Complex microbial inoculant for producing beta-glucuronidase and application of complex microbial inoculant in calculus bovis production

Abstract

The invention provides a complex microbial inoculant for producing beta-glucuronidase and application of the complex microbial inoculant in calculus bovis production, and relates to the technical field of microorganisms. The culture method of the complex microbial inoculant provided by the invention comprises the following steps: culturing lactobacillus ATCC 53103 in an initial culture medium to a logarithmic phase, sterilizing to obtain a complex culture medium, mixing the complex culture medium with nutritional ingredients, inoculating escherichia coli ATCC 25922 and riemerella CCUG 14215, and co-culturing to obtain the complex microbial inoculant. The complex microbial inoculant provided by the invention can generate a large amount of high-activity beta-glucuronidase, so that the efficiency of in-vitro production of calculus bovis can be effectively improved.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a composite bacterial agent for producing beta-glucuronidase and application thereof in bezoar production. Background Art

[0002] Bezoar is a precious Chinese herbal medicine with extremely high medicinal value. It plays an important role in the field of traditional Chinese medicine. It has significant therapeutic effects in treating fever, coma, stroke, epilepsy and convulsions. Its unique medicinal value and scarce production make it the focus of much attention in the medical field. At the same time, with the development of modern medicine, the medicinal value of bezoar has been continuously explored and expanded, and it also has broad prospects in new drug research and development and clinical treatment.

[0003] However, the production of natural bezoar is very effective, and the formation process of natural bezoar is extremely complex and long, relying on the specific physiological conditions of bovine animals, which makes the production of natural bezoar difficult to predict and control, and difficult to meet the huge market demand. Therefore, the State Food and Drug Administration has successively approved three natural bezoar substitutes: artificial bezoar, cultured bezoar and in vitro cultured bezoar. Among them, the efficacy of artificial bezoar is too different from that of natural bezoar. Cultivated bezoar requires raising a large number of bovine animals for cultivation, which is relatively inefficient. In vitro cultured bezoar has been widely used with the development of microbial technology.

[0004] The Chinese patent with publication number CN117562927A provides a preparation process for in vitro cultivation of bezoar, which uses β-glucuronidase and fermentation method to simulate the environment in which bezoar is produced in cattle bile, and cultivates bezoar in constant temperature, constant pH and constant oxygen. Among them, β-glucuronidase can specifically catalyze the hydrolysis reaction of glucuronic acid bond in bilirubin, thereby converting conjugated bilirubin into free bilirubin. This process is the core step of bezoar cultivation.

[0005] Therefore, β-glucuronidase is crucial in the cultivation process of bezoar. However, the above patent only selects suitable strains for high-density culture to increase the production of β-glucuronidase, which has an upper limit on production and is difficult to carry out large-scale production. Therefore, it is urgent to provide a solution to improve the above problem. Summary of the invention

[0006] The purpose of the present invention is to provide a composite bacterial agent for producing beta-glucuronidase and application of the composite bacterial agent in bezoar production.

[0007] In a first aspect, the present invention provides a composite bacterial agent for producing β-glucuronidase, wherein the culturing method of the composite bacterial agent comprises: culturing Lactobacillus ATCC 53103 in an initial culture medium to a logarithmic growth phase and then sterilizing to obtain a composite culture medium, mixing the composite culture medium with nutrients, and then inoculating Escherichia coli ATCC 25922 and Merrella sp. CCUG 14215 for co-culturing to obtain the composite bacterial agent.

[0008] In the culturing process of the composite bacterial agent provided by the present invention, lactobacillus ATCC 53103 is preliminarily cultured in the initial culture medium, and the metabolites of lactobacillus ATCC 53103 can be left in the composite culture medium, thereby facilitating the subsequent co-culturing of Escherichia coli ATCC 25922 and Merkel cell culture CCUG 14215. At the same time, in the co-culturing process of Escherichia coli ATCC25922 and Merkel cell culture CCUG 14215, Merkel cell culture CCUG 14215 can provide a powerful auxiliary environment for the growth of Escherichia coli ATCC25922, promote the absorption of the metabolites of lactobacillus ATCC 53103 by Escherichia coli ATCC 25922, and thus promote the production of β-glucuronidase by the composite bacterial agent.

[0009] Optionally, the viable count of Escherichia coli ATCC 25922 in the composite bacterial agent is 10 8 -10 9 CFU / mL, the viable count of the Merkel cell culture medium CCUG 14215 is 10 5 -10 7 By setting E. coli ATCC 25922 as the dominant bacterium, the growth competition of E. coli ATCC 25922 by Merrella sp. CCUG 14215 can be effectively avoided.

[0010] Optionally, the initial culture medium includes 8-10 g / L tryptone, 8-10 g / L beef extract, 3-6 g / L yeast extract, 15-20 g / L glucose, 1-2 g / L dimethyl hydrogen phosphate, 1-2 g / L potassium dihydrogen phosphate, and 1-2 g / L triammonium citrate.

[0011] Optionally, Lactobacillus ATCC 53103 is cultured in an initial culture medium at 35-40° C. in an anaerobic environment until the logarithmic growth phase, and the composite culture medium is prepared by centrifuging and collecting the supernatant after pasteurization.

[0012] Optionally, when the complex culture medium is mixed with nutrients, the nutrients include yeast extract, malt extract, tryptone, sodium chloride, vitamin B1, calcium salt, and glucose.

[0013] Optionally, after the composite culture medium is mixed with nutrients, it includes, by mass concentration, 6-10 g / L yeast extract, 8-10 g / L malt extract, 8-10 g / L tryptone, 8-10 g / L sodium chloride, 1-5 mg / L vitamin B1, 0.1-0.5 mg / L calcium salt and 1-5 g / L glucose.

[0014] Optionally, when inoculating Escherichia coli ATCC 25922 and Merkel cell culture CCUG 14215, the method includes: pre-inoculating Escherichia coli ATCC 25922 and culturing it at 35-40°C for 8-12 hours, and then inoculating Merkel cell culture CCUG 14215 and culturing it at 30-32°C, 5-8 vol.% oxygen content, and pH 6.0-6.5 for 10-12 hours to prepare the composite bacterial agent. By culturing Escherichia coli ATCC 25922 in advance, Escherichia coli ATCC 25922 can be grown alone and quickly in the composite culture medium, and Escherichia coli ATCC 25922 can be pre-adapted to the metabolites of lactobacillus, and after the subsequent addition of Merkel cell culture CCUG14215, the absorption and utilization of the metabolites of lactobacillus by Escherichia coli ATCC 25922 can be further promoted.

[0015] Optionally, the prepared composite bacterial agent is stored at 4°C.

[0016] In a second aspect, the present invention further provides a use of any of the above optional composite bacterial agents in bezoar production, wherein the composite bacterial agent is used to produce β-glucuronidase. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are 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. Unless otherwise defined, the technical terms or scientific terms used herein should be the common meanings understood by people with general skills in the field to which the present invention belongs.

[0018] The invention provides a composite bacterial agent, which can efficiently produce beta-glucuronidase with high enzyme activity. The culturing method comprises: culturing lactobacillus ATCC 53103 in an initial culture medium to a logarithmic growth phase and then sterilizing to obtain a composite culture medium; mixing the composite culture medium with nutrients, inoculating Escherichia coli ATCC 25922 and Merrella vulgaris CCUG 14215, and then co-culturing to obtain the composite bacterial agent.

[0019] In fact, during the cultivation of the composite bacterial agent, Lactobacillus ATCC 53103 can convert sugars into lactic acid through the glycolysis pathway, thereby adjusting the pH of the composite culture medium to a slightly acidic environment, thereby providing a suitable environment for the growth of Escherichia coli ATCC 25922. At the same time, Lactobacillus ATCC 53103 can transform the nutrients in the culture medium and excrete its own metabolites into the composite culture medium, which is beneficial to the utilization of metabolites by Escherichia coli ATCC 25922 and stimulates the production of β-glucuronidase.

[0020] In addition, during the cultivation process of the composite bacterial agent, when Escherichia coli ATCC 25922 and Bacillus subtilis CCUG 14215 are co-cultured, Bacillus subtilis CCUG 14215 can absorb and decompose the sugar components in the culture medium, thereby promoting the utilization of lipid substances by Escherichia coli ATCC 25922, which helps to metabolize and synthesize β-glucuronidase. At the same time, Bacillus subtilis CCUG14215 produces antioxidants, antibiotics and other biologically active substances in the metabolic process, which can effectively reduce oxidation in the culture environment, thereby providing a stable living environment for Escherichia coli ATCC 25922, allowing Escherichia coli ATCC 25922 to maintain a high activity, thereby better synthesizing β-glucuronidase. In addition, antioxidants can also prevent Escherichia coli ATCC25922 from oxidative damage and enhance the activity of β-glucuronidase.

[0021] Therefore, by culturing Lactobacillus ATCC 53103 in advance and collecting its metabolites, and co-culturing Escherichia coli ATCC 25922 and Merrella sp. CCUG 14215, it is beneficial to promote the growth of Escherichia coli ATCC 25922 and stimulate its production of β-glucuronidase.

[0022] In some embodiments, after the composite bacterial agent is prepared by co-cultivation, the number of viable Escherichia coli ATCC25922 in the composite bacterial agent is 10 8 -10 9 CFU / mL, the viable count of Merrella CCUG 14215 is 10 5 -10 7 In fact, by determining that Escherichia coli ATCC 25922 is in a dominant position in the composite bacterial agent, it is possible to promote the growth and metabolism of Escherichia coli ATCC 25922, and obtain sufficient nutrients to support its own metabolism through growth competition.

[0023] In fact, by adjusting the amount of Bacillus merkelii CCUG 14215 in the composite microbial agent, the enzymatic activity of Escherichia coli ATCC 25922 in producing β-glucuronidase can be adjusted when the composite microbial agent is producing β-glucuronidase. Specifically, when the content of Bacillus merkelii CCUG 14215 in the composite microbial agent is too low, it is difficult to achieve a good promotion effect during the culture process, and it is easy to be squeezed out by Escherichia coli ATCC 25922 and thus difficult to grow. When the content of Bacillus merkelii CCUG 14215 is too high, it is easy to over-compete with Escherichia coli ATCC 25922, affecting the growth metabolism of Escherichia coli ATCC 25922.

[0024] In some embodiments, after co-cultivation to obtain the composite bacterial agent, the live counts of Escherichia coli ATCC25922 and Merkel cell culture medium CCUG 14215 in the composite bacterial agent are detected. When the live count of Merkel cell culture medium CCUG 14215 does not meet the standard, an appropriate amount of Merkel cell culture medium CCUG 14215 can be added thereto. When the live count of Escherichia coli ATCC 25922 does not meet the standard, co-cultivation must be performed again.

[0025] In fact, when culturing Lactobacillus ATCC 53103, the initial culture medium used includes 8-10 g / L tryptone, 8-10 g / L beef extract, 3-6 g / L yeast extract, 15-20 g / L glucose, 1-2 g / L dimethyl hydrogen phosphate, 1-2 g / L potassium dihydrogen phosphate, and 1-2 g / L triammonium citrate. Specifically, the initial culture medium can use a liquid culture medium, which is necessary to be able to culture Lactobacillus ATCC 53103.

[0026] In fact, the culture products obtained after culturing in the initial culture medium include unconsumed tryptone, beef extract, yeast extract, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and triammonium citrate, as well as metabolites produced by the metabolism of Lactobacillus ATCC 53103 and the contents discharged from Lactobacillus ATCC 53103 after sterilization.

[0027] In some embodiments, when culturing Lactobacillus ATCC 53103, Lactobacillus ATCC 53103 is inoculated into a pre-sterilized initial culture medium, and transferred to an incubator at 35-40° C. for shake flask culture until the logarithmic growth phase of Lactobacillus ATCC 53103, and then pasteurized to kill Lactobacillus ATCC 53103, centrifuged and collected the supernatant to prepare a composite culture medium. In fact, when the initial culture medium is a solid culture medium, deionized water is added as a liquid medium after sterilization, and the culture medium is stirred and crushed, and then centrifuged and collected to prepare a composite culture medium.

[0028] In fact, when culturing Lactobacillus ATCC 53103, it can be activated by using a strain activation method commonly used in the art. In fact, Lactobacillus ATCC 53103 can be purchased from Hangzhou Baosai Biotechnology Co., Ltd.

[0029] In some embodiments, when the composite culture medium is mixed with nutrients, the nutrients include yeast extract, malt extract, tryptone, sodium chloride, vitamin B1, calcium salt, and glucose. In fact, the added nutrients are mainly used to supplement the nutrients consumed in the composite culture medium, thereby facilitating the growth and metabolism of Escherichia coli ATCC 25922 and Merrella sp. CCUG14215.

[0030] Specifically, after the composite culture medium is mixed with nutrients, the mass concentration includes 6-10 g / L yeast extract, 8-10 g / L malt extract, 8-10 g / L tryptone, 8-10 g / L sodium chloride, 1-5 mg / L vitamin B1, 0.1-0.5 mg / L calcium salt and 1-5 g / L glucose.

[0031] In fact, when inoculating Escherichia coli ATCC 25922 and Merkel cell culture CCUG 14215, it includes: pre-inoculating Escherichia coli ATCC 25922 and culturing it in an aerobic environment at 35-40°C for 8-12 hours, and then inoculating Merkel cell culture CCUG 14215 and co-culturing it at 30-32°C, 5-8 vol.% oxygen content, and pH 6.0-6.5 for 10-12 hours to obtain a composite bacterial agent. Specifically, after the composite bacterial agent is obtained, it is stored at a low temperature of 4°C. In fact, Merkel cell culture CCUG 14215 was purchased from Wuhan Warner Biotechnology Co., Ltd.

[0032] The present invention also provides an application of the composite bacterial agent in any of the above optional embodiments in the production of bezoar, wherein the composite bacterial agent is used to produce β-glucuronidase.

[0033] Example 1

[0034] This embodiment 1 provides a composite bacterial agent for producing β-glucuronidase, and the culturing method of the composite bacterial agent comprises the following steps:

[0035] S1. Activated Lactobacillus ATCC 53103 was added to 10 6cfu / g density was inoculated into the initial culture medium, and after shaking flask culture in an incubator in an anaerobic environment at 37°C for 5 hours, the culture was statically cultured until the logarithmic growth phase, and then sterilized at 120°C and a pressure of 2MPa, 20mL of deionized water was added and stirred, and then centrifuged at a speed of 300rpm to collect the supernatant to obtain a composite culture medium; wherein the initial culture medium includes 8g / L tryptone, 8g / L beef extract, 5g / L yeast extract, 17g / L glucose, 1g / L dimethyl hydrogen phosphate, 1g / L potassium dihydrogen phosphate, and 12g / L triammonium citrate;

[0036] S2, stirring and mixing the composite culture medium with yeast extract, malt extract, tryptone, sodium chloride, vitamin B1, calcium salt, and glucose, and adjusting the obtained culture solution to include 8 g / L yeast extract, 8 g / L malt extract, 10 g / L tryptone, 8 g / L sodium chloride, 3 mg / L vitamin B1, 0.3 mg / L calcium salt, and 3 g / L glucose in terms of mass concentration to prepare a mixed culture solution;

[0037] S3, inoculate Escherichia coli ATCC 25922 into the mixed culture solution in advance, culture it in an environment of 37°C and 2 vol.% O2 content for 10 hours, then inoculate Merkel cell culture medium CCUG 14215 and adjust the environmental parameters to 30°C, 5 vol.% O2 content, and pH 6.5, culture it for 48 hours, and then refrigerate it at 4°C to obtain a composite bacterial agent; wherein the composite bacterial agent includes 3.8×108 CFU / mL Escherichia coli ATCC 25922 and 2.4×10 6 CFU / mL of Merrelliella CCUG 14215.

[0038] Comparative Example 1

[0039] This comparative example 1 provides a composite bacterial agent for producing β-glucuronidase, and the culturing method of the composite bacterial agent comprises the following steps:

[0040] S1. Activated Lactobacillus ATCC 53103 was added to 10 6 cfu / g density was inoculated into the initial culture medium, and after shaking flask culture in an incubator in an anaerobic environment at 37°C for 5 hours, the culture was statically cultured until the logarithmic growth phase, and then sterilized at 120°C and a pressure of 2MPa, 20mL of deionized water was added and stirred, and then centrifuged at a speed of 300rpm to collect the supernatant to obtain a composite culture medium; wherein the initial culture medium includes 8g / L tryptone, 8g / L beef extract, 5g / L yeast extract, 17g / L glucose, 1g / L dimethyl hydrogen phosphate, 1g / L potassium dihydrogen phosphate, and 12g / L triammonium citrate;

[0041] S2, stirring and mixing the composite culture medium with yeast extract, malt extract, tryptone, sodium chloride, vitamin B1, calcium salt, and glucose, and adjusting the obtained culture solution to include 8 g / L yeast extract, 8 g / L malt extract, 10 g / L tryptone, 8 g / L sodium chloride, 3 mg / L vitamin B1, 0.3 mg / L calcium salt, and 3 g / L glucose in terms of mass concentration to prepare a mixed culture solution;

[0042] S3, inoculate Escherichia coli ATCC 25922 into the mixed culture solution in advance, culture it at 37°C and 2 vol.% O2 content environment for 22 hours, and then refrigerate it at 4°C to obtain a composite bacterial agent; wherein the composite bacterial agent includes 4.6×10 8 CFU / mL Escherichia coli ATCC 25922.

[0043] Comparative Example 2

[0044] Comparative Example 2 provides a composite bacterial agent for producing β-glucuronidase, and the culturing method of the composite bacterial agent comprises the following steps: inoculating Escherichia coli ATCC 25922 into a culture solution in advance, culturing for 12 hours in an environment of 37°C and 2 vol.% O2 content, then inoculating Merrella CCUG 14215 and adjusting the environmental parameters to 32°C, 5 vol.% O2 content, and pH 6.5, culturing for 12 hours, and refrigerating and storing at 4°C to obtain a composite bacterial agent; wherein the composite bacterial agent comprises 5.1×10 8 CFU / mL Escherichia coli ATCC 25922 and 3.5×10 6 CFU / mL of Merrelliella CCUG 14215; wherein the culture solution includes, by mass concentration, 8g / L yeast extract, 8g / L malt extract, 10g / L tryptone, 8g / L sodium chloride, 3mg / L vitamin B1, 0.3mg / L calcium salt and 3g / L glucose.

[0045] Comparative Example 3

[0046] This comparative example 3 provides a composite bacterial agent for producing β-glucuronidase, and the culturing method of the composite bacterial agent comprises the following steps:

[0047] S1, activated Lactobacillus CICC 6252 (purchased from China Industrial Microbiological Culture Collection Management Center) was added to 10 6cfu / g density was inoculated into the initial culture medium, and after shaking flask culture in an incubator in an anaerobic environment at 37°C for 5 hours, the culture was statically cultured until the logarithmic growth phase, and then sterilized at 120°C and a pressure of 2MPa, 20mL of deionized water was added and stirred, and then centrifuged at a speed of 300rpm to collect the supernatant to obtain a composite culture medium; wherein the initial culture medium includes 8g / L tryptone, 8g / L beef extract, 5g / L yeast extract, 17g / L glucose, 1g / L dimethyl hydrogen phosphate, 1g / L potassium dihydrogen phosphate, and 12g / L triammonium citrate;

[0048] S2, stirring and mixing the composite culture medium with yeast extract, malt extract, tryptone, sodium chloride, vitamin B1, calcium salt, and glucose, and adjusting the obtained culture solution to include 8 g / L yeast extract, 8 g / L malt extract, 10 g / L tryptone, 8 g / L sodium chloride, 3 mg / L vitamin B1, 0.3 mg / L calcium salt, and 3 g / L glucose in terms of mass concentration to prepare a mixed culture solution;

[0049] S3, inoculate Escherichia coli ATCC 25922 onto a solid culture medium in advance, culture it at 37°C and 2 vol.% O2 content for 10 h, then inoculate Merrella solani CCUG 14215 and adjust the environmental parameters to 30°C, 5 vol.% O2 content, pH 6.5, culture it for 12 h, and then refrigerate it at 4°C to obtain a composite bacterial agent; wherein the composite bacterial agent includes 4.2×10 8 CFU / mL Escherichia coli ATCC 25922 and 3.8×10 6 CFU / mL of Merrelliella CCUG 14215.

[0050] Comparative Example 4

[0051] This comparative example 4 provides a composite bacterial agent for producing β-glucuronidase, and the culturing method of the composite bacterial agent comprises the following steps:

[0052] S1, activated Lactobacillus CICC 6075 (purchased from China Industrial Microbiological Culture Collection Management Center) was added to 10 6 cfu / g density was inoculated into the initial culture medium, and after shaking flask culture in an incubator in an anaerobic environment at 37°C for 5 hours, the culture was statically cultured until the logarithmic growth phase, and then sterilized at 120°C and a pressure of 2MPa, 20mL of deionized water was added and stirred, and then centrifuged at a speed of 300rpm to collect the supernatant to obtain a composite culture medium; wherein the initial culture medium includes 8g / L tryptone, 8g / L beef extract, 5g / L yeast extract, 17g / L glucose, 1g / L dimethyl hydrogen phosphate, 1g / L potassium dihydrogen phosphate, and 12g / L triammonium citrate;

[0053] S2, stirring and mixing the composite culture medium with yeast extract, malt extract, tryptone, sodium chloride, vitamin B1, calcium salt, and glucose, and adjusting the obtained culture solution to include 8 g / L yeast extract, 8 g / L malt extract, 10 g / L tryptone, 8 g / L sodium chloride, 3 mg / L vitamin B1, 0.3 mg / L calcium salt, and 3 g / L glucose in terms of mass concentration to prepare a mixed culture solution;

[0054] S3, inoculate Escherichia coli ATCC 25922 into the mixed culture solution in advance, culture it in an environment of 37°C and 2 vol.% O2 content for 10 hours, then inoculate Merkel cell culture medium CCUG 14215 and adjust the environmental parameters to 30°C, 5 vol.% O2 content, and pH 6.5, culture it for 12 hours, and then refrigerate it at 4°C to obtain a composite bacterial agent; wherein the composite bacterial agent includes 4.8×108 CFU / mL Escherichia coli ATCC 25922 and 3.3×10 6 CFU / mL of Merrelliella CCUG 14215.

[0055] Comparative Example 5

[0056] This comparative example 5 provides a composite bacterial agent for producing β-glucuronidase, and the culturing method of the composite bacterial agent comprises the following steps:

[0057] S1, activated Lactobacillus CICC 23140 (purchased from China Industrial Microbiological Culture Collection Management Center) was added at 10 6 cfu / g density was inoculated into the initial culture medium, and after shaking flask culture in an incubator in an anaerobic environment at 37°C for 5 hours, the culture was statically cultured until the logarithmic growth phase, and then sterilized at 120°C and a pressure of 2MPa, 20mL of deionized water was added and stirred, and then centrifuged at a speed of 300rpm to collect the supernatant to obtain a composite culture medium; wherein the initial culture medium includes 8g / L tryptone, 8g / L beef extract, 5g / L yeast extract, 17g / L glucose, 1g / L dimethyl hydrogen phosphate, 1g / L potassium dihydrogen phosphate, and 12g / L triammonium citrate;

[0058] S2, stirring and mixing the composite culture medium with yeast extract, malt extract, tryptone, sodium chloride, vitamin B1, calcium salt, and glucose, and adjusting the obtained culture solution to include 8 g / L yeast extract, 8 g / L malt extract, 10 g / L tryptone, 8 g / L sodium chloride, 3 mg / L vitamin B1, 0.3 mg / L calcium salt, and 3 g / L glucose in terms of mass concentration to prepare a mixed culture solution;

[0059] S3, inoculate Escherichia coli ATCC 25922 into the mixed culture solution in advance, culture it in an environment of 37°C and 2 vol.% O2 content for 10 hours, then inoculate Merkel cell culture medium CCUG 14215 and adjust the environmental parameters to 30°C, 5 vol.% O2 content, and pH 6.5, culture it for 12 hours, and then refrigerate it at 4°C to obtain a composite bacterial agent; wherein the composite bacterial agent includes 5.2×108 CFU / mL Escherichia coli ATCC 25922 and 4.2×10 6 CFU / mL of Merrelliella CCUG 14215.

[0060] Comparative Example 6

[0061] This comparative example 6 provides a composite bacterial agent for producing β-glucuronidase, and the culturing method of the composite bacterial agent comprises the following steps:

[0062] S1. Activated Lactobacillus ATCC 53103 was added to 10 6 cfu / g density was inoculated into the initial culture medium, and after shaking flask culture in an incubator in an anaerobic environment at 37°C for 5 hours, the culture was statically cultured until the logarithmic growth phase, and then sterilized at 120°C and a pressure of 2MPa, 20mL of deionized water was added and stirred, and then centrifuged at a speed of 300rpm to collect the supernatant to obtain a composite culture medium; wherein the initial culture medium includes 8g / L tryptone, 8g / L beef extract, 5g / L yeast extract, 17g / L glucose, 1g / L dimethyl hydrogen phosphate, 1g / L potassium dihydrogen phosphate, and 12g / L triammonium citrate;

[0063] S2, stirring and mixing the composite culture medium with yeast extract, malt extract, tryptone, sodium chloride, vitamin B1, calcium salt, and glucose, and adjusting the obtained culture solution to include 8 g / L yeast extract, 8 g / L malt extract, 10 g / L tryptone, 8 g / L sodium chloride, 3 mg / L vitamin B1, 0.3 mg / L calcium salt, and 3 g / L glucose in terms of mass concentration to prepare a mixed culture solution;

[0064] S3, inoculate Escherichia coli ATCC 25922 into the mixed culture solution in advance, culture it in an environment of 37°C and 2 vol.% O2 content for 10 hours, then inoculate Merkel cell culture medium CGMCC No.6399 and adjust the environmental parameters to 30°C, 5 vol.% O2 content, and pH 6.5, culture it for 12 hours, and then refrigerate it at 4°C to obtain a composite bacterial agent; wherein the composite bacterial agent includes 3.8×108 CFU / mL Escherichia coli ATCC 25922 and 2.4×10 6 CFU / mL of Merrelliella CGMCC No.6399.

[0065] Comparative Example 7

[0066] This comparative example 7 provides a composite bacterial agent for producing β-glucuronidase, and the culturing method of the composite bacterial agent comprises the following steps:

[0067] S1. Activated Lactobacillus ATCC 53103 was added to 10 6 cfu / g density was inoculated into the initial culture medium, and after shaking flask culture in an incubator in an anaerobic environment at 37°C for 5 hours, the culture was statically cultured until the logarithmic growth phase, and then sterilized at 120°C and a pressure of 2MPa, 20mL of deionized water was added and stirred, and then centrifuged at a speed of 300rpm to collect the supernatant to obtain a composite culture medium; wherein the initial culture medium includes 8g / L tryptone, 8g / L beef extract, 5g / L yeast extract, 17g / L glucose, 1g / L dimethyl hydrogen phosphate, 1g / L potassium dihydrogen phosphate, and 12g / L triammonium citrate;

[0068] S2, stirring and mixing the composite culture medium with yeast extract, malt extract, tryptone, sodium chloride, vitamin B1, calcium salt, and glucose, and adjusting the obtained culture solution to include 8 g / L yeast extract, 8 g / L malt extract, 10 g / L tryptone, 8 g / L sodium chloride, 3 mg / L vitamin B1, 0.3 mg / L calcium salt, and 3 g / L glucose in terms of mass concentration to prepare a mixed culture solution;

[0069] S3, inoculate Escherichia coli ATCC 25922 into the mixed culture solution in advance, culture it in an environment of 37°C and 2 vol.% O2 content for 10 hours, then inoculate Merrella CGMCC No.6398 and adjust the environmental parameters to 30°C, 5 vol.% O2 content, pH 6.5, culture it for 12 hours, and store it in a refrigerator at 4°C to obtain a composite bacterial agent; wherein the composite bacterial agent includes 4.3×10 8 CFU / mL Escherichia coli ATCC 25922 and 3.5×10 6 CFU / mL of Merrelliella CGMCC No.6398.

[0070] Comparative Example 8

[0071] This comparative example 8 provides a composite bacterial agent for producing β-glucuronidase, and the culturing method of the composite bacterial agent comprises the following steps:

[0072] S1. Activated Lactobacillus ATCC 53103 was added to 10 6cfu / g density was inoculated into the initial culture medium, and after shaking flask culture in an incubator in an anaerobic environment at 37°C for 5 hours, the culture was statically cultured until the logarithmic growth phase, and then sterilized at 120°C and a pressure of 2MPa, 20mL of deionized water was added and stirred, and then centrifuged at a speed of 300rpm to collect the supernatant to obtain a composite culture medium; wherein the initial culture medium includes 8g / L tryptone, 8g / L beef extract, 5g / L yeast extract, 17g / L glucose, 1g / L dimethyl hydrogen phosphate, 1g / L potassium dihydrogen phosphate, and 12g / L triammonium citrate;

[0073] S2, stirring and mixing the composite culture medium with yeast extract, malt extract, tryptone, sodium chloride, vitamin B1, calcium salt, and glucose, and adjusting the obtained culture solution to include 8 g / L yeast extract, 8 g / L malt extract, 10 g / L tryptone, 8 g / L sodium chloride, 3 mg / L vitamin B1, 0.3 mg / L calcium salt, and 3 g / L glucose in terms of mass concentration to prepare a mixed culture solution;

[0074] S3, inoculate Escherichia coli ATCC 25922 into the mixed culture solution in advance, culture it in an environment of 37°C and 2 vol.% O2 content for 10 hours, then inoculate Merrella CGMCC No.6400 and adjust the environmental parameters to 30°C, 5 vol.% O2 content, pH 6.5, culture it for 12 hours, and store it in a refrigerator at 4°C to obtain a composite bacterial agent; wherein the composite bacterial agent includes 5.3×10 8 CFU / mL Escherichia coli ATCC 25922 and 8.2×10 6 CFU / mL of Merrelliella CGMCC No.6400.

[0075] Performance Testing

[0076] Pure Escherichia coli ATCC 25922 was used as a blank example, and after being cultured in a mixed culture solution with the composite bacterial agents prepared in Example 1 and Comparative Examples 1 to 8 for 36 hours, the enzyme activity of β-glucuronidase was detected using a β-glucuronidase detection kit. The results are shown in Table 1 below.

[0077] Pure Escherichia coli ATCC 25922 was used as a blank example, and was cultured in a mixed culture solution with the composite bacterial agents prepared in Example 1 and Comparative Examples 1 to 8. The time required to enter the logarithmic growth phase and the ratio of the maintenance time to the blank example were recorded, as shown in Table 2 below.

[0078] Table 1 Enzyme activity performance test data

[0079] Enzyme activity (U / L) Enzyme activity (U / L) Example 1 1384.6 Comparative Example 5 216.2 Comparative Example 1 286.5 Comparative Example 6 273.6 Comparative Example 2 198.6 Comparative Example 7 264.5 Comparative Example 3 231.8 Comparative Example 8 259.8 Comparative Example 4 253.4 Blank example 128.4

[0080] Table 2 The time taken to enter the logarithmic growth phase and the ratio of the duration

[0081]

[0082]

[0083] Although the embodiments of the present invention are described in detail above, it is obvious to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations are within the scope and spirit of the present invention as described in the claims. Moreover, the present invention described herein may have other embodiments and may be implemented or realized in a variety of ways.

Claims

1. A composite bacterial agent for producing β-glucuronidase, characterized in that: The culturing method of the composite bacterial agent comprises: culturing lactobacillus ATCC 53103 in an initial culture medium to a logarithmic growth phase and then sterilizing to obtain a composite culture medium; mixing the composite culture medium with nutrient components and then inoculating Escherichia coli ATCC 25922 and Merrella solani CCUG 14215 for co-culturing to obtain the composite bacterial agent.

2. The composite bacterial agent according to claim 1, characterized in that The viable count of Escherichia coli ATCC25922 in the composite bacterial agent is 10 8 -10 9 CFU / mL, the viable count of the Merkel cell culture medium CCUG 14215 is 10 5 -10 7 CFU / mL.

3. The composite bacterial agent according to claim 1, characterized in that The initial culture medium includes 8-10 g / L tryptone, 8-10 g / L beef extract, 3-6 g / L yeast extract, 15-20 g / L glucose, 1-2 g / L dipotassium hydrogen phosphate, 1-2 g / L potassium dihydrogen phosphate, and 1-2 g / L triammonium citrate.

4. The composite bacterial agent according to claim 3, characterized in that: Lactobacillus ATCC 53103 is cultured in an initial culture medium at 35-40° C. in an anaerobic environment until the logarithmic growth phase, and the composite culture medium is prepared by centrifuging and collecting the supernatant after pasteurization.

5. The composite bacterial agent according to claim 1, characterized in that: When the complex culture medium is mixed with nutrient components, the nutrient components include yeast extract, malt extract, tryptone, sodium chloride, vitamin B1, calcium salt and glucose.

6. The composite bacterial agent according to claim 5, characterized in that: After the composite culture medium is mixed with the nutrient components, the composite culture medium includes, by mass concentration, 6-10 g / L yeast extract, 8-10 g / L malt extract, 8-10 g / L tryptone, 8-10 g / L sodium chloride, 1-5 mg / L vitamin B1, 0.1-0.5 mg / L calcium salt and 1-5 g / L glucose.

7. The composite bacterial agent according to claim 1, characterized in that: When inoculating Escherichia coli ATCC 25922 and Merrella solani CCUG 14215 for co-culture, the method includes: pre-inoculating Escherichia coli ATCC 25922 and culturing it in an aerobic environment at 35-40° C. for 8-12 hours, and then inoculating Merrella solani CCUG 14215 and co-culturing it at 30-32° C., 5-8 vol.% oxygen content, and pH 6.0-6.5 for 48 hours to obtain the composite bacterial agent.

8. The composite bacterial agent according to claim 7, characterized in that: After the composite bacterial agent is prepared, it is stored at a low temperature of 4°C.

9. An application of the composite bacterial agent according to any one of claims 1 to 8 in bezoar production, characterized in that: The composite bacterial agent is used for producing beta-glucuronidase.

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