CN114651983B - Bacillus coagulans with uric acid-lowering and anti-oxidative abilities derived from shrimp paste, method and application - Google Patents

Abstract

The invention discloses bacillus coagulans with uric acid reducing and antioxidant capabilities, which is derived from shrimp paste, wherein the name of the strain is as follows: GH1-1, class name: bacillus coagulans (Bacillus coagulans), accession number: cgmccno.24236, date of preservation: 2022, 1 month 4, deposit unit: china general microbiological culture Collection center, north Chen Xi Lu No. 1, 3, the Korean region of Beijing, and the China general microbiological culture Collection center. The bacillus coagulans strain is screened from the traditional Chinese shrimp paste, has strong stress resistance and intestinal survival capability, and has good growth performance. Meanwhile, the composition, host immunity and metabolism of the microbiota can be regulated, so that the composition can be used for treating intestinal diseases.

Description

A Bacillus coagulans with uric acid-lowering and antioxidant capacity derived from shrimp paste, Method and application

technical field

The invention belongs to the technical field of microorganisms and fermented beverages, in particular to a bacillus coagulans with uric acid-lowering and anti-oxidation abilities derived from shrimp paste, a method and an application.

Background technique

Hyperuricemia is a metabolic syndrome caused by purine metabolic disorders. Under normal circumstances, it can be excreted through urine. Once the human body takes in more purine-containing substances or renal insufficiency occurs, the level of uric acid in the serum may change to a certain extent and show an upward trend. In recent years, with the rapid economic development and changes in lifestyle, the incidence of hyperuricemia in my country has increased significantly, and it is showing a younger trend. Hyperuricemia is not only a key risk factor for gout, but also closely related to kidney disease, hypertension, diabetes and cardiovascular disease. Therefore, hyperuricemia has become a growing public health problem. Effectively lowering uric acid levels is key to reducing the risk of gout and preventing complications. Most patients require long-term or even life-long uric acid-lowering drug therapy. However, long-term use of urate-lowering drugs is expensive and has certain side effects. Therefore, it is of great clinical and public health significance to actively search for safe, cheap and effective medicinal and edible products.

Probiotic food is a functional food that is beneficial to human health. It has the functions of improving intestinal microecological balance, regulating nutrient metabolism, and enhancing immune efficacy. It has huge commercial benefits and growing market share. Numerous studies have shown that probiotics have the potential to regulate purine and uric acid metabolism. However, the effect of probiotics has strain differences. Screening excellent probiotic strains with independent intellectual property rights suitable for the Chinese population from traditional Chinese food has important academic significance and economic and social value.

Protein is the basic substance of human body composition, accounting for about 18% of the total body weight. Drinking protein drinks is a healthy, convenient way to get more protein. Protein beverages are mainly divided into two categories: vegetable protein beverages and animal protein beverages. Compared with plant protein, the amino acid (especially the essential amino acid) composition of animal protein is more complete, the composition ratio is closer to the human body, and it is easier to be absorbed by the human body. At the same time, animal protein raw materials are often rich in vitamin B12, DHA, minerals and other important nutrients for the human body, while anti-nutrients such as lectins, saponins and phytic acid are less than plant protein raw materials. Therefore, as people pay more and more attention to health, the demand and development space of animal protein drinks are getting bigger and bigger. However, due to the relatively high production cost of animal protein beverages, at present, the protein beverages on the market are still dominated by vegetable proteins such as walnut kernels, almonds, and peanuts. Animal protein beverages are basically limited to dairy products, and more new types of beverages are urgently needed. product.

Shrimp paste, as some traditional fermented food, is one of the seasonings commonly used in my country’s coastal areas, East Asia, and Southeast Asia. However, it has been difficult to promote in other regions, and the application population is relatively limited. It is also rich in high salt, which is not ideal in terms of health. . Studies have found that: in the traditional production process of shrimp paste, the main flora in shrimp paste include Bacillus, Lactobacillus, Pediococcus, Saccharomyces, etc. Sterilization is rarely carried out in the preparation process of shrimp paste, so , often these natural symbiotic microorganisms survive.

Through searching, no patent publications related to the patent application of the present invention have been found.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, and provide a strain of Bacillus coagulans with uric acid-lowering and anti-oxidative abilities derived from shrimp paste, a method and an application.

The technical scheme that the present invention solves technical problem adopts is:

A Bacillus coagulans with uric acid-lowering and anti-oxidation abilities derived from shrimp paste, the name of the strain is: GH1-1, the classification name is: Bacillus coagulans (Bacillus coagulans), and the preservation number is: CGMCC NO.24236 , Preservation date: January 4, 2022, Preservation unit: General Microbiology Center of China Microbiological Culture Collection Management Committee, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

Further, the strain has strong purine metabolism and xanthine oxidase XOD inhibition ability.

Further, the strain has strong antioxidant capacity.

Further, the strain is derived from traditional shrimp paste in Binzhou, Shandong.

The shrimp meat protein fermented beverage obtained by using the above-mentioned Bacillus coagulans fermentation.

Further, the beverage eliminates the disadvantages of high purine and poor taste of shrimp meat, and has strong antioxidant activity; or, the beverage has the ability to inhibit xanthine oxidase XOD.

The fermentation method of the aforementioned shrimp meat protein fermented beverage, the steps are as follows:

(1) Processing of raw materials: Clean the fresh shrimps, remove their shells, soak them in water at 65-75°C for 30 minutes, then dry them overnight, crush them, pass them through a 100-mesh sieve, and store them for later use;

(2) Degreasing: Mix the raw materials obtained in step (1) with absolute ethanol, add excess ethanol, stand at 50-60°C for 1 hour, stir once every 20 minutes, repeat degreasing twice, dry to remove absolute ethanol ;

(3) Enzymolysis: use papain for enzymolysis, then boil to inactivate the enzyme, centrifuge at 6000r/min for 10min to remove the enzyme, and take the supernatant;

(4) Activated carbon treatment: add activated carbon to the enzymatic hydrolysis solution in (3) to remove bitterness, astringency and deodorization, centrifuge at 8000r/min for 10min, take the supernatant, and centrifuge again at 8000r/min for 10min, take the supernatant;

(5) Sterilization: Place the enzymatic hydrolysis solution in step (4) in a water bath at 62-65°C, pasteurize it for 30 minutes and then cool it down;

(6) compounding: add pasteurized fresh pure lemon juice to the enzymatic hydrolysis solution in step (5) for compounding;

⑺Fermentation: Add the strain liquid of Bacillus coagulans GH1-1 to the compound solution in step 6 for fermentation;

(8) Blending: Add flavoring agents to the fermented liquid in step (7) for blending to obtain the shrimp meat protein fermented beverage.

Further, the mixing mass ratio of raw material and absolute ethanol in the step (2) is 1:6;

The enzymatic hydrolysis condition in the step (3) is to use papain, at pH=7.2, 60°C, with a substrate concentration of 4%, an enzyme dosage of 6000U/g, and react for 6-8h;

In the step (4), the amount of activated carbon added is 15g/L, and the treatment conditions are 50-60°C for 30-40min;

The composite volume ratio of enzymolysis solution and fresh pure lemon juice in the step (6) is 3:7;

The fermentation conditions in the step (7) are: 3% inoculum amount, 12 hours of shaking table fermentation at 37° C.; the OD ₆₀₀ of the seed liquid in the step (7) = 1-2;

The flavoring agent used in the step (8) is steviol glycoside, and its final concentration is 1 g/L.

The above-mentioned application of Bacillus coagulans in animal protein fermented beverages.

The beneficial effects that the present invention obtains are:

1. The present invention screens out a strain of Bacillus coagulans from traditional Chinese shrimp paste. This strain has strong stress resistance and intestinal survival ability, and has good growth performance. At the same time, it can have a therapeutic effect on intestinal diseases by regulating the composition of microbiota, host immunity and metabolism.

2. The bacterial strain GH1-1 studied in the present invention has strong anti-oxidation, purine metabolism and ability to inhibit xanthine oxidase.

3. The shrimp meat protein fermented beverage of the present invention has been systematically optimized, and a shrimp protein beverage rich in protein and other nutrients and more in line with the taste of the public has been prepared.

4. The beverage of the present invention is an animal protein fermented beverage. Compared with a vegetable protein fermented beverage, the amino acid (especially essential amino acid) composition integrity of animal protein is higher, the composition ratio is closer to the human body, and it is easier to be absorbed by the human body. The beverage of the invention has stable quality, rich nutrition and good taste and flavor.

5. It is found through anti-oxidation experiments that the beverage of the present invention has a very high DPPH scavenging rate and the DPPH scavenging rate after fermentation is significantly improved, indicating that more antioxidant substances are produced during the fermentation process. Therefore, this live bacteria drink can be used as a new type of anti-oxidation functional drink.

Through the xanthine oxidase (XOD) activity inhibition experiment, it is found that the beverage of the present invention has higher XOD inhibition ability, which is obviously improved after fermentation.

The beverage of the invention is rich in protein content, and has a strong DPPH clearance rate and an inhibition rate to xanthine oxidase (XOD), suggesting that it has a good antioxidant effect. This study provides a new option for the development of deep processing of shrimp meat.

6. The fermentation strain of the present invention is Bacillus coagulans (Bacillus coagulans) GH1-1 isolated and purified from traditional shrimp paste in Binzhou, Shandong Province. Microscopic examination and 16SrDNA sequence analysis have identified the strain as Bacillus coagulans GH1-1. The present invention uses traditional shrimp paste as raw material, screens out probiotics that can better metabolize shrimp meat, and then through systematic optimization and analysis, researches and develops a probiotic shrimp meat protein fermented beverage suitable for modern production .

7. The present invention separates the probiotics in the traditional shrimp paste, screens out the dominant strains and uses them to ferment the shrimp meat protein. Then optimize the fermentation conditions, and invent a shrimp protein fermented beverage with stable quality, rich nutrition, and excellent taste and flavor.

Description of drawings

Fig. 1 is the microscopic examination result figure of GH1-1 among the present invention;

Fig. 2 is the phylogenetic tree diagram of GH1-1 in the present invention;

Fig. 3 is the growth curve diagram of GH1-1 in the present invention;

Fig. 4 is the detection diagram of purine metabolism ability of GH1-1 in the present invention; wherein, a is the liquid phase diagram of the standard product, two peaks are respectively hypoxanthine and guanine from left to right, and the total peak area is 2342221; b is GH1 -1 Metabolic liquid phase diagram, the two peaks are xanthine and hypoxanthine, the total peak area is 310238;

Fig. 5 is beverage DPPH clearance measurement result figure among the present invention;

Fig. 6 is a graph showing the results of determination of XOD inhibition ability of beverages in the present invention.

Detailed ways

In order to better understand the present invention, the present invention will be further described in detail below in conjunction with the examples, but the protection scope of the present invention is not limited to the range indicated by the examples.

The raw materials used in the present invention, if no special instructions, are conventional commercially available products, the methods used in the present invention, if no special instructions, are conventional methods in this area, and the quality of each material used in the present invention is Normal use quality.

A Bacillus coagulans with uric acid-lowering and anti-oxidation abilities derived from shrimp paste, the name of the strain is: GH1-1, the classification name is: Bacillus coagulans (Bacillus coagulans), and the preservation number is: CGMCC NO.24236 , Preservation date: January 4, 2022, Preservation unit: General Microbiology Center of China Microbiological Culture Collection Management Committee, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

Preferably, the strain has strong purine metabolism and xanthine oxidase XOD inhibition ability.

Preferably, the strain has strong antioxidant capacity.

Preferably, the strain is derived from traditional shrimp paste in Binzhou, Shandong.

The shrimp meat protein fermented beverage obtained by using the above-mentioned Bacillus coagulans fermentation.

Preferably, the beverage eliminates the disadvantages of high purine and poor taste of shrimp meat, and has strong antioxidant activity; or, the beverage has the ability to inhibit xanthine oxidase XOD.

The fermentation method of the aforementioned shrimp meat protein fermented beverage, the steps are as follows:

(1) Processing of raw materials: Clean the fresh shrimps, remove their shells, soak them in water at 65-75°C for 30 minutes, then dry them overnight, crush them, pass them through a 100-mesh sieve, and store them for later use;

(2) Degreasing: Mix the raw materials obtained in step (1) with absolute ethanol, add excess ethanol, stand at 50-60°C for 1 hour, stir once every 20 minutes, repeat degreasing twice, dry to remove absolute ethanol ;

(3) Enzymolysis: use papain for enzymolysis, then boil to inactivate the enzyme, centrifuge at 6000r/min for 10min to remove the enzyme, and take the supernatant;

(4) Activated carbon treatment: add activated carbon to the enzymatic hydrolysis solution in (3) to remove bitterness, astringency and deodorization, centrifuge at 8000r/min for 10min, take the supernatant, and centrifuge again at 8000r/min for 10min, take the supernatant;

(5) Sterilization: Place the enzymatic hydrolysis solution in step (4) in a water bath at 62-65°C, pasteurize it for 30 minutes and then cool it down;

(6) compounding: add pasteurized fresh pure lemon juice to the enzymatic hydrolysis solution in step (5) for compounding;

⑺Fermentation: Add the strain liquid of Bacillus coagulans GH1-1 to the compound solution in step 6 for fermentation;

(8) Blending: Add flavoring agents to the fermented liquid in step (7) for blending to obtain the shrimp meat protein fermented beverage.

Preferably, the mixing mass ratio of raw material and absolute ethanol in the step (2) is 1:6;

The enzymatic hydrolysis condition in the step (3) is to use papain, at pH=7.2, 60°C, with a substrate concentration of 4%, an enzyme dosage of 6000U/g, and react for 6-8h;

In the step (4), the amount of activated carbon added is 15g/L, and the treatment conditions are 50-60°C for 30-40min;

The composite volume ratio of enzymolysis solution and fresh pure lemon juice in the step (6) is 3:7;

The fermentation conditions in the step (7) are: 3% inoculum amount, 12 hours of shaking table fermentation at 37° C.; the OD ₆₀₀ of the seed liquid in the step (7) = 1-2;

The flavoring agent used in the step (8) is steviol glycoside, and its final concentration is 1 g/L.

The above-mentioned application of Bacillus coagulans in animal protein fermented beverages.

Specifically, the relevant preparation and detection are as follows:

A screening of probiotics in shrimp paste, and the use of the bacteria to ferment the shrimp meat protein beverage can be carried out according to the following production steps:

⑴ Preliminary isolation, screening and identification of strains in traditional Chinese shrimp paste under laboratory conditions, and culture in MRS broth medium;

⑵Bacillus with strong acid-producing ability was screened out through calcium-dissolving circle and microscopic examination, and then determined to be Bacillus coagulans through 16S rDNA sequence analysis;

(3) Through the determination of the purine metabolism ability, it is determined that the strain has a strong purine metabolism ability, which can reduce the discomfort of the shrimp meat protein drink to patients with hyperuricemia, and can be used for the development of uric acid-lowering related products;

(4) Processing of raw materials: Clean the fresh shrimps, remove their shells, soak them in water at 65°C for 30 minutes, then dry them overnight, crush them, pass them through a 100-mesh sieve, and store them for later use;

⑸ Degreasing: Mix the raw materials with absolute ethanol at a ratio of 1:6, let stand at 50°C for 1 hour, stir once every 20 minutes, and repeat the degreasing twice;

(6) Enzymolysis: use papain at pH = 7.2, 60°C, according to substrate concentration of 4%, enzyme dosage of 6000U/g, react for 6 hours for enzymolysis, then boil to inactivate the enzyme, centrifuge, and take the supernatant;

⑺Activated carbon treatment: add 15g/L activated carbon to the enzymatic hydrolysis solution in ⑹, treat at 55°C for 30 minutes to remove bitterness, astringency and deodorization, centrifuge, and take the supernatant;

(8) Sterilization: Place the enzymatic hydrolysis solution in (7) in a water bath at 65°C, pasteurize it for 30 minutes and then cool it down;

⑼ Compounding: Add pasteurized fresh lemon juice to the enzymatic hydrolyzate in ⑻ to compound at a ratio of 3:7;

⑽Fermentation: Ferment the compound solution in ⑼ with 3% inoculum at 37°C for 12 hours;

⑾Preparation: Add 1g/L steviol glycoside as a flavoring agent to the fermented liquid in ⑽ to obtain the shrimp meat protein fermented beverage.

⑿Determination of protein content: Use Bio-Rad protein quantification kit to detect protein content: Precisely pipette 4 μL of reagent and 200 μL of sample into the well plate under dark conditions, and react for 15 minutes in the dark, use a microplate reader to measure its OD ₅₉₅ value, and standard curve comparing.

⒀Determination of DPPH clearance rate: Take 1mL of distilled water, DPPH, and sample in a 5mL centrifuge tube, shake well, and keep it in the dark for 30 minutes, then measure the absorbance at 517nm, calculate the clearance rate according to the formula and compare it with the vitamin C standard curve control.

⒁ Determination of XOD inhibitory ability: Use the five reagents in the xanthine oxidase (XOD) assay kit to add distilled water, samples before and after fermentation, and react in a water bath at 37°C for 20 minutes to measure their OD ₅₃₀ . The OD ₅₃₀ value is directly proportional, so the OD ₅₃₀ value represents the enzyme activity, and the inhibition rate is calculated.

Example 1

Sample source: A shrimp paste specialty store in Binzhou Economic Development Zone, Shandong Province.

Under sterile conditions, take 1g of shrimp paste fermented for 3, 5, 8, and 12 months and mix them in 1mL of sterile water. After gradient dilution, take 100μL of samples from each gradient and spread evenly on MRS solids with calcium carbonate added. On the medium plate, cultured anaerobically at 37°C for 48h. The white round colonies with calcium-dissolving circles were selected in turn, streaked and purified on the MRS solid plate, and cultured in an anaerobic box at 37°C for 48 hours. The bacterial solution was mixed with 60% glycerol at a volume ratio of 3:1 and stored at -80°C for later use.

The formula and conditions of MRS solid medium are: peptone 10g, beef extract 10g, yeast powder 5g, glucose 20g, anhydrous sodium acetate 5g, Tween-80 1mL, diamine citrate 2g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.58g, 0.25g manganese sulfate, 20g calcium carbonate, 18g agar, 1000mL distilled water, pH6.5～6.7, 115℃, sterilize for 15min.

Example 2

Morphological identification of strains:

The morphological characteristics of the strains were detected by Gram staining. The results are shown in Figure 1, the bacterial strains screened by the present invention belong to Gram-positive bacteria, the morphology is rod-shaped, and spores can be seen in microscopic examination.

Example 3

Molecular biological identification of strains:

The 16S rDNA of the strain was extracted by PCR and sequenced, and the DNA sequence was obtained after splicing, and the BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi) provided by NCBI was used to search the Genbank database for homology to the sequence Gene sequences with higher sex were used to build a tree using the Neighbor Joining method of MEGA-7 software, and the built tree was saved in Newick format. Then use iTOL (https://itol.embl.de/) to beautify the gene sequence phylogenetic tree. The results showed that its 16S rDNA was as high as 97.71% similar to Bacillus coagulans strain wy01. The phylogenetic tree shown in Figure 2 shows that this strain is in family I together with five other strains of Bacillus coagulans. Combined with the morphological characteristics and physiological and biochemical characteristics of the strain, the strain was identified as Bacillus coagulans and named It is Bacillus coagulans GH1-1, and it is preserved in the China General Microorganism Culture Collection Management Center with the preservation number CGMCC24236.

Example 4

Optimization of process parameters:

The activated bacterial suspension was inoculated into the protein solution, and the fermentation time, inoculation amount, and steviol glycoside addition amount were used as the influencing factors, and the sensory evaluation was used as the index, and a single factor experiment was carried out.

Determination of fermentation time: control the inoculation amount to 3%, steviol glycoside addition to 1.25g/L, ferment the compound beverage for 8, 10, 12, 14, and 16 hours respectively, and determine the fermentation time based on sensory evaluation.

Determination of the inoculum amount: control the fermentation time to 12h, add steviol glycoside to 1.25g/L, and ferment the compound beverage according to the inoculum amount of 1%, 2%, 3%, 4%, and 5% respectively, and use the sensory evaluation Determine the inoculum size for the indicator.

Determination of the amount of steviol glycoside added: control the inoculum amount to 3%, and the fermentation time is 12 hours, add 0.75, 1, 1.25, 1.5, 1.75 g/L stevioside respectively, ferment the compound beverage, and determine the stevioside by sensory evaluation Add amount.

On the basis of the single factor experiment, taking the fermentation time, inoculum amount, and steviol glycoside addition as the influencing factors, the two levels with the highest scores were obtained, an L ₄ (2 ³ ) orthogonal experiment was designed, and the sensory evaluation was used as the index to optimize the Optimum process parameters.

A fixed 10 people will score the organizational state, smell, taste and other indicators of the comprehensive sample, and the average value after removing the lowest and highest scores will be the final score of the product. See Table 1 for scoring criteria.

Table 1 Sensory scoring standard table

The results showed that the taste was the best when the fermentation time was 12h, the inoculation amount was 3%, and the addition amount of steviol glycoside was 1g/L.

Example 5

Detection of growth curve and purine metabolism ability of the strain:

In order to better determine the appropriate fermentation time, 40 μL of the activated strain was cultured in MRS liquid medium at 37°C for 24 hours, and the OD ₆₀₀ value was measured every 2 hours to draw the growth curve with the uninoculated medium as the control. As shown in Figure 3, the strain was in the incubation period from 0 to 4 hours, in the logarithmic growth period from 4 hours to 16 hours, and reached the stable period at 16 hours, with a maximum OD ₆₀₀ of about 1.8. Because the strains in the middle and late stages of logarithmic growth have the advantages of fast growth and uniformity among individuals, they can grow close to synchronously when used as fermented seeds to reproduce offspring, so that the fermentation metabolism is normal and the cycle is shortened, so the seed culture time is 12h.

Due to the high content of purine in shrimp meat, there are certain potential concerns about promoting hyperuricemia and gout in the development of beverages and other foods. Screening a probiotic strain that can metabolize purine can not only eliminate the risk of high purine in beverages, but also play a probiotic role in the intestinal tract. Since this strain is isolated from shrimp paste and has strong ability to ferment and metabolize raw shrimp meat, we also analyzed its purine metabolism ability: the standard substances of guanine and hypoxanthine and the strains that were screened were metabolized The latter samples were tested for their ability to metabolize guanine and xanthine using LC-LFD. The results are shown in Figure 4, compared with Figure 4 (a) and (b), it was found that B. coagulans GH1-1 can metabolize guanine and hypoxanthine well, and convert them into xanthine and hypoxanthine, and the total purine The amount was reduced by 86.75%. Therefore, using this strain to ferment beverages can effectively reduce the possibility of purine in shrimp protein, and this strain may have the potential to metabolize purine in the intestine after entering the intestine.

Example 6

A preparation method of a shrimp meat protein fermented beverage, comprising the following methods:

(1) Processing of raw materials: Clean the fresh shrimp, remove the shell, soak in water at 65°C for 30 minutes, then dry overnight, crush, pass through a 100-mesh sieve, and store for later use;

(2) Degreasing: Mix the raw materials with absolute ethanol at a mass ratio of 1:6, let stand at 50°C for 1 hour, and stir once every 20 minutes, repeat the degreasing twice, dry to remove absolute ethanol;

(3) Enzymolysis: Use papain at pH = 7.2, 60°C, with a substrate concentration of 4% and an enzyme dosage of 6000U/g, react for 6 hours for enzymolysis, then boil to inactivate the enzyme, centrifuge, and take the supernatant;

(4) Activated carbon treatment: Add activated carbon 15g/L to the enzymolysis solution in (3), treat at 55°C for 30 minutes to remove bitterness, astringency and deodorization, centrifuge at 8000r/min for 10min, take the supernatant, and centrifuge again at 8000r/min for 10min, take the supernatant ;

(5) Sterilization: Place the enzymatic solution in (4) in a water bath at 65°C, pasteurize it for 30 minutes and then cool it down;

⑹ Compounding: add the enzymatic hydrolyzate in ⑸ and pasteurized fresh lemon juice to compound according to the volume ratio of 3:7;

⑺Fermentation: Inject Bacillus coagulans GH1-1 into the compound solution in ⑹ with 3% inoculation amount, the OD ₆₀₀ of the seed solution is 1.5, and ferment on a shaking table at 37°C for 12 hours;

(8) Blending: Add 1g/L steviol glycosides as a flavoring agent to the fermentation broth in (7) and blend to obtain a shrimp meat protein fermented beverage.

The shrimp meat protein fermented drink prepared in this example has good uniformity and a characteristic aroma of lemon. And the shrimp meat protein fermented beverage prepared in the present embodiment is sweet and sour, and has a pleasant flavor.

Example 7

Protein content of shrimp meat protein fermented drink:

The protein content was detected by the Bio-Rad protein quantification kit: 4 μL of reagent and 200 μL of sample were precisely pipetted into the well plate under dark conditions, and reacted for 15 minutes in the dark, and the OD ₅₉₅ value was measured with a microplate reader, and compared with the standard curve. The protein content in the beverage was determined to be 2.44g/100mL, while the protein content of common protein drinks in the market except milk is generally around 0.5g/100mL.

Example 8

Shrimp protein fermented drink DPPH scavenging capacity:

Take 1mL of distilled water, DPPH, and samples in a 5mL centrifuge tube, shake well, and keep it in the dark for 30 minutes, then measure the absorbance at 517nm, calculate the clearance rate according to formula (1) and compare it with the vitamin C standard curve:

DPPH clearance rate = [1-(A _X -A _X0 )/A ₀ ]×100% (1)

Among them, A _X : 1.0mL sample + 1.0ml DPPH + 1.0mL distilled water

A ₀ : 1.0mL DPPH+2.0mL distilled water

_AX0 : 3.0 mL absolute ethanol.

The results are shown in Figure 5, the unfermented beverage DPPH clearance rate is 66.61%, after comparing with the vitamin C standard curve, the result shows that it is equivalent to vitamin C of 859.13 μ mol/L; The beverage DPPH clearance rate after fermentation is 80.03%, and After comparing the standard curve of vitamin C, the result shows that the vitamin C is equivalent to 914.47μmol/L. It can be seen that after fermentation, the antioxidant capacity of the beverage can be increased by 22.14%. Compared with the DPPH clearance rate of about 50% in the current market lemon-containing beverage, the beverage has increased by about 60%.

Example 9

Shrimp protein fermented drink XOD inhibitory ability:

Use the five reagents in the xanthine oxidase (XOD) assay kit to add distilled water, samples before and after fermentation, and react in a water bath at 37°C for 20 minutes to measure their OD ₅₃₀ . Since the XOD activity is directly proportional to the OD ₅₃₀ value, Therefore, the OD ₅₃₀ value was used to represent the enzyme activity, and the inhibition rate was calculated.

The results are shown in Figure 6. It is calculated that the unfermented beverage can inhibit 53.11% of the XOD activity, and it can reach 66.67% after fermentation, and the ability to inhibit the XOD activity is increased by 25.53%. At this time, the solubility of the sample is 1.3%.

Although the embodiments of the present invention are disclosed for the purpose of illustration, those skilled in the art will understand that various alternatives, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims, therefore However, the scope of the present invention is not limited to the content disclosed in the embodiments.

Claims (8)

1. A Bacillus coagulans with uric acid-lowering and antioxidant capacity derived from shrimp paste, characterized in that: the name of the strain is: GH1-1, the classification name is: Bacillus coagulans ( Bacillus coagulans ), and the preservation number For: CGMCCNO.24236, preservation date: January 4, 2022, preservation unit: General Microbiology Center of China Microbiological Culture Collection Management Committee, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. 2. The bacillus coagulans with uric acid-lowering and anti-oxidative ability derived from shrimp paste according to claim 1, characterized in that: said bacterial strain has strong purine metabolism and xanthine oxidase XOD inhibitory ability. 3. The Bacillus coagulans with uric acid-lowering and antioxidant capacity derived from shrimp paste according to claim 1, characterized in that: said bacterial strain has strong antioxidant capacity. 4. The Bacillus coagulans with uric acid-lowering and antioxidant capacity derived from shrimp paste according to any one of claims 1 to 3, characterized in that: the strain is derived from traditional shrimp paste in Binzhou, Shandong. 5. The shrimp meat protein fermented beverage obtained by fermentation of Bacillus coagulans as described in any one of claims 1 to 4. 6. the fermentation method of shrimp protein fermented beverage as claimed in claim 5, is characterized in that: the steps are as follows: (1) Processing of raw materials: Clean the shrimp and remove the shell, soak in water at 65~75°C for 30 minutes, then dry overnight, crush, pass through a 100-mesh sieve, and store for later use; (2) Degreasing: Mix the raw materials obtained in step (1) with absolute ethanol, add excess ethanol, let stand at 50~60°C for 1 hour, stir once every 20 min, repeat degreasing twice, dry to remove anhydrous ethanol; (3) Enzymolysis: use papain for enzymolysis, then boil to inactivate the enzyme, centrifuge at 6000 r/min for 10 min to remove the enzyme, and take the supernatant; (4) Activated carbon treatment: Add activated carbon to the enzymatic hydrolysis solution in (3) to remove bitterness, astringency and deodorization, centrifuge at 8000 r/min for 10 min, take the supernatant, and centrifuge again at 8000 r/min for 10 min, take the supernatant; (5) Sterilization: Place the enzymatic solution in step (4) in a water bath at 62-65°C, pasteurize it for 30 minutes and then cool it down; (6) compounding: add pasteurized pure lemon juice to the enzymatic hydrolysis solution in step (5) for compounding; ⑺Fermentation: Add the strain liquid of Bacillus coagulans GH1-1 to the compound solution in step 6 for fermentation; (8) Blending: Add flavoring agents to the fermented liquid in step (7) for blending to obtain the shrimp meat protein fermented beverage. 7. The fermentation method of shrimp meat protein fermented beverage according to claim 6, is characterized in that: the mixing mass ratio of raw material and absolute ethanol is 1:6 in the described step (2); The enzymatic hydrolysis condition in the step (3) is to use papain, at pH=7.2, 60°C, the substrate concentration is 4%, the enzyme dosage is 6000 U/g, and the reaction is 6~8 h; In the step (4), the amount of activated carbon added is 15 g/L, and the treatment conditions are 30 to 40 min at 50 to 60 ° C; The compound volume ratio of enzymolysis solution and pure lemon juice in the step (6) is 3:7; The fermentation conditions in the step (7) are: 3% inoculum amount, 12 hours of shaking table fermentation at 37°C; the OD ₆₀₀ of the seed solution in the step (7) = 1-2; The flavoring agent used in the step (8) is steviol glycoside, and its final concentration is 1 g/L. 8. The application of Bacillus coagulans in animal protein fermented beverages as claimed in any one of claims 1 to 4.