Preparation method and application of probiotic fermentation product based on periplaneta americana

Abstract

The invention relates to the technical field of preparation of periplaneta americana fermentation products, in particular to a preparation method and application of a probiotic fermentation product based on periplaneta americana, and the preparation method comprises the following steps: S1, preparing periplaneta americana defatted powder; s2, adding distilled water and a glucose solution into the American cockroach degreased powder, sterilizing at high temperature, and cooling to room temperature to obtain a fermentation substrate; s3, according to the sequence of lactococcus lactis subsp. Lactis, lactobacillus helveticus and saccharomyces cerevisiae, the bacterial suspension of the corresponding strain is added into the fermentation substrate in sequence, and a sample inoculated with the bacterial suspension is obtained; and S4, culturing the sample inoculated with the bacterium suspension at the temperature of 30-33 DEG C for 68-72 hours to obtain the probiotic fermentation product based on the periplaneta americana. Through mixed fermentation of microbial strains, active substances are better released, and the antioxidant activity of a periplaneta americana fermentation product is remarkably improved.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of American cockroach fermentation products, and in particular to a preparation method and application of a probiotic fermentation product based on American cockroach. Background Art

[0002] American cockroaches are widely distributed in China and have a long history of being used as medicine. The American cockroaches are rich in bioactive substances, and their main chemical components include pheromones, proteins, fatty acids and their esters, amino acids, alkaloids, alkanes, polysaccharides, isoflavones, cockroach oils and polypeptides. The American cockroach extract has antibacterial, anti-inflammatory, antioxidant, analgesic, antipyretic and anti-tumor medicinal values, and has good application prospects in the medical field. Kangfuxin Liquid, a Chinese patent medicine prepared from the alcohol extract of its dried insect body, is widely used in clinical practice.

[0003] With the continuous improvement of people's health awareness, microbial fermentation technology as a green biotransformation technology has gradually become a research hotspot. Microbial fermentation technology can promote the biotransformation of compounds in the substrate, stimulate the production of bioactive compounds (these compounds may initially be lacking or present in small amounts in the unprocessed substrate), reduce the content of anti-nutritional components such as protease inhibitors, phytic acid, urease, and oxalic acid, and improve the microbial safety of edible insects. It may control the growth of harmful microorganisms in insect products, improve flavor, extend shelf life, and improve the nutritional value of biomass and biological functional properties such as antioxidant, antibacterial, anti-aging activity, anti-diabetic and anti-atherosclerotic activity.

[0004] The study of antioxidant activity has always had an indelible impact in the fields of food, cosmetics, pharmacology and medicine. Antioxidants play an important role in the development of many chronic diseases, such as cardiovascular disease, aging, heart disease, anemia, cancer, and inflammation. Oxidative stress is an inevitable part of the wound healing process, especially in the inflammatory stage. Therefore, antioxidant activity is also considered to be one of the basic characteristics of an ideal wound dressing. It can be seen that the medicinal value of American cockroach is inseparable from its antioxidant activity.

[0005] Insect bioactive peptides have antioxidant, antihypertensive and antibacterial effects. Proteins, peptides, and amino acids are the ingredients that account for a relatively high proportion of American cockroach medicines. The antioxidant activity of edible insect protein hydrolysates or peptides is relatively high compared to other food protein hydrolysates. Fermentation is a widely studied technology for producing bioactive peptides from food sources. Compared with enzymatic hydrolysis technology, the benefits of microbial fermentation are: a high diversity of microbial proteases can produce peptides of different sizes and sequences, and therefore have greater biological activity; a high level of protease activity, a whole set of microbial-specific proteases rather than just one protease, which allows the peptide to be fully released from the precursor protein, thereby shortening the production cycle; because expensive commercial enzymes are not used, the fermentation process is cheaper than enzymatic hydrolysis and the culture cost is relatively low. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, the object of the present invention is to provide a preparation method and application of a probiotic fermentation product based on American cockroaches.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for preparing a probiotic fermentation product based on American cockroaches comprises the following steps:

[0009] S1. Preparation of defatted powder of American cockroach: After the dried adult American cockroach is crushed, the fat is defatted by Soxhlet extraction, the insect powder is dried, and the defatted powder of American cockroach is obtained by passing through an 80-mesh sieve;

[0010] S2. Add distilled water to the defatted powder of American cockroach, add 5-10% glucose solution, sterilize at high temperature, and cool to room temperature to obtain a fermentation substrate;

[0011] S3. In a clean bench, 5-8% of the bacterial suspension of the corresponding strains of Lactococcus lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae were added to the fermentation substrate in the order of Lactococcus lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae, and the time interval for addition was 24h to obtain a sample of the inoculated bacterial suspension;

[0012] S4. The sample of the inoculated bacterial suspension was cultured in a shaker at a temperature of 30-33 ° C for 68-72 h to obtain a probiotic fermentation product based on American cockroaches;

[0013] Wherein, the ratio of the bacterial suspensions corresponding to Lactococcus lactis subsp. lactis, Lactobacillus helveticus and Saccharomyces cerevisiae is 3:2:1.

[0014] Preferably, in step S3, the preparation of the bacterial suspension of Lactococcus lactis subsp. lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae comprises the following steps:

[0015] S101. Lactococcus lactis subsp. lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae are cultured under appropriate conditions for 24-48 hours to obtain respective mixed solutions and are marked;

[0016] S102. Take 5-8 mL of the mixture and put it into a conical flask containing 100 mL of liquid culture medium, place it in a shaker at 165-180 rpm, dilute the concentrated bacterial suspension appropriately to make its optical density value between 0.1 and 0.65, and obtain the time when the respective single colonies and the respective bacterial suspensions are in the logarithmic growth phase;

[0017] S103. The respective single colonies were placed in a shaker for cultivation. Lactococcus lactis subsp. lactis was cultured for 16 h, Lactobacillus helveticus was cultured for 9 h, and Saccharomyces cerevisiae was cultured for 28 h. The supernatant was then removed by centrifugation, and the precipitate was washed with PBS solution and resuspended with sterile water to a final concentration of 10 7 CFU / mL, and obtain the corresponding bacterial suspensions of Lactococcus lactis subsp. lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae.

[0018] Preferably, in step S101, the respective suitable conditions are: Lactobacillus helveticus adopts MRS medium, the culture temperature is 37°C, and the pH value is 6.5; Lactococcus lactis subsp. lactis adopts M17 medium, the culture temperature is 30°C, and the pH value is 7.0; Saccharomyces cerevisiae adopts YPD medium, the culture temperature is 28°C, and the pH value is 5.6.

[0019] Preferably, in step S103, the components of the PBS solution are: 0.24-0.3 parts of potassium dihydrogen phosphate, 3.6-4 parts of disodium hydrogen phosphate dodecahydrate, 8-10 parts of sodium chloride, 0.2-0.4 parts of potassium chloride and 800-1000 parts of distilled water.

[0020] Preferably, in step S103, the rotation speed of the shaking incubator is 165-180 rpm.

[0021] Preferably, in step S103, the centrifugal speed is 10000-12000 rpm and the temperature is 3-5°C.

[0022] Preferably, in step S2, the ratio of American cockroach defatted powder to distilled water is 1:8-10.

[0023] Preferably, in step S2, the temperature of high temperature sterilization is 121-130° C. and the time is 15-20 min.

[0024] Preferably, in step S4, the rotation speed of the shaking table is 165-180 rpm.

[0025] An application of the probiotic fermentation product of American cockroach prepared according to the preparation method in anti-oxidation.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The present invention uses microbial fermentation technology, which is green and safe compared to chemical synthesis. It not only has no side effects of artificial synthetic products, but also is accompanied by the improvement of biochemical properties and the consumption of toxic components of inherent raw materials.

[0028] 2. Compared with single-strain fermentation and mixed fermentation of two bacteria, the polypeptide content and antioxidant activity of the American cockroach product obtained by mixed fermentation of three bacteria in the present invention are significantly improved.

[0029] 3. The present invention releases active substances better through mixed fermentation of microbial strains, and significantly improves the antioxidant activity of the American cockroach fermentation product. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The present invention is a preparation step of the probiotic fermentation product based on American cockroach;

[0031] Figure 2 The results of determination of polypeptide content (a), DPPH free radical scavenging rate (b), superoxide anion free radical scavenging rate (c) and superoxide dismutase activity (d) of the fermentation products of American cockroach defatted powder fermented by different bacterial species;

[0032] Figure 3 The results of the determination of polypeptide content (a), DPPH free radical scavenging rate (b), superoxide anion free radical scavenging rate (c) and superoxide dismutase activity (d) of the fermentation products of American cockroach defatted powder fermented by three bacteria in different compound ratios;

[0033] Figure 4 Effects of the order of bacterial addition on the polypeptide content (a), DPPH free radical scavenging rate (b), superoxide anion free radical scavenging rate (c) and superoxide dismutase activity (d) of the fermentation product of American cockroach defatted powder. DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] See also Figure 1-4 , the present invention provides a technical solution:

[0036] Example 1

[0037] A method for preparing a probiotic fermentation product based on American cockroach, characterized in that it comprises the following steps:

[0038] S1. Preparation of defatted powder of American cockroach: After the dried adult American cockroach is crushed, the fat is defatted by Soxhlet extraction, the insect powder is dried, and the defatted powder of American cockroach is obtained by passing through an 80-mesh sieve;

[0039] S2. Add distilled water to the defatted powder of American cockroaches at a ratio of 1:8, add 5% glucose solution, sterilize at 121°C for 15 minutes, and cool to room temperature to obtain a fermentation substrate;

[0040] S3. In a clean bench, 5% of the bacterial suspension of the corresponding species was added to the fermentation substrate in the order of Lactococcus lactis subsp. lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae, at a time interval of 24h, wherein Lactococcus lactis subsp. lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae were added in a ratio of 3:2:1 to obtain a sample of the inoculated bacterial suspension;

[0041] S4. The sample of the inoculated bacterial suspension was cultured at 30°C in a shaker at 165 rpm for 68 h to obtain a probiotic fermentation product based on American cockroaches;

[0042] S5. The fermentation product was centrifuged at 7500 rpm and 3°C for 10 min, and the obtained fermentation supernatant was filtered through a 0.22 μm filter membrane and used to determine the polypeptide content and in vitro antioxidant activity.

[0043] The preparation of the bacterial suspension of Lactococcus lactis subsp. lactis, Lactobacillus helveticus and Saccharomyces cerevisiae comprises the following steps:

[0044] S101. Lactococcus lactis subsp. lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae are cultured under appropriate conditions for 24 h to obtain respective mixed solutions and are marked;

[0045] S102. Take 5 mL of the mixture and put it into a conical flask containing 100 mL of liquid culture medium, place it in a shaker at 165 rpm, dilute the concentrated bacterial suspension appropriately to make its optical density value 0.1, and obtain the time when the respective single colonies and the respective bacterial suspensions are in the logarithmic growth phase;

[0046] S103. The individual colonies were placed in a shaker and cultured at 165 rpm. Lactococcus lactis subsp. lactis was cultured for 16 h, Lactobacillus helveticus was cultured for 9 h, and Saccharomyces cerevisiae was cultured for 28 h. The supernatant was removed after centrifugation at 10,000 rpm and 3°C. The precipitate was washed with PBS solution and resuspended with sterile water to a final concentration of 10 7 CFU / mL, and obtain the corresponding bacterial suspensions of Lactococcus lactis subsp. lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae.

[0047] This example is further named FPAG-10 and FPAG-16.

[0048] Example 2

[0049] A method for preparing a probiotic fermentation product based on American cockroach, characterized in that it comprises the following steps:

[0050] S1. Preparation of defatted powder of American cockroach: After the dried adult American cockroach is crushed, the fat is defatted by Soxhlet extraction, the insect powder is dried, and the defatted powder of American cockroach is obtained by passing through an 80-mesh sieve;

[0051] S2. American cockroach defatted powder was added to distilled water at a ratio of 1:10, and 10% glucose solution was added, sterilized at 130°C for 20 minutes, and cooled to room temperature to obtain a fermentation substrate;

[0052] S3. In a clean bench, 8% of the bacterial suspension of the corresponding strains was added to the fermentation substrate in the order of Lactococcus lactis subsp. lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae, at a time interval of 24h, wherein Lactococcus lactis subsp. lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae were added in a ratio of 3:2:1 to obtain a sample of the inoculated bacterial suspension;

[0053] S4. The sample of the inoculated bacterial suspension was cultured at 33°C in a shaker at 180 rpm for 72 h to obtain a probiotic fermentation product based on American cockroaches;

[0054] S5. The fermentation product was centrifuged at 8000 rpm and 5°C for 15 min, and the obtained fermentation supernatant was filtered through a 0.22 μm filter membrane and used to determine the polypeptide content and in vitro antioxidant activity.

[0055] The preparation of the bacterial suspension of Lactococcus lactis subsp. lactis, Lactobacillus helveticus and Saccharomyces cerevisiae comprises the following steps:

[0056] S101. Lactococcus lactis subsp. lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae were cultured under appropriate conditions for 48 h to obtain respective mixed solutions and marked;

[0057] S102. Take 8 mL of the mixture and put it into a conical flask containing 100 mL of liquid culture medium, place it in a shaker at 180 rpm, dilute the concentrated bacterial suspension appropriately to make its optical density value 0.65, and obtain the time when the respective single colonies and the respective bacterial suspensions are in the logarithmic growth phase;

[0058] S103. The individual colonies were placed in a shaker and cultured at 180 rpm. Lactococcus lactis subsp. lactis was cultured for 16 h, Lactobacillus helveticus was cultured for 9 h, and Saccharomyces cerevisiae was cultured for 28 h. The supernatant was removed after centrifugation at 12,000 rpm and 5°C. The precipitate was washed with PBS solution and resuspended with sterile water to a final concentration of 10 7 CFU / mL, and obtain the corresponding bacterial suspensions of Lactococcus lactis subsp. lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae.

[0059] Example 3

[0060] A method for preparing a probiotic fermentation product based on American cockroach, characterized in that it comprises the following steps:

[0061] S1. Preparation of defatted powder of American cockroach: After the dried adult American cockroach is crushed, the fat is defatted by Soxhlet extraction, the insect powder is dried, and the defatted powder of American cockroach is obtained by passing through an 80-mesh sieve;

[0062] S2. American cockroach defatted powder was added to distilled water at a ratio of 1:9, and 7% glucose solution was added, sterilized at 125°C for 17 minutes, and cooled to room temperature to obtain a fermentation substrate;

[0063] S3. In a clean bench, 6% of the bacterial suspension of the corresponding strains of Lactococcus lactis subsp. lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae were added to the fermentation substrate in the order of Lactococcus lactis subsp. lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae, at a time interval of 24h, wherein the addition ratio of Lactococcus lactis subsp. lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae was 3:2:1 to obtain a sample of the inoculated bacterial suspension;

[0064] S4. The sample of the inoculated bacterial suspension was cultured at 31°C in a shaker at 170 rpm for 70 h to obtain a probiotic fermentation product based on American cockroaches;

[0065] S5. The fermentation product was centrifuged at 7550 rpm and 4°C for 12 min, and the obtained fermentation supernatant was filtered through a 0.22 μm filter membrane and used to determine the polypeptide content and in vitro antioxidant activity.

[0066] The preparation of the bacterial suspension of Lactococcus lactis subsp. lactis, Lactobacillus helveticus and Saccharomyces cerevisiae comprises the following steps:

[0067] S101. Lactococcus lactis subsp. lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae were cultured under appropriate conditions for 36 h to obtain respective mixed solutions and marked;

[0068] S102. Take 6 mL of the mixture and put it into a conical flask containing 100 mL of liquid culture medium, place it in a shaker at 170 rpm, dilute the concentrated bacterial suspension appropriately to make its optical density value 0.4, and obtain the time when the respective single colonies and the respective bacterial suspensions are in the logarithmic growth phase;

[0069] S103. The individual colonies were placed in a shaker and cultured at 170 rpm. Lactococcus lactis subsp. lactis was cultured for 16 h, Lactobacillus helveticus was cultured for 9 h, and Saccharomyces cerevisiae was cultured for 28 h. The supernatant was removed after centrifugation at 11000 rpm and 4°C. The precipitate was washed with PBS solution and resuspended with sterile water to a final concentration of 10 7 CFU / mL, and obtain the corresponding bacterial suspensions of Lactococcus lactis subsp. lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae.

[0070] Example 4

[0071] A method for preparing a probiotic fermentation product based on American cockroach, characterized in that it comprises the following steps:

[0072] S1. Preparation of defatted powder of American cockroach: After the dried adult American cockroach is crushed, the fat is defatted by Soxhlet extraction, the insect powder is dried, and the defatted powder of American cockroach is obtained by passing through an 80-mesh sieve;

[0073] S2. American cockroach defatted powder was added to distilled water at a ratio of 1:9, and 9% glucose solution was added, sterilized at 126°C for 19 minutes, and cooled to room temperature to obtain a fermentation substrate;

[0074] S3. In a clean bench, 7% of the bacterial suspension of the corresponding strains was added to the fermentation substrate in the order of Lactococcus lactis subsp. lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae, at a time interval of 24h, wherein Lactococcus lactis subsp. lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae were added in a ratio of 3:2:1 to obtain a sample of the inoculated bacterial suspension;

[0075] S4. The sample of the inoculated bacterial suspension was cultured at 32°C in a shaker at 175 rpm for 71 h to obtain a probiotic fermentation product based on American cockroaches;

[0076] S5. The fermentation product was centrifuged at 7800 rpm and 4°C for 14 min, and the obtained fermentation supernatant was filtered through a 0.22 μm filter membrane and used to determine the polypeptide content and in vitro antioxidant activity.

[0077] The preparation of the bacterial suspension of Lactococcus lactis subsp. lactis, Lactobacillus helveticus and Saccharomyces cerevisiae comprises the following steps:

[0078] S101. Lactococcus lactis subsp. lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae were cultured under appropriate conditions for 40 h to obtain their respective mixed solutions and marked;

[0079] S102. Take 7 mL of the mixture and put it into a conical flask containing 100 mL of liquid culture medium, place it in a shaker at 175 rpm, dilute the concentrated bacterial suspension appropriately to make its optical density value 0.55, and obtain the time when the respective single colonies and the respective bacterial suspensions are in the logarithmic growth phase;

[0080] S103. The individual colonies were placed in a shaker and cultured at 175 rpm. Lactococcus lactis subsp. lactis was cultured for 16 h, Lactobacillus helveticus was cultured for 9 h, and Saccharomyces cerevisiae was cultured for 28 h. The supernatant was removed after centrifugation at 11500 rpm and 4°C. The precipitate was washed with PBS solution and resuspended with sterile water to a final concentration of 10 7 CFU / mL, and obtain the corresponding bacterial suspensions of Lactococcus lactis subsp. lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae.

[0081] Comparative Example 1

[0082] Comparative Example 1 is different from Example 1 in that the three bacterial species are changed to two, namely, Lactobacillus helveticus and Lactococcus lactis subsp. lactis, and the addition ratio is 1:1. This comparative example is further named FPAG-1, and the remaining steps are exactly the same in Comparative Example 1 and Example 1.

[0083] Comparative Example 2

[0084] Comparative Example 2 is different from Example 1 in that the three bacterial species are changed to two, namely, Lactococcus lactis subsp. lactis and Saccharomyces cerevisiae, and the addition ratio is 1:1. This comparative example is further named FPAG-2, and the remaining steps are exactly the same in Comparative Example 2 and Example 1.

[0085] Comparative Example 3

[0086] Comparative Example 3 is different from Example 1 in that the three bacterial species are changed to two, namely, Lactobacillus helveticus and Saccharomyces cerevisiae, and the addition ratio is 1:1. This comparative example is further named FPAG-3, and the remaining steps are exactly the same in Comparative Example 3 and Example 1.

[0087] Comparative Example 4

[0088] Comparative Example 4 is different from Example 1 in that the ratio of Lactobacillus helveticus: Lactococcus lactis subsp. lactis: Saccharomyces cerevisiae is 1:1:1, and this comparative example is further named FPAG-4. The remaining steps in Comparative Example 4 and Example 1 are exactly the same.

[0089] Comparative Example 5

[0090] Comparative Example 5 is different from Example 1 in that no bacteria are added, and the comparative examples are further named Control-1, Control-2, Control-3, and Control-4. The remaining steps are exactly the same in Comparative Example 5 and Example 1.

[0091] Comparative Example 6

[0092] Comparative Example 6 is different from Example 1 in that the ratio of Lactobacillus helveticus: Lactococcus lactis subsp. lactis: Saccharomyces cerevisiae is 1:1:1, and this comparative example is further named FPAG-5. The remaining steps in Comparative Example 6 and Example 1 are exactly the same.

[0093] Comparative Example 7

[0094] Comparative Example 7 is different from Example 1 in that the ratio of Lactobacillus helveticus: Lactococcus lactis subsp. lactis: Saccharomyces cerevisiae is 1:2:2, and this comparative example is further named FPAG-6. The remaining steps in Comparative Example 7 and Example 1 are exactly the same.

[0095] Comparative Example 8

[0096] Comparative Example 8 is different from Example 1 in that the ratio of Lactobacillus helveticus: Lactococcus lactis subsp. lactis: Saccharomyces cerevisiae is 1:3:3, and this comparative example is further named FPAG-7. The remaining steps in Comparative Example 8 and Example 1 are exactly the same.

[0097] Comparative Example 9

[0098] Comparative Example 9 is different from Example 1 in that the ratio of Lactobacillus helveticus: Lactococcus lactis subsp. lactis: Saccharomyces cerevisiae is 2:1:2, and this comparative example is further named FPAG-8. The remaining steps in Comparative Example 9 and Example 1 are exactly the same.

[0099] Comparative Example 10

[0100] Comparative Example 10 is different from Example 1 in that the ratio of Lactobacillus helveticus: Lactococcus lactis subsp. lactis: Saccharomyces cerevisiae is 2:2:3, and this comparative example is further named FPAG-9. The remaining steps in Comparative Example 10 and Example 1 are exactly the same.

[0101] Comparative Example 11

[0102] Comparative Example 11 is different from Example 1 in that the ratio of Lactobacillus helveticus: Lactococcus lactis subsp. lactis: Saccharomyces cerevisiae is 3:1:3, and this comparative example is further named FPAG-11. The remaining steps in Comparative Example 11 and Example 1 are exactly the same.

[0103] Comparative Example 12

[0104] Comparative Example 12 is different from Example 1 in that the ratio of Lactobacillus helveticus: Lactococcus lactis subsp. lactis: Saccharomyces cerevisiae is 3:2:1, and this comparative example is further named FPAG-12. The remaining steps are exactly the same in Comparative Example 12 and Example 1.

[0105] Comparative Example 13

[0106] Comparative Example 13 is different from Example 1 in that the ratio of Lactobacillus helveticus: Lactococcus lactis subsp. lactis: Saccharomyces cerevisiae is 3:3:2, and this comparative example is further named FPAG-13. The remaining steps in Comparative Example 13 and Example 1 are exactly the same.

[0107] Comparative Example 14

[0108] Comparative Example 14 is different from Example 1 in that the order of adding the three strains is Lactobacillus helveticus, Lactococcus lactis subsp. lactis, and Saccharomyces cerevisiae, and this comparative example is further named FPAG-14. The remaining steps are exactly the same in Comparative Example 14 and Example 1.

[0109] Comparative Example 15

[0110] Comparative Example 15 is different from Example 1 in that the order of adding the three strains is Lactobacillus helveticus, Saccharomyces cerevisiae, and Lactococcus lactis subsp. lactis, and this comparative example is further named FPAG-15. The remaining steps are exactly the same in Comparative Example 15 and Example 1.

[0111] Comparative Example 16

[0112] Comparative Example 16 is different from Example 1 in that the order of adding the three strains is Lactococcus lactis subsp. lactis, Saccharomyces cerevisiae, and Lactobacillus helveticus, and this comparative example is further named FPAG-17, and the remaining steps are exactly the same in Comparative Example 16 and Example 1.

[0113] Comparative Example 17

[0114] Comparative Example 17 is different from Example 1 in that the order of adding the three strains is Saccharomyces cerevisiae, Lactococcus lactis subsp. lactis, and Lactobacillus helveticus, and this comparative example is further named FPAG-18, and the remaining steps are exactly the same in Comparative Example 17 and Example 1.

[0115] Comparative Example 18

[0116] Comparative Example 18 is different from Example 1 in that the order of adding the three strains is Saccharomyces cerevisiae, Lactobacillus helveticus, and Lactococcus lactis subsp. lactis, and this comparative example is further named FPAG-19, and the remaining steps are exactly the same in Comparative Example 18 and Example 1.

[0117] Performance Test:

[0118] 1. Determination of polypeptide content

[0119] Add 5 mL of fermentation supernatant to an equal volume of 10% trichloroacetic acid, mix thoroughly, let stand at room temperature for 20 minutes, centrifuge at 4000 rpm for 15 minutes, and retain the supernatant for the determination of polypeptide content. Add 0, 2, 4, 6, 8, 12, 16, 20 μl of BSA (bovine serum albumin) standard to a 96-well plate, and add PBS to make up to 20 μl. Dilute the sample appropriately and add 20 μl to the sample well of the 96-well plate. Add 200 μl of the prepared Folin phenol A reagent (dissolve 1g Na2CO3 in 50mL 0.2mol / L NaOH, then dissolve 0.5g CuSO4·5H2O in 100mL 1% sodium tartrate solution, then mix 50mL of the former with 1mL of the latter, prepare it before use) to each well, shake it gently, mix it, and leave it at room temperature for 10 minutes. Add 20 μl of Folin phenol B reagent (Solarbio, Beijing, China) to each well, mix it quickly, and leave it at 37℃ for 30 minutes. Use an ELISA reader to measure A650 and calculate the polypeptide content. The calculation formula is as follows:

[0120] X=(C×V×β) / m

[0121] X: polypeptide content in fermentation supernatant (mg / g)

[0122] C: Peptide concentration in the standard curve (mg / mL)

[0123] V: volume of fermentation supernatant used for determination (mL), 5 mL in this experiment

[0124] β: dilution factor, in this experiment it is 50 / 5×2=20

[0125] m: initial mass of fermentation sample (mg).

[0126] The test results are shown in Table 2.

[0127] 2. Determination of DPPH free radical scavenging ability in antioxidant activity assay

[0128] The DPPH free radical scavenging ability was determined by referring to the method of (Chen et al., 2021) with some modifications. 3 mL of the diluted fermentation supernatant was mixed with 3 mL of DPPH (2,2-biphenyl-1-picrylhydrazyl)-anhydrous ethanol solution (0.1 mol / L), and the mixture was reacted at room temperature in the dark for 1 h. The absorbance was immediately measured at 517 nm. The DPPH free radical scavenging ability (%) was calculated as follows:

[0129] DPPH free radical scavenging ability (%) = (1-A1 / A0) × 100

[0130] Where A0 is the blank absorbance and A1 is the sample absorbance.

[0131] The test results are shown in Table 2.

[0132] 3. Determination of superoxide anion free radical scavenging ability in antioxidant activity assay

[0133] Take a 1.5mL brown centrifuge tube, add 300μl NADH (936μmol / L, nicotinamide adenine dinucleotide, reduced coenzyme I disodium salt), then add 300μl NBT (336μmol / L, nitro blue tetrazolium chloride), then add 30μl fermentation supernatant, mix well, finally add PMS (120μmol / L, N-methylphenazine methyl sulfate), mix well, and immediately measure the absorbance at 560nm after 5 minutes at room temperature away from light. The calculation formula is as follows:

[0134] Superoxide anion free radical scavenging ability (%) = (1-A1 / A0) × 100

[0135] Where A0 is the blank absorbance and A1 is the sample absorbance.

[0136] The test results are shown in Table 2.

[0137] 4. Detection of superoxide dismutase activity in antioxidant activity assay

[0138] Superoxide dismutase activity was detected using a Solebol kit. Before the assay, the working solutions of reagents 1, 3, and 4 were placed in a 37°C water bath for more than 5 minutes, and the reagents were added to the 96-well plate according to the following table:

[0139] Table 1: Sample loading table for superoxide dismutase activity assay

[0140] Reagent Name (μl) Determination tube Control tube Blank tube 1 Blank tube 2 sample 20 20 / / Reagent 1 45 45 45 45 Reagent 2 working solution 20 / 20 / Reagent 3 35 35 35 35 Distilled water 70 90 90 110 Reagent 4 working solution 10 10 10 10

[0141] Mix thoroughly, place in a 37°C water bath for 30 min, and measure the absorbance at 450 nm. The calculation formula is as follows:

[0142] Inhibition percentage X = [(A1-A2)-(A3-A4)] / (A1-A2)*100

[0143] Superoxide dismutase activity test (U / mL) = [X / (1-X)*V1] / V2*β

[0144] X: Inhibition percentage (%)

[0145] A1: Blank 1 absorbance

[0146] A2: Blank 2 absorbance

[0147] A3: Sample tube absorbance

[0148] A4: Absorbance of sample control tube

[0149] V1: total reaction volume, 0.2 mL

[0150] V2: The volume of sample added to the reaction system, 0.02 mL

[0151] β: sample dilution factor.

[0152] The test results are shown in Table 2 below:

[0153] The determination results of polypeptide content (mg / g), DPPH free radical scavenging rate (%), superoxide anion free radical scavenging rate (%) and superoxide dismutase activity (U / mL) of fermentation products in different groups.

[0154]

[0155]

[0156] Depend on Figure 2 As shown in Table 2, compared with the control group (Conture-1), the mixed fermentation of microbial strains significantly improved the polypeptide content, DPPH free radical scavenging ability, superoxide anion free radical scavenging ability, and superoxide dismutase activity of the American cockroach fermentation product. Among them, the FPAG-4 group, i.e., the compounding ratio of Lactobacillus helveticus: Lactococcus lactis subsp. lactis: Saccharomyces cerevisiae, was 1:1:1, and the values ​​of polypeptide content, DPPH free radical scavenging ability, superoxide anion free radical scavenging ability, and superoxide dismutase activity were significantly higher than those of other fermentation groups, which were 114.01±7.32mg / g, 54.84±0.32%, 47.77±0.73%, and 31.32±1.61U / mL, respectively. Therefore, three kinds of bacteria were selected for mixed fermentation.

[0157] Subsequently, the optimal ratio of the three bacteria for mixed fermentation was explored, and the results were as follows: Figure 3As shown in Table 2, when the composite ratio of Lactobacillus helveticus: Lactococcus lactis subsp. lactis: Saccharomyces cerevisiae was 2:3:1, i.e., the FPAG-10 group, the values ​​of polypeptide content, DPPH free radical scavenging ability, superoxide anion free radical scavenging ability, and superoxide dismutase activity of the American cockroach fermentation product were significantly higher than those of other fermentation groups, which were 195.53±4.49mg / g, 85.29±0.09%, 93.05±0.35%, and 30.39±1.92U / mL, respectively, so this composite ratio was selected.

[0158] Finally, in the optimization experiment of the order of adding bacteria, Figure 4 As shown in Table 2, when the FPAG-16 group, i.e., the order of strain addition was "Lactococcus lactis subsp. lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae", the values ​​of polypeptide content, DPPH free radical scavenging ability, superoxide anion free radical scavenging ability, and superoxide dismutase activity of the American cockroach fermentation product were significantly higher than those of other fermentation groups, which were 162.57±6.77 mg / g, 85.69±0.19%, 94.32±0.11%, and 46.52±1.06 U / mL, respectively, so this strain addition order was selected.

[0159] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a probiotic fermentation product based on American cockroach, characterized in that: The following steps are involved: S1. Preparation of defatted powder of American cockroach: After the dried adult American cockroach is crushed, the fat is defatted by Soxhlet extraction, the insect powder is dried, and the defatted powder of American cockroach is obtained by passing through an 80-mesh sieve; S2. Add distilled water to the defatted powder of American cockroach, add 5-10% glucose solution, sterilize at high temperature, and cool to room temperature to obtain a fermentation substrate; S3. In a clean bench, 5-8% of the bacterial suspension of the corresponding strains of Lactococcus lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae were added to the fermentation substrate in the order of Lactococcus lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae, and the time interval for addition was 24h to obtain a sample of the inoculated bacterial suspension; S4. The sample of the inoculated bacterial suspension was cultured in a shaker at a temperature of 30-33 ° C for 68-72 h to obtain a probiotic fermentation product based on American cockroaches; Wherein, the ratio of the bacterial suspensions corresponding to Lactococcus lactis subsp. lactis, Lactobacillus helveticus and Saccharomyces cerevisiae is 3:2:

1.

2. The method for preparing a probiotic fermentation product based on American cockroach according to claim 1, characterized in that: In step S3, the preparation of the bacterial suspension of Lactococcus lactis subsp. lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae comprises the following steps: S101. Lactococcus lactis subsp. lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae are cultured under appropriate conditions for 24-48 h to obtain respective mixed solutions and marked; S102. Take 5-8 mL of the mixture and put it into a conical flask containing 100 mL of liquid culture medium, place it in a shaker at 165-180 rpm, dilute the concentrated bacterial suspension appropriately to make its optical density value between 0.1 and 0.65, and obtain the time when the respective single colonies and the respective bacterial suspensions are in the logarithmic growth phase; S103. The respective single colonies were placed in a shaker for cultivation. Lactococcus lactis subsp. lactis was cultured for 16 h, Lactobacillus helveticus was cultured for 9 h, and Saccharomyces cerevisiae was cultured for 28 h. The supernatant was then removed by centrifugation, and the precipitate was washed with PBS solution and resuspended with sterile water to a final concentration of 10 7 CFU / mL, and obtain the corresponding bacterial suspensions of Lactococcus lactis subsp. lactis, Lactobacillus helveticus, and Saccharomyces cerevisiae.

3. The method for preparing a probiotic fermentation product based on American cockroach according to claim 2, characterized in that: In step S101, the respective suitable conditions are: Lactobacillus helveticus adopts MRS medium, the culture temperature is 37°C, and the pH value is 6.5; Lactococcus lactis subsp. lactis adopts M17 medium, the culture temperature is 30°C, and the pH value is 7.0; Saccharomyces cerevisiae adopts YPD medium, the culture temperature is 28°C, and the pH value is 5.

6.

4. The method for preparing a probiotic fermentation product based on American cockroach according to claim 2, characterized in that: In step S103, the components of the PBS solution are: 0.24-0.3 parts of potassium dihydrogen phosphate, 3.6-4 parts of disodium hydrogen phosphate dodecahydrate, 8-10 parts of sodium chloride, 0.2-0.4 parts of potassium chloride and 800-1000 parts of distilled water.

5. The method for preparing a probiotic fermentation product based on American cockroach according to claim 2, characterized in that: In step S103, the rotation speed of the shaking incubator is 165-180 rpm.

6. The method for preparing a probiotic fermentation product based on American cockroach according to claim 2, characterized in that: In step S103, the centrifugal speed is 10000-12000 rpm and the temperature is 3-5°C.

7. The method for preparing a probiotic fermentation product based on American cockroach according to claim 1, characterized in that: In step S2, the ratio of the defatted powder of American cockroach to distilled water is 1:8-10.

8. The method for preparing a probiotic fermentation product based on American cockroach according to claim 1, characterized in that: In step S2, the temperature of high temperature sterilization is 121-130°C and the time is 15-20 minutes.

9. The method for preparing a probiotic fermentation product based on American cockroach according to claim 1, characterized in that: The rotation speed of the shaking table in step S4 is 165-180 rpm.

10. Use of the probiotic fermentation product of American cockroach obtained by the preparation method according to any one of claims 1 to 9 in anti-oxidation.

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