Preparation method and application of ginseng leavening for improving content of converted ginsenoside CK

Abstract

The invention relates to the technical field of fermentation engineering, and particularly discloses a preparation method and application of a ginseng leavening for improving the content of transformed ginsenoside CK. The preparation method comprises the following fermentation conditions: 1) performing carbon source and nitrogen source combined regulation on ginseng root hair fermentation liquor; (2) inoculating the three probiotics into the ginseng root hair fermentation liquor in stages, and carrying out synergistic fermentation; 3) regulating and controlling the dissolved oxygen level in stages; 4) regulating and controlling fermentation conditions by stages; the carbon source comprises fructo-oligosaccharide, inulin and xylooligosaccharide; the nitrogen source comprises soybean protein isolate and pea protein; the probiotics comprise strains of the genus Lactobacillus, the genus Bifidobacterium and the genus Saccharomyces. According to the method, the yield of ginsenoside CK in the ginseng leavening can be remarkably increased, the fermentation efficiency is improved, and the fermentation period is shortened.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fermentation engineering, and specifically discloses a preparation method and application of ginseng fermentation product with improved content of converted ginsenoside CK. BACKGROUND

[0002] Ginsenoside CK is a main metabolite of natural diol-type ginsenoside converted by intestinal microorganisms. It has become a research hotspot due to its stronger biological activity and unique therapeutic effect than natural ginsenoside, and its low-toxicity and high-efficiency anti-tumor, immune-regulating and neuroprotective activities. With the development of related technologies, breakthroughs have been made in its preparation process, and it has shown important practical significance in the fields of medicine, cosmetics, etc.

[0003] Since ginsenoside CK has not been detected in total ginsenosides, it is currently mainly obtained by hydrolysis of the original ginsenoside. This type of preparation method relies on the supply of ginsenoside resources. Due to the fact that wild ginseng resources have been basically exhausted, and artificial cultivation of ginseng faces problems such as long growth cycle, diseases and pests, and continuous cropping obstacles, the supply, quality and safety of ginsenoside resources are all facing challenges.

[0004] The preparation of ginsenoside CK by biotransformation technology has attracted great attention in recent years, and the mainstream schemes include: (1) microbial fermentation method (such as using Escherichia coli, Saccharomyces cerevisiae, Aspergillus, etc.), (2) enzyme catalysis method (using β-glucosidase or recombinant enzyme), (3) plant cell culture method (such as ginseng adventitious root or hairy root culture); Among them, the enzyme catalysis method has poor stability, free enzymes are easy to inactivate in high temperature or organic solvents, and the reuse rate is low; the plant cell culture method has a long production cycle and low efficiency; relatively speaking, the microbial fermentation method is more suitable for industrial production, but the microbial fermentation technology generally has the defect of low yield of ginsenoside CK, and there is still a great technical bottleneck in how to improve the yield of CK. SUMMARY

[0005] In order to solve the problems of low fermentation conversion efficiency and insufficient yield of traditional ginsenoside CK, the present application provides a preparation method and application of ginseng fermentation product with improved content of converted ginsenoside CK.

[0006] To solve the above technical problems, the technical scheme provided by the present application is: A preparation method of ginseng fermentation product with improved content of converted ginsenoside CK, characterized in that the following fermentation conditions are included: 1) carbon source and nitrogen source combination regulation of ginseng root hair fermentation broth; 2) inoculating three kinds of probiotics into the ginseng root hair fermentation liquor in stages for synergistic fermentation; 3) regulating the dissolved oxygen level in stages; 4) regulating the fermentation conditions in stages; The carbon source includes fructo-oligosaccharide, inulin and xylo-oligosaccharide; the nitrogen source includes soybean protein isolate and pea protein; the probiotics include strains of Lactobacillus, Bifidobacterium and Saccharomyces; Preferably, in the ginseng root hair fermentation liquor, the carbon source includes fructo-oligosaccharide 2-3 g / L, xylo-oligosaccharide 1-1.5 g / L and inulin 1-2 g / L; the synergistic combination of the above carbon sources not only promotes the growth of various probiotics and increases the viable bacterial count in the fermentation liquor, but also improves the antioxidant activity and acid production capacity of the fermentation liquor, and the inoculation of three kinds of probiotics in stages can greatly improve the ability and efficiency of fermentation conversion of CK, and greatly improve the yield of CK.

[0007] Preferably, in the ginseng root hair fermentation liquor, the nitrogen source includes soybean protein isolate 1-2 g / L and pea protein 0.5-1 g / L; although the cost of soybean protein isolate is relatively low, the methionine content is relatively low, and the nutritional components are not balanced, and the complementary of amino acids can be realized by synergistic combination with pea protein, so as to promote the growth and metabolism of microorganisms, thereby improving the yield of effective components CK in the fermentation product.

[0008] Preferably, the ratio of the probiotics is: 35-45% of Lactobacillus, 25-35% of Bifidobacterium and 25-35% of Saccharomyces, based on 100% of the total inoculation amount of probiotics; Preferably, the strain of Lactobacillus is selected from Lactobacillus plantarum; Preferably, the strain of Bifidobacterium is selected from Bifidobacterium longum; Preferably, the strain of Saccharomyces is selected from Saccharomyces cerevisiae.

[0009] The total ginsenoside content of ginseng root hair is significantly higher than that of main root and stem and leaf, and is rich in diol-type ginsenosides such as Rb1 and Rd, which are direct precursors of microbial conversion of CK, and the characteristics of small fiber structure conducive to microbial adhesion and penetration make it particularly suitable for microbial fermentation conversion. In addition, the polysaccharides and amino acids in ginseng root hair can promote microbial growth and enhance the activity of key enzymes such as β-glucosidase, thereby promoting saponin biotransformation. Therefore, the preparation of ginsenoside CK by biotransformation with ginseng root hair as the fermentation raw material is an ideal synthesis path.

[0010] The present application is found in continuous research that the ginsenoside CK yield can be significantly improved by inoculating the ginseng root hair fermentation liquor with strains of Lactobacillus, Bifidobacterium and Saccharomycete in stages, and under the regulation of specific carbon source and nitrogen source combination, which has important significance for providing safe and reliable ginsenoside CK bioconversion technology.

[0011] As a preferred embodiment of the present application, the probiotics are inoculated in stages in the order of strains of Lactobacillus, Bifidobacterium and Saccharomycete; Preferably, the strain of Lactobacillus is inoculated at the beginning of fermentation, the strain of Bifidobacterium is inoculated at 18-40h of fermentation, and the strain of Saccharomycete is inoculated at 48h-72h of fermentation.

[0012] The strains of Lactobacillus and Bifidobacterium can secrete various glycosidases (such as β-glucosidase), which can convert the main components of ginsenosides (such as Rb1, Rg1) into ginsenoside CK, while the strains of Saccharomycete can further decompose the cell wall of ginseng and release more active ingredients during fermentation, and also promote the conversion of ginsenosides. The present application uses strains of Lactobacillus, Bifidobacterium and Saccharomycete for staged and synergistic fermentation, which not only can improve the bioavailability, but also is conducive to improving the fermentation efficiency and the yield of ginsenoside CK.

[0013] As a preferred embodiment of the present application, the ginseng root hair fermentation liquor comprises ginseng root hair extract 0.5-2g / L and glucose 20-30g / L; Preferably, the ginseng root hair extract is obtained by crushing ginseng root hair as raw material and then solvent extraction; More preferably, the specific method of solvent extraction is as follows: the cleaned and dried ginseng root hair is crushed to 20-40 mesh, 60-80vol% ethanol aqueous solution is added according to the solid-liquid mass ratio of 1:(6-8), and reflux extraction is carried out at 60-80℃ for 1.5-2h each time, the filtrates are combined, and vacuum reduction is carried out at 35-55℃ until there is no alcohol smell, to obtain the ginseng root hair extract.

[0014] As a preferred embodiment of the present application, the ginseng root hair fermentation liquor further comprises basic salts, preferably MgSO40.3-0.6g / L and KH2PO41-3g / L; Preferably, the initial pH of the ginseng root hair fermentation liquor is 6.0-6.5.

[0015] As a preferred embodiment of the present application, the initial stage of fermentation is regulated to be aerobic, preferably controlling the dissolved oxygen concentration to be 2-6 mg / L; at 12-18 h of fermentation, it is regulated to be anaerobic, preferably controlling the dissolved oxygen concentration to be ≤0.2 mg / L; at 48-72 h of fermentation, it is regulated to be micro-aerobic, preferably controlling the dissolved oxygen concentration to be 0.5-1 mg / L.

[0016] As a preferred embodiment of the present application, during the fermentation stage of inoculating strains of Lactobacillus and Bifidobacterium, the fermentation temperature is adjusted to be 35-38℃; during the fermentation stage of inoculating strains of Saccharomyces, the fermentation temperature is adjusted to be 28-32℃.

[0017] As a preferred embodiment of the present application, during the fermentation process, the pH is allowed to naturally drop to 4.5-6.0; if the pH drops too much, 0.1-0.2 g / L of sodium carbonate is used for fine adjustment.

[0018] As a preferred embodiment of the present application, before the probiotics are inoculated into the ginseng root hair fermentation liquor, they are first activated in a shake flask culture medium, and then the activated seed culture liquor is inoculated for fermentation. The shake flask activation culture can use the culture medium commonly used in the field according to the type of probiotics or make conventional adjustments, which will not be repeated here. The inoculation amount of the seed culture liquor is generally 2-10% of the volume of the fermentation liquor, but it should be noted that the ratio of the three probiotic culture liquors should be as much as possible within the range defined above.

[0019] As a preferred embodiment of the present application, when the content of the fermentation product ginsenoside CK reaches the peak value, the fermentation is terminated, the bacterial bodies are centrifuged and separated, and the fermentation liquor is collected to obtain the ginseng fermentation product.

[0020] The present application also provides the use of the ginseng fermentation product prepared by the method described above in the fields of food, biology and medicine.

[0021] The advantages of the present application over the prior art are as follows: 1. The three-source carbon-nitrogen matrix is created, and the combination of fructooligosaccharides / inulin / xylooligosaccharides can improve the symbiotic stability of the bacterial community, and the combination of soybean protein isolate / pea protein can realize amino acid complementation, both of which can synergistically improve the ability and efficiency of CK fermentation conversion.

[0022] 2. Three kinds of probiotics are inoculated for fermentation in stages, and through functional complementation of the bacterial community and precise regulation of the metabolic environment, the CK conversion efficiency is significantly improved.

[0023] 3. Through the regulation of dissolved oxygen and fermentation conditions in stages, the energy metabolism pathway of the bacterial community is optimized and the accumulation of by-products is reduced, thereby reducing energy metabolism loss. DETAILED DESCRIPTION

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0025] Unless otherwise specified, the raw materials and reagents used in the following embodiments of the present invention can be obtained through commercial channels. Among them, the ginseng rootlets were sampled from 5-year-old ginseng produced in Fusong County, Jilin Province. The total saponin content was identified as 68.32 mg / g (ginseng rootlets) by HPLC-MS, and the ginsenoside CK content was 0.38 mg / g (ginseng rootlets).

[0026] Lactobacillus plantarum: ZKAW02, Zhongke E Microbial Technology Co., Ltd. Bifidobacterium longum: ZKAWO7, Zhongke E Microbial Technology Co., Ltd. Saccharomyces cerevisiae: SN, Hubei Angel Yeast Co., Ltd. In the following embodiments of the present invention, the strains were all obtained by activating the strains to obtain seed activation solution, which was then inoculated into ginseng root fermentation broth for fermentation production, wherein: Preparation of Lactobacillus plantarum seed activation solution: MRS liquid medium, activation culture temperature 37℃, culture time 24h, shake flask culture; Preparation of Bifidobacterium longum seed activation solution: MRS liquid medium, activation culture temperature 37℃, culture time 24h, anaerobic culture; Preparation of Saccharomyces cerevisiae seed activation solution: YPD liquid medium, activation culture temperature 30℃, culture time 12h, shake culture.

[0027] The analysis / control methods involved in the following embodiments of the present invention are as follows: (1) Saponin content: HPLC-MS analysis (2) Enzyme activity assay: PNPG method to test β-glucosidase activity (3) pH / temperature: Online Fermac 360 monitoring system In the following embodiments of the present invention, the activation and fermentation production are carried out in an environment isolated from external microorganisms, and the activation culture medium and fermentation broth are sterilized in advance. This is a routine operation and will not be described in detail. Example 1

[0028] A method for preparing ginseng fermentation product with increased content of ginsenoside CK includes the following steps: S1, clean the ginseng root hairs, dry at 60℃ to moisture content <8%, crush into powder, pass through 20 mesh sieve; add the powder to 60vol% ethanol aqueous solution according to material liquid mass ratio 1:7, reflux extraction at 60℃ for 1.5h each time, combine the filtrate, vacuum reduce pressure at 45℃ until no alcohol smell, obtain ginseng root hair extract.

[0029] Mix the ginseng root hair extract, glucose, and water to obtain ginseng root hair fermentation broth, which contains ginseng root hair extract 2g / L and glucose 25g / L.

[0030] S2, add combined carbon source to the ginseng root hair fermentation broth, which contains fructo-oligosaccharide 2.5g / L, xylo-oligosaccharide 1.5g / L, and inulin 1.5g / L; continue to add combined nitrogen source, which contains soybean protein isolate 2g / L and pea protein 0.5g / L; additionally add inorganic salt, which contains MgSO4 0.5g / L and KH2PO4 2.5g / L, adjust the initial pH to 6.0; S3, add the prepared ginseng root hair fermentation broth to the fermentation tank, monitor the tank temperature in line to stabilize at 35℃, control the dissolved oxygen concentration to be 2mg / L, start inoculating Lactobacillus plantarum seed activation liquid, start fermentation; when the fermentation is carried out for 12h, adjust the dissolved oxygen concentration to be ≤0.2mg / L; S4, after the fermentation is carried out for 18h, maintain the fermentation temperature at 35℃ and the dissolved oxygen concentration at ≤0.2mg / L, start inoculating Bifidobacterium longum seed activation liquid, continue fermentation; S5, after the fermentation is carried out for 50h, adjust the fermentation temperature to 32℃, at the same time adjust the dissolved oxygen concentration to 1mg / L, start inoculating Saccharomyces cerevisiae seed activation liquid, continue fermentation; Taking the total inoculation amount of probiotics as 100%, the ratio of the inoculation amount of probiotics in the three stages is: 35% Lactobacillus plantarum, 35% Bifidobacterium longum, and 30% Saccharomyces cerevisiae; S5, when the fermentation is carried out for 96h, the content of the product ginsenoside CK reaches the peak value, terminate the fermentation, centrifugal separate the bacterial body, collect the fermentation broth, obtain ginseng fermentation product.

[0031] Detect and analyze the content of total saponins and ginsenoside CK after fermentation, and convert it into the relative content with respect to each g of substrate, i.e. ginseng root hair, unit mg / g, record in Table 1; additionally, monitor the β-glucosidase activity in the whole fermentation process, record the peak activity in Table 1. Example 2

[0032] A preparation method of ginseng fermentation product for improving the content of converted ginsenoside CK, specifically comprising the following steps: S1, clean the ginseng root hairs, dry at 60℃ hot air to moisture content <8%, crush into powder, pass through 40 mesh sieve; add the powder according to the solid-liquid mass ratio of 1:6 into 70vol% ethanol aqueous solution, reflux extraction at 80℃ for 2h each time, combine the filtrate, vacuum reduce pressure at 35℃ until no alcohol smell, obtain ginseng root hair extract.

[0033] Mix the ginseng root hair extract, glucose, and water to obtain ginseng root hair fermentation broth, which contains 0.5g / L of ginseng root hair extract and 20g / L of glucose.

[0034] S2, add combined carbon source to the ginseng root hair fermentation broth, which contains 3g / L of fructooligosaccharide, 1g / L of xylooligosaccharide, and 1.5g / L of inulin; continue to add combined nitrogen source, which contains 1g / L of soybean protein isolate and 1g / L of pea protein; additionally add inorganic salt, which contains 0.4g / L of MgSO4 and 3g / L of KH2PO4, and adjust the initial pH to 6.5; S3, add the prepared ginseng root hair fermentation broth to the fermentation tank, monitor the tank temperature online to stabilize at 38℃, control the dissolved oxygen concentration to be 6mg / L, start inoculating the Lactobacillus plantarum seed activation liquid, and start fermentation; when the fermentation is carried out for 14h, adjust the dissolved oxygen concentration to be ≤0.2mg / L; S4, after the fermentation is carried out for 24h, maintain the fermentation temperature at 38℃ and the dissolved oxygen concentration at ≤0.2mg / L, start inoculating the Bifidobacterium longum seed activation liquid, and continue fermentation; S5, after the fermentation is carried out for 60h, adjust the fermentation temperature to 30℃, at the same time adjust the dissolved oxygen concentration to 0.5mg / L, start inoculating the Saccharomyces cerevisiae seed activation liquid, and continue fermentation; Taking the total inoculation amount of probiotics as 100%, the proportion of the inoculation amount of probiotics in the three stages is: 45% of Lactobacillus plantarum, 30% of Bifidobacterium longum, and 25% of Saccharomyces cerevisiae; S5, when the fermentation is carried out for 98h, the content of the product ginsenoside CK reaches the peak value, the fermentation is terminated, the bacterial bodies are centrifuged and separated, the fermentation broth is collected, and the ginseng fermentation product is obtained.

[0035] The contents of total saponins and ginsenoside CK after fermentation are detected and analyzed, and are converted into the relative content with respect to each g of substrate, i.e. ginseng root hair, unit mg / g, which is recorded in Table 1; in addition, the β-glucosidase activity in the fermentation process is monitored throughout, and the peak activity is recorded in Table 1. Example 3

[0036] A preparation method of a ginseng fermentation product for increasing the content of converted ginsenoside CK, specifically comprising the following steps: S1, clean the ginseng root hairs, dry at 60℃ to moisture content <8%, crush into powder, pass through 30 mesh sieve; add 70vol% ethanol aqueous solution to the powder according to the material-liquid mass ratio of 1:8, reflux extraction at 60℃ for 2h each time, combine the filtrate, vacuum reduce pressure at 45℃ until no alcohol smell, obtain ginseng root hair extract.

[0037] Mix the ginseng root hair extract, glucose, and water to obtain ginseng root hair fermentation liquor, which contains 1g / L of ginseng root hair extract and 30g / L of glucose.

[0038] S2, add combined carbon source to the ginseng root hair fermentation liquor, which contains 2g / L of fructooligosaccharide and 1g / L of xylooligosaccharide; continue to add combined nitrogen source, which contains 1.5g / L of soybean protein isolate and 0.8g / L of pea protein; additionally add inorganic salt, which contains 0.6g / L of MgSO4 and 1g / L of KH2PO4, adjust the initial pH to 6.5; S3, add the prepared ginseng root hair fermentation liquor to the fermentation tank, monitor the tank temperature to be stable at 36℃ online, control the dissolved oxygen concentration to be 4mg / L, start inoculating Lactobacillus plantarum seed activation liquid, start fermentation; when the fermentation is carried out for 18h, adjust the dissolved oxygen concentration to be ≤0.2mg / L; S4, after the fermentation is carried out for 40h, keep the fermentation temperature at 36℃ and the dissolved oxygen concentration at ≤0.2mg / L, start inoculating Bifidobacterium longum seed activation liquid, continue fermentation; S5, after the fermentation is carried out for 72h, adjust the fermentation temperature to be 28℃ and the dissolved oxygen concentration to be 0.6mg / L, start inoculating Saccharomyces cerevisiae seed activation liquid, continue fermentation; Taking the total inoculation amount of probiotics as 100%, the proportion of the inoculation amount of probiotics in the three stages is: 40% of Lactobacillus plantarum, 25% of Bifidobacterium longum, and 35% of Saccharomyces cerevisiae; S5, when the fermentation is carried out for 102h, the content of the product ginsenoside CK reaches the peak value, terminate the fermentation, centrifugal separate the bacterial body, collect the fermentation liquor, obtain ginseng fermentation product.

[0039] Detect and analyze the contents of total ginsenoside and ginsenoside CK after fermentation, and convert them into relative contents with respect to each g of substrate, i.e. ginseng root hair, unit mg / g, which are recorded in Table 1; additionally, monitor the β-glucosidase activity in the whole fermentation process, and record the peak activity in Table 1. Example 4

[0040] A preparation method of ginseng fermentation product for increasing the content of converted ginsenoside CK, specifically comprising the following steps: S1. Ginseng rootlets are cleaned and dried in hot air at 60℃ until the moisture content is <8%. They are then pulverized into powder and passed through a 20-mesh sieve. The powder is added to an 80 vol% ethanol aqueous solution at a mass ratio of 1:6, and refluxed at 70℃ for 1.5 h each time. The filtrates are combined and vacuum reduced at 55℃ until there is no alcohol odor, thus obtaining ginseng rootlet extract.

[0041] Ginseng root extract, glucose, and water were mixed to obtain ginseng root fermentation liquid containing 2 g / L ginseng root extract and 25 g / L glucose.

[0042] S2. Add a combination of carbon sources to the ginseng root fermentation broth, making it contain 3 g / L of fructooligosaccharides, 1.5 g / L of xylooligosaccharides, and 1 g / L of inulin; continue to add a combination of nitrogen sources, making it contain 1.8 g / L of soy protein isolate and 0.6 g / L of pea protein; additionally add inorganic salts, making it contain 0.3 g / L of MgSO4 and 2 g / L of KH2PO4, and adjust the initial pH to 6.0; S3. Add the prepared ginseng root fermentation liquid to the fermentation tank, monitor the temperature inside the tank online to ensure it is stable at 37℃, control the dissolved oxygen concentration at 3 mg / L, start inoculating with Lactobacillus plantarum seed activation liquid, and start fermentation; after 16 hours of fermentation, adjust the dissolved oxygen concentration to ≤0.2 mg / L. S4. After 30 hours of fermentation, maintain the fermentation temperature at 37℃ and the dissolved oxygen concentration at ≤0.2 mg / L, then inoculate with Bifidobacterium longum seed activation solution and continue fermentation. S5. After 55 hours of fermentation, adjust the fermentation temperature to 28℃ and simultaneously adjust the dissolved oxygen concentration to 1 mg / L. Start inoculating with the brewing yeast seed activation solution and continue fermentation. With the total probiotic inoculation amount as 100%, the proportion of probiotic inoculation amount in the three stages is: 35% Lactobacillus plantarum, 35% Bifidobacterium longum, and 30% Saccharomyces cerevisiae. S5. After 97 hours of fermentation, the content of ginsenoside CK in the product was detected to have reached its peak. Fermentation was then terminated, the bacterial cells were separated by centrifugation, and the fermentation broth was collected to obtain ginseng fermentation product.

[0043] The contents of total saponins and ginsenoside CK after fermentation were detected and analyzed, and converted into relative contents per gram of substrate, i.e., ginseng rootlets, in mg / g, and recorded in Table 1. In addition, the β-glucosidase activity during the fermentation process was monitored throughout, and the peak activity was recorded in Table 1.

[0044] Comparative Example 1 A method for preparing ginseng fermentation product with increased content of ginsenoside CK includes the following steps: S1. Clean the ginseng rootlets and dry them in hot air at 60℃ until the moisture content is <8%. Crush them into powder and pass them through a 20-mesh sieve. Add the powder to a 60 vol% ethanol aqueous solution at a mass ratio of 1:7 and reflux extract at 60℃ for 1.5 hours each time. Combine the filtrates and vacuum reduce the pressure at 45℃ until there is no alcohol smell to obtain the ginseng rootlet extract.

[0045] Ginseng root extract, glucose, and water were mixed to obtain ginseng root fermentation liquid containing 2 g / L ginseng root extract and 25 g / L glucose.

[0046] S2. Add a combination of carbon sources to the ginseng root fermentation broth, containing 2.5 g / L fructooligosaccharides, 1.5 g / L xylooligosaccharides, and 1.5 g / L inulin; continue adding a combination of nitrogen sources, containing 2 g / L soy protein isolate and 0.5 g / L pea protein; additionally add inorganic salts, containing 0.5 g / L MgSO4 and 2.5 g / L KH2PO4, and adjust the initial pH to 6.0. S3. Add the prepared ginseng root fermentation liquid to the fermentation tank, monitor the temperature inside the tank online to stabilize at 32℃, control the dissolved oxygen concentration to 1 mg / L, and inoculate with Lactobacillus plantarum seed activation liquid, Bifidobacterium longum seed activation liquid, and Saccharomyces cerevisiae seed activation liquid in a one-time inoculation ratio of 35%, 35%, and 30% to start fermentation. S4. After 24 hours of fermentation, adjust the fermentation temperature to 35℃ and simultaneously adjust the dissolved oxygen concentration to ≤0.2 mg / L, and continue fermentation. S5. After 130 hours of fermentation, the content of ginsenoside CK in the product was detected to have reached its peak. Fermentation was then terminated, the cells were separated by centrifugation, and the fermentation broth was collected to obtain ginseng fermentation product.

[0047] The contents of total saponins and ginsenoside CK after fermentation were detected and analyzed, and converted into relative contents per gram of substrate, i.e., ginseng rootlets, in mg / g, and recorded in Table 1. In addition, the β-glucosidase activity during the fermentation process was monitored throughout, and the peak activity was recorded in Table 1.

[0048] Comparative Example 2 The ginseng ferment was prepared using a method that was essentially the same as in Example 1, except that xylooligosaccharides were not added to the combined carbon source.

[0049] After 106 hours of fermentation, the content of ginsenoside CK was detected to have reached its peak. Fermentation was then terminated, the bacterial cells were separated by centrifugation, and the fermentation broth was collected to obtain ginseng fermentation product.

[0050] The contents of total saponins and ginsenoside CK after fermentation were detected and analyzed, and converted into relative contents per gram of substrate, i.e., ginseng rootlets, in mg / g, and recorded in Table 1. In addition, the β-glucosidase activity during the fermentation process was monitored throughout, and the peak activity was recorded in Table 1.

[0051] Comparative Example 3 The ginseng ferment was prepared using a method essentially the same as in Example 1, except that inulin was not added to the combined carbon source.

[0052] After 102 hours of fermentation, the content of ginsenoside CK was detected to have reached its peak. Fermentation was then terminated, the bacterial cells were separated by centrifugation, and the fermentation broth was collected to obtain ginseng fermentation product.

[0053] The contents of total saponins and ginsenoside CK after fermentation were detected and analyzed, and converted into relative contents per gram of substrate, i.e., ginseng rootlets, in mg / g, and recorded in Table 1. In addition, the β-glucosidase activity during the fermentation process was monitored throughout, and the peak activity was recorded in Table 1.

[0054] Comparative Example 4 The ginseng ferment was prepared using a method essentially the same as in Example 1, except that pea protein was not added to the combined nitrogen source.

[0055] After 114 hours of fermentation, the content of ginsenoside CK was detected to have reached its peak. Fermentation was then terminated, the bacterial cells were separated by centrifugation, and the fermentation broth was collected to obtain ginseng fermentation product.

[0056] The contents of total saponins and ginsenoside CK after fermentation were detected and analyzed, and converted into relative contents per gram of substrate, i.e., ginseng rootlets, in mg / g, and recorded in Table 1. In addition, the β-glucosidase activity during the fermentation process was monitored throughout, and the peak activity was recorded in Table 1.

[0057] Table 1. Analysis and Detection Results

[0058] As can be seen from the test results in Table 1, the embodiment scheme provided by the present invention not only significantly improves the CK fermentation yield compared to Comparative Examples 1-4, but also greatly enhances the peak enzyme activity of β-glucosidase. Calculations show that, compared to the scheme in Comparative Example 1 where all three probiotics were added at once, Example 1 increased the CK content by more than 15% and the peak enzyme activity by 34%, which not only plays an important role in increasing the CK content in ginseng fermentation products but also helps to shorten the fermentation cycle and reduce production costs. Furthermore, a comparison between Example 1 and Comparative Examples 2-4 shows that the controlled combination of carbon and nitrogen sources in the fermentation broth of the present invention is particularly important for improving CK yield and the peak enzyme activity of β-glucosidase, unexpectedly achieving a high CK production capacity increase under the optimized synergistic formulation.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing ginseng fermentation products with increased content of ginsenoside CK, characterized in that... The fermentation conditions include the following: 1) Regulate the combination of carbon and nitrogen sources in the fermentation broth of ginseng roots; 2) Three types of probiotics were inoculated into the ginseng root fermentation liquid in stages for synergistic fermentation; 3) Adjust dissolved oxygen levels in stages; 4) Adjust fermentation conditions in stages; The carbon source includes fructooligosaccharides, inulin, and xylooligosaccharides; the nitrogen source includes soy protein isolate and pea protein; the probiotics include strains of Lactobacillus, Bifidobacterium, and Saccharomyces. Preferably, the carbon source in the ginseng root fermentation broth includes 2-3 g / L of fructooligosaccharides, 1-1.5 g / L of xylooligosaccharides, and 1-2 g / L of inulin; Preferably, the nitrogen source in the ginseng root fermentation broth includes 1-2 g / L of soy protein isolate and 0.5-1 g / L of pea protein; Preferably, the probiotic formulation is: 35-45% Lactobacillus, 25-35% Bifidobacterium, and 25-35% Saccharomyces, with the total probiotic inoculation amount being 100%. Preferably, the strain of Lactobacillus is selected from Lactobacillus plantarum; Preferably, the strain of Bifidobacterium is selected from Bifidobacterium longum; Preferably, the yeast strain is selected from Saccharomyces cerevisiae.

2. The method for preparing ginseng fermentation product with increased content of ginsenoside CK according to claim 1, characterized in that, The probiotics were inoculated in stages according to the order of Lactobacillus, Bifidobacterium, and Saccharomyces strains. Preferably, the Lactobacillus strain is inoculated at the beginning of fermentation, the Bifidobacterium strain is inoculated 18-40 hours after fermentation, and the yeast strain is inoculated 48-72 hours after fermentation.

3. The method for preparing ginseng fermentation product with increased content of ginsenoside CK according to claim 1, characterized in that, The ginseng root fermentation liquid contains 0.5-2 g / L of ginseng root extract and 20-30 g / L of glucose; Preferably, the ginseng root extract is obtained by pulverizing ginseng rootlets and then extracting them with a solvent. More preferably, the solvent extraction method is as follows: the cleaned and dried ginseng rootlets are crushed to 20-40 mesh, and 60-80 vol% ethanol aqueous solution is added at a material-to-liquid mass ratio of 1:(6-8). The mixture is then refluxed at 60-80°C for 1.5-2 h each time. The filtrates are combined and vacuum reduced at 35-55°C until no alcohol odor is detected, thus obtaining the ginseng rootlet extract.

4. A method for preparing ginseng fermentation product with increased content of ginsenoside CK according to any one of claims 1-3, characterized in that, The ginseng root fermentation liquid also contains basic salts, preferably 0.3-0.6 g / L of MgSO4 and 1-3 g / L of KH2PO4; Preferably, the initial pH of the ginseng root fermentation broth is 6.0-6.

5.

5. A method for preparing ginseng fermentation product with increased content of ginsenoside CK according to any one of claims 1-3, characterized in that, The initial fermentation stage is controlled as aerobic, with the dissolved oxygen concentration preferably controlled at 2–6 mg / L; during the 12–18 h fermentation stage, the fermentation is controlled as anaerobic, with the dissolved oxygen concentration preferably controlled at ≤0.2 mg / L; and during the 48–72 h fermentation stage, the fermentation is controlled as microaerobic, with the dissolved oxygen concentration preferably controlled at 0.5–1 mg / L.

6. A method for preparing ginseng fermentation product with increased content of ginsenoside CK according to any one of claims 1-3, characterized in that, During the fermentation stage inoculated with Lactobacillus and Bifidobacterium strains, the fermentation temperature was adjusted to 35-38℃; during the fermentation stage inoculated with Yeast strains, the fermentation temperature was adjusted to 28-32℃.

7. A method for preparing ginseng fermentation product with increased content of ginsenoside CK according to any one of claims 1-3, characterized in that, During fermentation, allow the pH to drop naturally to 4.5-6.0; if the pH drops too much, adjust it by adding 0.1-0.2 g / L of sodium carbonate.

8. A method for preparing ginseng fermentation product with increased content of ginsenoside CK according to any one of claims 1-3, characterized in that, Before the probiotics are inoculated into the ginseng root fermentation liquid, they are first activated in a shake flask culture medium, and then the activated seed culture liquid is fermented and inoculated.

9. A method for preparing ginseng fermentation product with increased content of ginsenoside CK according to any one of claims 1-3, characterized in that, When the content of ginsenoside CK in the fermentation product reaches its peak, the fermentation is terminated, the bacterial cells are separated by centrifugation, and the fermentation broth is collected to obtain the ginseng fermentation product.

10. The application of a ginseng ferment obtained by the method according to any one of claims 1-9 in the food, biological, and pharmaceutical fields.

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