared with the compound bacterial agent F-05 is the highest.

[0092] 3. Perform RP-HPLC reverse-phase high-performance liquid chromatography separation on the sea cucumber peptides prepared with the compound bacterial agent F-05, and determine the components of sea cucumber protein peptides by LC-MS / MS; Through computer simulation and in vitro determination experiments, a total of 3 sea cucumber protein peptides with tyrosinase activity were screened: WEPPPTGPGSP, GPIDGLP, RPDIPGPR.

[0093] Perform detection on the tyrosinase inhibition rate of the prepared sea cucumber protein peptides: WEPPPTGPGSP, GPIDGLP, RPDIPGPR. The method is as follows:

[0094] Take four centrifuge tubes A, B, C, and D, and add 0.25 mL of L-tyrosine and 0.25 mL of PBS to each. Add 0.25 mL of the sample solution to tubes B and D. Add 0.25 mL of PBS solution to tubes A and C. Vortex and mix the 4 tubes evenly, and incubate at 37 °C for 10 minutes. Then add 0.25 mL of enzyme solution to tubes C and D, and add the same volume of PBS buffer to tubes A and B to make up the test sample volume. Keep the four tubes at 37 °C and continue the incubation reaction for 20 minutes. Take 200 μL of the above test solution and add it to a 96-well plate, and measure the absorbance at 475 nm with an enzyme-labeled instrument.

[0095] Calculation of tyrosinase activity inhibition rate:

[0096] Tyrosinase activity inhibition rate % = ((C - A) - (D - B)) / (C - A) * 100%.

[0097] The results show that the tyrosinase activity inhibition rates of the sea cucumber protein peptides: WEPPPTGPGSP, GPIDGLP, RPDIPGPR are 75%, 79%, and 87% respectively.

[0098] Example 3: Brewing of sea cucumber peptide craft beer

[0099] The specific steps are as follows:

[0100] (1) Gelatinization: Add 0.5% oatmeal, 20 mL of α-thermostable amylase (20,000 U / ml), and water to the gelatinization pot, and raise the temperature step by step (1 °C / min) to 98 °C to obtain mash.

[0101] (2) Weigh the following raw materials by weight percentage: 12% Canadian barley malt, 45% Australian barley malt, 43% wheat malt, add them to a crusher for crushing to obtain a crushed mixture;

[0102] Add water to the saccharification pot. When the temperature rises to 40°C, add the above-mentioned crushed and mixed materials (add citric acid with a mass fraction of 0.08%, lactic acid with a mass fraction of 0.007%, calcium sulfate with a mass fraction of 0.09%. After 5 minutes, continue to add xylanase at 0.15 IU / g and β-glucan complex enzyme at 1.5 IU / g), keep warm for 10 minutes, then raise the temperature to 50°C and keep warm for 20 minutes. At this time, transfer the mash from the mash pot to the saccharification pot, stir, raise the temperature to 68°C, and keep warm for 30 minutes; after passing the iodine test, raise the temperature to 76°C to end the saccharification;

[0103] Pump the saccharified liquid after saccharification to the filter, filter to obtain clear and bright wort; after filtration, let it stand for 3 minutes, then circulate and clarify for 5 - 10 minutes until the turbidity ≤ 30 EBC (no large particles), and collect the original juice;

[0104] Wash the grains: Wash the grains three times. The temperature of the wash water is 72 - 76°C, and the pH = 5.4 - 5.6. After three times of washing the grains, combine the collected wort. The residual sugar in the washed grains is above 1.5°P; combine the original juice and the wort after washing the grains;

[0105] (3) Add calcium sulfate with a mass fraction of 0.09% and calcium chloride with a mass fraction of 0.004% to the wort collected in step (2), stir evenly. After the clarified wort is heated to boiling, add hops in three portions. After boiling for 10 minutes, add SAAZ fragrant hops with a mass fraction of 0.15% of the wort mass. After boiling for 20 minutes, add SAAZ fragrant hops with a mass fraction of 0.5% of the wort mass again. Continue to boil for 20 minutes, and finally add SAAZ fragrant hops with a mass fraction of 0.2% of the wort mass, and continue to boil for 10 minutes, then cool down.

[0106] (4) Precipitation: Cool to 18°C and perform swirling precipitation for 15 minutes, then filter.

[0107] (5) Aerate the filtered wort obtained in step (4), with the aeration amount of 9 - 10 mg / L, and the addition amount of imported ale yeast at the full tank is 10 - 11 million cells / mL;

[0108] (6) Primary fermentation: Transfer the wort added with ale yeast to the fermentation tank, seal the tank for fermentation (control the temperature at 18 - 20°C). When the diacetyl content is 0.09 mg / L and the fermentation degree is 63 - 65%, the primary fermentation ends;

[0109] Secondary fermentation: Add the sea cucumber peptide prepared in Example 2 to the fermentation tank, with the addition amount of 1 mg / kg according to the mass fraction; after addition, cool and store.

[0110] The cold storage temperature is -1.0 ± 0.3°C until filtering the wine. After filling the tank, there is no pressure control, and it naturally rises to 0.08 - 0.09 Mpa and maintains a constant pressure until filtering the wine;

[0111] (7) Filtering the sake

[0112] After cold storage for 45 - 60 days, centrifugation is carried out using a centrifuge. The number of yeast cells in the clear sake after filtering is 8 million - 10 million / mL, and the temperature of the clear sake is -1.0 to -0.5 °C.

[0113] (8) Canning: After the clear sake sampling is qualified, it undergoes filling, capping, sterilization, and then is packed and stored in the warehouse.

[0114] Example 4: Brewing of sea cucumber peptide craft beer

[0115] The specific brewing method is the same as that of Example 3, except that in step (6), after the main fermentation is completed, sea cucumber peptides prepared in Example 2 are not added to the fermentation tank.

[0116] Example 5: Experimental results

[0117] The protein contents in the beers prepared in Example 3 and Example 4 are respectively detected, and the results are shown in Table 3.

[0118] Table 3: Protein contents in beers obtained by adding different sea cucumber peptides

[0119] Added bacterial agent Protein content in beer (g / 100g) Blank (Example 4) 0.53 Compound bacterial agent F-01 (1:1) 1.53 Compound bacterial agent F-05 (2:1) 2.13 Leuconostoc mesenteroides subsp. mesenteroides bacterial suspension 0.92 Lactiplantibacillus plantarum bacterial suspension 0.89

[0120] The detection results show that compared with ordinary beer, the beer of this invention has significantly higher protein content and also higher polypeptide content. It has the characteristics of large profit margin and good economic benefits, and is beneficial for wide promotion.

[0121] Example 6: Brewing of sea cucumber peptide craft beer

[0122] The specific brewing method is the same as that of Example 3, except that in step (6), after the main fermentation is completed, sea cucumber protein peptides prepared in Example 2: WEPPPTGPGSP, GPIDGLP, RPDIPGPR (which can be directly chemically synthesized) are not added to the fermentation tank. According to the mass fraction, the addition amount is 1 mg / kg; after addition, cooling and cold storage are carried out.

[0123] Sea cucumber peptide craft beer is prepared.

[0124] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and decorations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A preparation method of sea cucumber peptide, characterized in that, The method includes the following steps: adding water to sea cucumbers, steaming, cooling and standing still, then using a wall breaker to break the walls to obtain a sea cucumber wall-breaking liquid; adding a microbial inoculant to the prepared sea cucumber wall-breaking liquid and fermenting to prepare sea cucumber peptides. The preparation method of the microbial inoculant is as follows: 1) Prepare a solid sea cucumber culture medium: add agar and glucose to the sea cucumber wall-breaking liquid; 2) Inoculate Leuconostoc mesenteroides subsp. mesenteroides or Lactobacillus plantarum into the solid sea cucumber culture medium, after cultivation, obtain a single colony, inoculate the obtained single colony into a new solid sea cucumber culture medium, repeat the above steps, and after culturing for 30 to 60 generations, obtain a single colony after subculture; 3) Respectively inoculate the single colonies obtained after subculture in step 2) into a liquid sea cucumber culture medium for cultivation to obtain a fermentation broth; centrifuge the prepared fermentation broth, collect the bacterial cells and add sterile water to prepare a bacterial suspension; the microbial inoculant is: a Leuconostoc mesenteroides subsp. mesenteroides bacterial suspension, a Lactobacillus plantarum bacterial suspension, or a mixture prepared by mixing the Leuconostoc mesenteroides subsp. mesenteroides bacterial suspension and the Lactobacillus plantarum bacterial suspension according to a volume ratio of 1 to 5:1 to 5.

2. The preparation method according to claim 1, wherein The sea cucumber wall-breaking liquid is prepared by adding water to sea cucumbers. The addition amount of the water is added according to a weight ratio of 1:5 to 6, boiled for 30 to 50 minutes, cooled and stood still for 1 to 1.5 hours, then the whole system is poured into a wall breaker, broken by the wall breaker, and passed through a 200 to 300 mesh sieve to obtain the sea cucumber wall-breaking liquid. Preferably, in step 1), add 1.5-2% (w / w) agar and 5-8% (w / w) glucose to the sea cucumber wall-breaking liquid to prepare a solid sea cucumber culture medium. Preferably, step 2) is that the cultivation method is: respectively inoculate Leuconostoc mesenteroides subsp. mesenteroides or Lactobacillus plantarum into the solid sea cucumber culture medium, cultivate in a constant temperature incubator at 35 to 37 °C for 10 to 12 hours, select a single colony with a strain diameter greater than 1.5 mm and inoculate it into a new solid sea cucumber culture medium, repeat the above steps, and after culturing for 30 to 60 generations, respectively obtain a single colony of Leuconostoc mesenteroides subsp. mesenteroides or Lactobacillus plantarum after subculture. Preferably, in step 3), the cultivation is carried out at 35 to 37 °C and a rotation speed of 150-200 r / min for 16 to 18 hours to prepare a seed liquid; inoculate the prepared seed liquid according to an inoculation amount of 5% to 10% by volume, and cultivate at 35 to 37 °C and a rotation speed of 150-200 r / min for 20 to 22 hours, with the pH controlled at 6.5 to 7.0; obtain a fermentation broth; after centrifuging the prepared fermentation broth, collect the bacterial cells. Preferably, in step 3), in the bacterial suspension, the total bacterial concentration is: 1-5×10 6 CFU / ml; Preferably, the microbial inoculant is a mixture prepared by mixing the prepared Leuconostoc mesenteroides subsp. mesenteroides bacterial suspension and the Lactobacillus plantarum bacterial suspension according to a volume ratio of 1:1 or 2:

1.

3. The preparation method according to claim 1 or 2, characterized in that, Inoculate the microbial inoculant according to a ratio of 3 to 5% (v / v) into the sea cucumber wall-breaking liquid obtained in step (1), and cultivate at 35 to 37 °C and a rotation speed of 150-200 r / min for at least 20 hours, with the pH controlled at 6.5 to 7.0; prepare sea cucumber peptides.

4. A sea cucumber peptide, characterized in that, The sea cucumber peptide is prepared by the method according to any one of claims 1 to 3.

5. The sea cucumber peptide according to claim 4, wherein The sea cucumber peptide includes WEPPPTGPGSP peptide, GPIDGLP peptide and / or RPDIPGPR peptide.

6. Use of the method according to any one of claims 1 to 3, or the sea cucumber peptide prepared by the method according to any one of claims 1 to 3, or the sea cucumber peptide according to claim 4 or 5 in the preparation of craft beer.

7. Use of the WEPPPTGPGSP peptide, GPIDGLP peptide or RPDIPGPR peptide according to claim 5 in the preparation of a tyrosinase inhibitor or in the preparation of a whitening cosmetic.

8. A preparation method of sea cucumber peptide craft beer, characterized in that, The method comprises the following steps: (1) Add oatmeal with a mass fraction of 0.5-1.0%, 20-40 mL of α-thermostable amylase, and water into a mashing pot, and stepwise heat up to 98 °C at a heating rate of 1-5 °C / min to obtain mash; (2) Add 10-15 parts of Canadian barley malt, 40-45 parts of Australian barley malt, and 35-40 parts of wheat malt according to the mass fraction, add them into a crusher for crushing to obtain a crushed mixture; Add water into a saccharification pot. When the temperature rises to 40-45 °C, add the above-mentioned crushed mixture. At the same time, add citric acid with a mass fraction of 0.08-0.1%, lactic acid with a mass fraction of 0.007-0.01%, calcium sulfate with a mass fraction of 0.09-0.1%, xylanase with a mass fraction of 0.15-0.2 IU / g, and β-glucan complex enzyme with a mass fraction of 1.5-2.0 IU / g; keep warm for 10-15 min, then heat up to 50-55 °C, keep warm for 15-20 minutes, then introduce the mash in the mashing pot into the saccharification pot, stir, heat up to 68-72 °C, and keep warm for 25-30 minutes; after passing the iodine test, heat up to 70-80 °C and then end saccharification; filter the saccharified liquid after saccharification is completed to collect the original juice; after sparging, combine the collected wort. After sparging, the residual sugar in the sparged grains is above 1.5 °P; combine the original juice and the wort after sparging; (3) Add calcium sulfate and calcium chloride to the wort collected in step (2), stir evenly. After heating the clarified wort to boiling, add hops in three portions. After boiling for 10-15 min, add SAAZ fragrant hops with a mass fraction of 0.1%-0.25% of the wort mass. After boiling for 20-25 min, add hops with a mass fraction of 0.5%-0.75% of the wort mass again. Boil for 20-25 min, and finally add SAAZ fragrant hops with a mass fraction of 0.1%-0.3% of the wort mass, continue to boil for 5-10 min, and then cool down; (4) After precipitation and filtration, aerate the wort, with the aeration amount of 9-10 mg / L, and the addition amount of full-tank brewing yeast is 10-11 million / mL; (5) Primary fermentation: Transport the wort added with brewing yeast to a fermentation tank, seal the tank for fermentation (control the temperature at 18-20 °C). When the diacetyl content is 0.09 mg / L and the fermentation degree is 63-65%, the primary fermentation ends; After the main fermentation is completed, add sea cucumber peptides prepared by the method described in any one of claims 1 to 3 or the sea cucumber peptides described in claim 4 or 5 to the fermentation tank, and the addition amount is 1 to 2%; after the addition, cool down and store in cold. After cold storage for 45 - 60 days, centrifuge with a centrifuge, and the number of yeasts in the filtered wine is 800 - 10 million / mL; After cold storage for 45 - 60 days, centrifuge with a centrifuge, and then package the finished product.

9. The preparation method according to claim 8, characterized in that, The hops is SAAZ hops; the enzyme activity of the α - high temperature resistant amylase is 20,000 U / ml.

10. A sea cucumber peptide craft beer prepared by the preparation method described in claim 8 or 9.

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