Streptococcus thermophilus with rare ginsenoside conversion effect and application of streptococcus thermophilus

Abstract

The invention belongs to the technical field of biological medicines, and relates to streptococcus thermophilus with a rare ginsenoside conversion effect and application of the streptococcus thermophilus. The strain is Streptococcus thermophilus 17140, the Streptococcus thermophilus 17140 is preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on September 23, 2024, and the preservation number of the Streptococcus thermophilus 17140 is GDMCC NO.65173. The invention further discloses a preparation method of the Streptococcus thermophilus 17140. The strain disclosed by the invention has good functions of fermenting and converting various ginsenosides and notoginsenoside and generating rare ginsenosides.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to a thermophilic streptococcus with rare ginsenoside conversion function and application thereof. Background Art

[0002] Ginseng is a traditional precious medicine with a long history of medicinal use. Modern research shows that ginsenosides are important medicinal substances in ginseng and have significant pharmacological activity. Studies have shown that ginsenosides are metabolized into secondary saponins such as Rg3, Rg5, Rh2, Rh3, CK, protopanaxadiol, Rg2, Rh1, and protopanaxatriol, which have less sugar and smaller molecular weight, under the action of biological enzymes and intestinal bacteria in the body. Because these secondary saponins are extremely low in ginseng or do not occur under natural conditions, they are also called rare ginsenosides. Compared with natural ginsenosides, rare ginsenosides are deglycosylated, making ginsenosides smaller in size, more permeable, easier to pass through cell membranes, and more bioavailable. They are the main active form of ginseng to play a medical and health function. However, due to the differences in individual intestinal microorganisms, the conversion efficiency of rare ginsenosides in the body varies. Therefore, converting ginsenosides in vitro into rare ginsenosides that are more conducive to human absorption and reducing individual differences caused by human factors is crucial to improving the utilization efficiency of ginseng and ensuring the full and stable performance of its medicinal effects.

[0003] Domestic and foreign scholars mainly prepare rare ginsenosides by physical, chemical or biological methods. The physical method mainly involves heating natural ginsenosides to undergo deglycosylation reaction to form rare ginsenosides. However, the physical method for converting saponins has problems such as high reaction temperature, high energy consumption, and low saponin content. The chemical method for converting ginsenosides includes acid hydrolysis and alkaline hydrolysis. The 3- and 20-position glycosidic bonds in the parent nucleus structure of ginsenosides are unbalanced. The 3-position glycosidic bond is easily broken under neutral conditions, and the 20-position glycosidic bond is easily broken under acid or alkaline conditions. Therefore, it is difficult to effectively and accurately control the breaking of glycosidic bonds by chemical methods, and it is easy to generate by-products. In addition, there are problems such as violent reactions, difficult to control the degree of hydrolysis, difficult purification, and environmental pollution during the reaction process. Biotransformation is a process that uses microorganisms or enzymes as catalysts to achieve conversion. Biotransformation has unique advantages in the preparation of rare ginsenosides because of its good regioselectivity and mild and specific reaction. Enzymatic conversion can use commercial enzyme preparations, which are easy to operate, but are expensive, and have problems such as inconvenient enzyme sources and high costs, so their application is limited. Microbial transformation mainly utilizes intestinal bacteria, food-grade microorganisms, soil microorganisms, plant endophytes, etc. Since microorganisms themselves grow exponentially, the fermentation speed is fast; there is no need to separate and purify the enzyme, which is more convenient than enzymatic transformation; the production cost is lower than physical transformation and chemical transformation.

[0004] Probiotics are microbial additives that have beneficial effects on the host by improving the balance of intestinal microorganisms. They have a variety of physiological effects on the body that cannot be matched by other normal physiological flora, including: adjusting intestinal flora; improving protein and vitamin metabolism; preventing constipation; producing antibiotics; alleviating lactose intolerance; treating liver damage; reducing the incidence of colon cancer; anti-tumor; enhancing the immune system; lowering cholesterol; treating urinary tract infections, etc. However, there is a problem of limited conversion pathways in the conversion of ginsenosides by probiotics, that is, a single probiotic or a combination of probiotics cannot be converted to generate some rare ginsenosides, such as Lactobacillus bulgaricus cannot be converted to generate rare ginsenosides Rh2 and F2, Lactobacillus plantarum 581 cannot be converted to generate rare ginsenoside Rh2, and Lactobacillus plantarum atcc8014, Lactobacillus plantarum wcsf1, Lactococcus lactis atcc11454, Lactococcus lactis nz3900 and Lactobacillus delbrueckii or their combination cannot be used to convert and obtain rare ginsenosides F2 and CK. Meanwhile, thermophilic streptococci are commonly used probiotics for the preparation of fermented dairy products, but there is no report that thermophilic streptococci can be transformed into rare ginsenosides. Therefore, further development and utilization of probiotics, especially thermophilic streptococci to transform ginsenosides can not only obtain highly active rare ginsenosides, but also the fermentation liquid does not contain any substances harmful to the human body and nature, and can be directly used in the production of medicines or foods, reducing the occurrence of biological pollution. Summary of the invention

[0005] The present invention aims to provide a thermophilic streptococcus capable of transforming rare ginsenosides and its application.

[0006] A strain of Streptococcus thermophilus with the ability to transform rare ginsenosides, the strain is Streptococcus thermophilus 17140, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on September 23, 2024, and the deposit number is: GDMCC NO.65173.

[0007] An application of the thermophilic streptococcus having the function of transforming rare ginsenosides, and an application of the thermophilic streptococcus in transforming rare ginsenosides.

[0008] The rare ginsenosides are ginsenoside F2, Rg3, Rh2 and compound K (CK).

[0009] A thermophilic streptococcus agent with rare ginsenoside conversion function, the agent contains the thermophilic streptococcus.

[0010] The bacterial agent is a fermentation liquid, a crude enzyme liquid, a culture supernatant or a subculture liquid containing the strain.

[0011] The fermentation broth is obtained by inoculating a single bacterial colony into a fermentation medium for fermentation and culturing;

[0012] The obtained fermentation liquid is separated and the obtained bacteria are resuspended, and then crushed and centrifuged to remove the debris to obtain the crude enzyme solution;

[0013] The fermentation liquid is centrifuged and the supernatant is collected to obtain a culture supernatant;

[0014] The obtained fermentation liquid is further subcultured to obtain the subculture liquid.

[0015] Further, the fermentation broth is obtained by inoculating thermophilic Streptococcus 17140 in a culture medium and culturing and activating the culture medium until the OD 600 =0.6-0.8, inoculate in a fermentation tank at a ratio of 1-30%, and culture at 37°C and 150rpm until OD 600 >0.8, that is;

[0016] The fermentation liquid obtained above is centrifuged at 3000-12000 rpm to remove the bacterial cells, and the obtained supernatant;

[0017] The culture obtained by the above centrifugation was resuspended in 50-1000 mL of PBS buffer, ultrasonically disrupted in an ice-water bath, and centrifuged to remove bacterial fragments to obtain a crude enzyme solution.

[0018] The use of the bacterial agent in the transformation and preparation of rare ginsenosides; or, the use of the bacterial agent in the preparation of dairy products containing rare ginsenosides.

[0019] A method for preparing rare ginsenosides comprises inoculating the strain or the bacterial agent into a culture system to obtain a culture solution, and then adding a ginseng extract into the culture solution for further culture, thereby converting the ginsenosides in the ginseng extract to obtain the rare ginsenosides.

[0020] The strain or bacterial agent is inoculated into the culture system and cultured at 32°C to 47°C and pH 5.0 to 6.5 for 48 hours to 150 hours.

[0021] A method for preparing a dairy product containing rare ginsenosides comprises activating the strain or the bacterial agent respectively, adding ginseng extract to the system after activation and mixing, adding milk to the system after mixing for culturing, and obtaining the dairy product containing rare ginsenosides.

[0022] A method for preparing a dairy product containing rare ginsenosides comprises activating the strain or the bacterial agent respectively, mixing the mixture with probiotics, inoculating the mixture into a culture system containing milk, and adding ginseng extract for fermentation culture, so as to obtain a dairy product containing rare ginsenosides.

[0023] The above-mentioned ginseng extract is dammarane-type triterpenoid saponins, ginsenosides or notoginseng saponins, specifically: dammarane-type triterpenoid saponins are ginsenosides Rb1, Rb2, Rd, Rc, notoginseng saponins D, Fa, K, R4, T, L, O, P, Q, S, Fc, Fe, or ginseng extract containing glycol group saponins in ginsenosides or notoginseng saponins, American ginseng extract, notoginseng extract, japonicus extract, ginseng stem and leaf extract, American ginseng stem and leaf extract, notoginseng stem and leaf extract, and japonicus stem and leaf extract.

[0024] The advantages of the present invention are:

[0025] The thermophilic streptococcus 17140 of the present invention is obtained by screening from sauerkraut fermentation liquid. After being cultured, the ability of thermophilic streptococcus 17140 to transform rare ginsenosides is significantly improved, and the ability to transform and generate rare ginsenosides F2, Rh2, Rg3 and CK is 3.34 times, 2.4 times, 2.39 times and 2.24 times that of the 60th generation thermophilic streptococcus screened, respectively; based on ginsenoside Rb1, the molar conversion rate of rare ginsenosides reaches more than 38%. The thermophilic streptococcus is a commonly used probiotic. Using it as a fermentation bacterium can not only achieve a high rare ginsenoside conversion rate, but also achieve a conversion rate of nearly 85% with ginsenoside monomers as substrates, and its fermentation product can be used for medicine and food production without further treatment, and is environmentally friendly, free of biological pollution, and suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The embodiment of the present invention provides the effect of conversion time on the conversion of rare ginsenosides by Streptococcus thermophilus 17140 using ginsenoside Rb1 as substrate; wherein, CK: ginsenoside compound K; Rh2: ginsenoside Rh2; Rg3: ginsenoside Rg3; Rb1: ginsenoside Rb1.

[0027] Figure 2 The embodiment of the present invention provides the effect of pH on the transformation of rare ginsenosides by Streptococcus thermophilus 17140 using ginsenoside Rb1 as substrate; wherein, CK: ginsenoside compound K; Rh2: ginsenoside Rh2; Rg3: ginsenoside Rg3; Rb1: ginsenoside Rb1.

[0028] Figure 3 The embodiment of the present invention provides the effect of conversion temperature on the conversion of rare ginsenosides by Streptococcus thermophilus 17140 using ginsenoside Rb1 as substrate; wherein, CK: ginsenoside compound K; Rh2: ginsenoside Rh2; Rg3: ginsenoside Rg3; Rb1: ginsenoside Rb1.

[0029] Figure 4The thermophilic Streptococcus 17140 provided in an embodiment of the present invention transforms ginsenoside Rb1 in milk culture medium to produce rare ginsenosides; wherein, CK: ginsenoside compound K; Rh2: ginsenoside Rh2; Rg3: ginsenoside Rg3; Rb1: ginsenoside Rb1; 1: milk culture medium + ginsenoside Rb1: 2: milk culture medium.

[0030] Figure 5 The thermophilic Streptococcus 17140 provided in the embodiment of the present invention transforms the ginseng total saponin extract to increase the content of rare ginsenosides; wherein, CK: ginsenoside compound K; Rh2: ginsenoside Rh2; Rg3: ginsenoside Rg3; Rb1: ginsenoside Rb1; 1: milk culture medium + ginsenoside Rb1: 2: milk culture medium.

[0031] Figure 6 The invention provides a high-content rare ginsenoside acid cheese prepared by using thermophilic Streptococcus 17140. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. It should be clear to those skilled in the art that the following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0033] Example 1

[0034] Screening of Streptococcus thermophilus 17140:

[0035] The traditional method was used to make Chinese cabbage sauerkraut. The fermented cabbage sample was collected after fermentation for about 45 days. 1 mL of the fermented cabbage sample was added with sterile distilled water and diluted in a 10-fold gradient to prepare a dilution multiple of 10. -1 ~10 -9 A series of concentrations of diluted bacterial solution were prepared. 0.1 mL was applied to MRS solid separation medium and cultured at 37°C for 48 hours. Single colonies with typical characteristics of lactic acid bacteria were selected and repeatedly streaked on the medium until they were found to be pure strains under microscopic examination. Pure strains were inoculated onto MRS solid medium for esculin by plate coating method and cultured at 37°C for 48 hours. The strains whose medium color or colony color turned brown-black were selected. The selected strains had the ability to hydrolyze esculin to produce esculinogen.

[0036] The above-screened strains were determined to be Gram-positive and catalase-negative.

[0037] The above-screened strains were cultured in MRS medium for 48 hours, then centrifuged to remove the bacteria, ginsenoside Rb1 was added to the supernatant, and the culture was incubated at 37°C for 72 hours to screen out strains that can transform and produce ginsenosides Rg3, Rh2 and CK.

[0038] Then, the obtained strain was further domesticated into probiotics by carbon source stress method, that is, the strain was inoculated into 1 mL of MRS liquid medium at 1% and cultured at 37°C overnight. 1 / 3 of the bacterial liquid was discarded, and an equal volume of MRS medium without glucose was added, and then an appropriate amount of ginseng extract (the final concentration of ginsenosides was 1 mg·mL -1 ), 37°C, 200rpm, shake culture for 56h. Repeat the above operation for directional domestication. In order to meet the demand for its own carbon source, probiotics need to obtain the carbon source required for their own growth through enzymatic hydrolysis of saponins. Long-term directional evolution cultivation forces the expression of ginsenoside-related enzymes in the strain to increase, and directional cultivation of strain 17140 with high ginsenoside conversion ability is obtained.

[0039] The obtained strain 17140 was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on September 23, 2024. The address of the depository is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The deposit number is: GDMCC NO.65173; the Latin name is Streptococcus thermophilus.

[0040] MRS solid separation medium composition: peptone 10.0g, Tween-80 1.0mL, beef extract 10.0g, sodium acetate 5.0g, yeast extract 5.0g, dipotassium hydrogen phosphate 2.0g, diammonium hydrogen citrate 2.0g, magnesium sulfate 0.58g, glucose 20.0g, manganese sulfate 0.26g, agar powder 20.0g, dissolve each component in 1L distilled water and sterilize at 121℃ for 30min.

[0041] The composition of the solid culture medium for esculin MRS is as follows: 1.0 g of esculin, 0.5 g of ferric citrate, 10.0 g of peptone, 1.0 mL of Tween-80, 10.0 g of beef extract, 5.0 g of sodium acetate, 5.0 g of yeast extract, 2.0 g of dipotassium hydrogen phosphate, 2.0 g of diammonium hydrogen citrate, 0.58 g of magnesium sulfate, 20.0 g of glucose, 0.26 g of manganese sulfate, and 20.0 g of agar powder. Dissolve each component in 1 L of distilled water and sterilize at 121°C for 30 min.

[0042] The thermophilic streptococcus 17140 of the present invention has the following biological characteristics:

[0043] Morphological characteristics: The strain is Gram-positive, arranged singly, and spherical.

[0044] Culture characteristics: After the strain was cultured at 37℃ on MRS solid separation medium for 48 hours, the colonies were round, medium-sized, raised, slightly white, moist, and had neat edges.

[0045] Genetic characteristics (strain 16S rRNA gene sequence): The 16S rRNA gene sequence of strain 17140 is 1433 bp long and the sequence is as follows.

[0046]

[0047] Example 2 Transformation of Ginsenoside Rb1 by Streptococcus thermophilus 17140

[0048] 1) Preparation of fermentation liquid of Streptococcus thermophilus 17140

[0049] The thermophilic Streptococcus 17140 stored in MRS solid medium was transferred to 10 mL of MRS liquid medium and cultured at 37 °C for about 12 h until OD 600 =0.6 for activation. After activation, inoculate into 50 mL MRS liquid culture medium at a volume percentage of 2%, culture in a constant temperature shaking incubator at 37°C for 48 hours, centrifuge the culture solution and take the supernatant. Add the supernatant to an Amicon Ultra-15ml50kDa ultrafiltration tube, centrifuge at 7000×g, 4°C for 20 minutes, and obtain the fermentation conversion liquid of Streptococcus thermophilus 17140.

[0050] 2) Effect of transformation time on transformation efficiency

[0051] Take 4 portions of 500 μL fermentation conversion liquid, add 1 mg of ginsenoside Rb1, adjust pH to 5.5 with acetic acid-sodium acetate buffer, mix well, and place in a constant temperature incubator at 37°C for fermentation for 12, 24, 48 and 72 hours. After the fermentation is completed, centrifuge at 10,000×g for 15 minutes, collect the supernatant, mix n-butanol with the supernatant in a ratio of 1:1 (v / v), and sonicate for 10 minutes. After standing, take the n-butanol layer, repeat three times, combine the n-butanol layers, place them in a rotary evaporator to recover the solvent until dry, and add methanol to re-dissolve. Use a spotting capillary to absorb the standard and sample, and spot them on the thin layer chromatography plate. The developing solvent is chloroform: methanol: water = 65:35:10 (v / v). Take the lower layer, and after the chromatography plate is developed, take it out, evaporate the solvent, spray 10% sulfuric acid-ethanol color developer, and color at 105°C for 5 minutes (see Figure 1 ). The result is Figure 1 As shown, Rg3 was generated when the fermentation conversion time was 24 h. When the conversion time reached 48 h, Rg3, Rh2 and CK were detectable in the product, and Rb1 was completely converted.

[0052] 3) Effect of pH on transformation efficiency

[0053] Take 4 portions of 500 μL fermentation conversion liquid, add 1 mg of ginsenoside Rb1 respectively, and adjust the pH of each sample to 4.5, 5.0, 5.5, 6.0 and 6.5 with acetic acid-sodium acetate buffer, mix well and place in a constant temperature incubator at 37°C for 48 hours. After the fermentation is completed, centrifuge at 10000×g for 15 minutes, collect the supernatant, mix n-butanol with the supernatant in a ratio of 1:1 (v / v), and sonicate for 10 minutes. After standing, take the n-butanol layer, repeat three times, combine the n-butanol layers, place them in a rotary evaporator to recover the solvent until dry, and add methanol to re-dissolve. Use a spotting capillary to absorb the standard and sample, and spot it on the thin layer chromatography plate. The developing solvent is chloroform: methanol: water = 65:35:10 (v / v), take the lower layer, and after the chromatography plate is developed, take it out, evaporate the solvent, spray 10% sulfuric acid-ethanol color developer, and color at 105°C for 5 minutes (see Figure 2 ). The result is Figure 2 As shown, when the pH is between 5.0 and 6.5, the fermentation conversion liquid can convert the substrate ginsenoside Rb1 to produce ginsenosides Rh2, Rg3, and CK. When the pH is 5.5, ginsenoside Rb1 is almost completely converted.

[0054] 4) Effect of conversion temperature on conversion efficiency

[0055] Take 4 portions of 500 μL fermentation conversion liquid, add 1 mg of ginsenoside Rb1, adjust pH to 5.5 with acetic acid-sodium acetate buffer, mix well and place in a constant temperature incubator at 27°C, 32°C, 37°C, 42°C and 47°C for 48 hours. After the fermentation is completed, centrifuge at 10,000×g for 15 minutes, collect the supernatant, mix n-butanol with the supernatant in a ratio of 1:1 (v / v), and sonicate for 10 minutes. After standing, take the n-butanol layer, repeat three times, combine the n-butanol layers, place them in a rotary evaporator to recover the solvent until dry, and add methanol to re-dissolve. Use a spotting capillary to absorb the standard and sample, and spot on the thin layer chromatography plate. The developing solvent is chloroform: methanol: water = 65:35:10 (v / v). Take the lower layer, and after the chromatography plate is developed, take it out, evaporate the solvent, spray 10% sulfuric acid-ethanol color developer, and color at 105°C for 5 minutes. The results are as follows Figure 3 As shown, the fermentation conversion liquid can convert ginsenoside Rb1 to Rh2, Rg3, and CK at a fermentation temperature of 32°C to 47°C. When the conversion temperature is 37°C, the substrate Rb1 is basically completely converted.

[0056] MRS solid separation medium composition: peptone 10.0g, Tween-80 1.0mL, beef extract 10.0g, sodium acetate 5.0g, yeast extract 5.0g, dipotassium hydrogen phosphate 2.0g, diammonium hydrogen citrate 2.0g, magnesium sulfate 0.58g, glucose 20.0g, manganese sulfate 0.26g, agar powder 20.0g, dissolve each component in 1L distilled water and sterilize at 121℃ for 30min.

[0057] The composition of MRS solid separation medium: peptone 10.0 g, Tween-80 1.0 mL, beef extract 10.0 g, sodium acetate 5.0 g, yeast extract 5.0 g, dipotassium hydrogen phosphate 2.0 g, diammonium hydrogen citrate 2.0 g, magnesium sulfate 0.58 g, glucose 20.0 g, manganese sulfate 0.26 g. Dissolve each component in 1 L of distilled water and sterilize at 121°C for 30 min.

[0058] Example 3: Transformation of ginsenoside Rb1 by Streptococcus thermophilus 17140 in milk culture medium to produce rare ginsenosides

[0059] Pure milk was diluted with sterile water at a ratio of 1:3 (v / v), and the Streptococcus thermophilus 17140 bacterial solution (OD 600 =0.6), inoculation amount 2% (volume percentage), placed in a 37°C constant temperature incubator at 200rpm for 48h. Take 5mL of the above fermentation broth and add 0.5mg of ginsenoside Rb1, adjust the initial pH to 5.8 with acetic acid-sodium acetate buffer, mix evenly and place in a 37°C constant temperature incubator at 200rpm for 56h. Add 6mL of acetonitrile to terminate the reaction, mix evenly and ultrasonicate for 30min, centrifuge and take the supernatant, repeat three times, combine the supernatants, place in a rotary evaporator to recover the solvent until dry, and add 1mL of methanol to dissolve. Use a spotting capillary to absorb the standard and sample, and spot it on the thin layer chromatography plate. The developing solvent is chloroform: methanol: water = 65:35:10 (v / v), take the lower layer, and after the chromatography plate is developed, take it out, evaporate the solvent, spray 10% sulfuric acid-ethanol color developer, and color at 105°C for 5min (see Figure 4 ). The result is Figure 4 As shown, Streptococcus thermophilus 17140 can transform ginsenoside Rb1 into rare ginsenosides CK, Rg3, and Rh2 in milk culture medium.

[0060] Example 4: Transformation of ginsenoside extract by Streptococcus thermophilus 17140 to increase the content of rare ginsenosides

[0061] Take 1g of red ginseng powder (passed through a No. 4 sieve), weigh it accurately, put it in a Soxhlet extractor, add 50mL of chloroform and heat it under reflux for 3h, discard the chloroform solution, evaporate the solvent from the residue, move it into a 100mL conical flask together with the filter paper tube, accurately add 50mL of water-saturated n-butanol, plug it tightly, leave it overnight, ultrasonically treat it (power 250W, frequency 50kHz) for 30min, filter it, and evaporate the filtrate in an evaporating dish. Using vanillin-sulfuric acid as a colorimetric agent, the total ginsenoside content in the ginsenoside extract was determined by colorimetry at 544nm. The total ginsenoside content was 79% based on ginsenoside Rb1.

[0062] Pure milk was diluted with sterilized water at a ratio of 1:3 (v / v), and the thermophilic Streptococcus 17140 solution (OD 600 =0.6), placed in a 37°C constant temperature incubator at 200rpm and cultured for 48h. Take 5mL of the above fermentation broth and add 4mg of ginseng total saponin extract, adjust the initial pH to 5.8, mix evenly and place in a 37°C constant temperature incubator at 200rpm and culture for 56h. Add an equal volume of acetonitrile to terminate the conversion reaction, mix evenly with the fermentation broth and ultrasonicate for 30min, centrifuge and take the supernatant, repeat three times, combine the supernatants, place in a rotary evaporator to spin dry the solvent, and add 1mL of methanol to dissolve. Use a spotting capillary to absorb the standard and sample, and spot it on the thin layer chromatography plate. The developing solvent is chloroform: methanol: water = 65:35:10 (v / v). Take the lower layer. After the chromatography plate is developed, take it out, evaporate the solvent, spray 10% sulfuric acid-ethanol color developer, and color at 105°C for 5min (see Figure 5 ). The result is Figure 5 As shown, compared with the unconverted ginsenoside extract, Streptococcus thermophilus 17140 can transform ginsenosides and significantly increase the content of ginsenosides CK, Rg3, and Rh2.

[0063] HPLC was used to detect the contents of Rg3, Rh2 and CK before and after transformation. The results showed that the content of Rg3 in the total ginsenoside extract before transformation was 17.85 μg mL -1 , Rh2 content is 14.76μg·mL -1 CK was not detected; after transformation by thermophilic Streptococcus, the contents of Rg3, Rh2 and CK were 36.62 μg·mL -1 , 42.56 μg·mL -1 and 29.97 μg mL -1 Compared with before transformation, the contents of Rg3 and Rh2 increased by 105.13% and 188.21%, respectively.

[0064] Example 5 Subculture broth of Streptococcus thermophilus 17140

[0065] Weigh an appropriate amount of red ginseng powder, use water as the extraction solvent, extract 4 times at a solid-liquid ratio of 1:30 (g:mL), each time for 2 hours to obtain a red ginseng water extract. The total ginsenoside content in the red ginseng water extract was determined by colorimetry to be 4.03%. The HPLC method showed that the obtained red ginseng water extract did not contain rare ginsenoside Rh2, but only contained trace amounts of rare ginsenoside F2.

[0066] Take thermophilic Streptococcus stored at -80℃, streak it on MRS solid culture medium, and culture it at 37℃ for 20h. Pick a single colony and place it in 3mL of MRS liquid culture medium. Culture it at 37℃, 200rpm, shake it overnight to activate the strain. Take the activated bacterial solution at a 1% (volume percentage) inoculation amount and add it to 1mL of MRS liquid culture medium for secondary activation as the subsequent subculture bacterial solution. Centrifuge the subculture bacterial solution at 4℃, 3000rpm for 1min, discard 1 / 3 volume of the culture solution, add an equal volume of MRS culture medium without glucose, and then add red ginseng water extract until the final concentration of total ginsenosides is 1mg·mL -1 , 37°C, 200 rpm, shake culture for 56 h. Centrifuge at 4°C, 3000 rpm for 1 min, discard 1 / 3 of the culture medium, add an equal volume of MRS medium without glucose, and supplement with red ginseng water extract until the final concentration of total ginsenosides is about 1 mg·mL -1 , 37℃, 200rpm, shaking culture for 56h. Each 56h culture was defined as one generation, and a total of 140 generations were directed evolution cultured. 60, 80, 100, 120 and 140 generations of directed cultured thermophilic Streptococcus were retained. The increase in ginsenoside F2 and the production of ginsenoside Rh2 were used as indicators to evaluate the transformation efficiency of thermophilic Streptococcus cultivated by directed evolution. The production of ginsenoside Rh2 and the increase in F2 produced by thermophilic Streptococcus cultivated by directed evolution for 60 to 140 generations are shown in Table 1.

[0067] Table 1 Detection of the ability of Streptococcus thermophilus 17140 to produce rare ginsenosides after 60-140 generations of directed evolution

[0068]

[0069] Through directed evolution, the ability of the 140th generation strain to transform into F2 was 223% of the 60th generation strain in terms of the increase in ginsenoside F2, an increase of 123%; the ability of the 140th generation strain to transform into Rh2 was 160% of the 60th generation strain in terms of the generation of ginsenoside Rh2, an increase of 60%. Using the carbon source stress method, the conversion rate of rare ginsenosides of Streptococcus thermophilus 17140 can be significantly improved through directed evolution.

[0070] Example 6 Application of Streptococcus thermophilus 17140 in the preparation of high-content rare ginsenoside cheese

[0071] Take the thermophilic Streptococcus 17140 stored at -80℃, streak it on MRS solid medium, and culture it at 37℃ for 16h. Pick a single colony and place it in 3mL MRS liquid medium. Culture it at 37℃, 200rpm, shake overnight. Inoculate the bacterial solution with 1% (volume percentage) inoculation into 5mL MRS liquid medium, shake and culture for 5-6h until OD600 =0.6. Centrifuge 5 mL of bacterial solution at 4°C, 2900×g for 15 min, discard the supernatant, and resuspend the cells in sterile water to OD 600 =0.6, and 0.2 mol·L -1 Sodium phosphate buffer (1.22 g NaH2PO4·2H2O and 4.37 g Na2HPO4·12H2O were weighed, dissolved in distilled water and fixed to 100 mL) to adjust the pH of the bacterial solution to 5.8. The above bacterial solution was inoculated at 5% (volume percentage) of the total reaction volume, and the red ginseng water extract obtained in Example 5 was added until the final concentration of ginsenosides was 2 mg·mL -1 At the same time, add pasteurized fresh milk with a final concentration of 50%, and culture at 37°C and 200rpm under anaerobic conditions for 144 hours. Under sterile conditions, add 15% white sugar, mix well, and continue to ferment for 12 hours.

[0072] The content of rare ginsenosides in yogurt was determined by HPLC. The contents of ginsenosides F2, Rg3, Rh2, and CK were 43.32 μg mL -1 , 275.16μg·mL -1 , 26.67 μg·mL -1 and 23.9 μg mL -1 , the content of F2 increased by 36.21%, the content of Rg3 increased by 139.74%, and the content of Rh2 increased by 142.96%. No CK was detected in the ginsenoside extract before fermentation. The number of lactic acid bacteria in yogurt was detected by MRS solid medium culture counting, and 5×10 6 CFU·mL -1 .

[0073] The prepared yogurt is milky white, colorful, uniform, does not separate after long-term storage, and has good flavor ( Figure 6 ).

[0074] Example 7 Preparation of ginseng fermented milk rich in rare ginsenosides by using Streptococcus thermophilus 17140 together with Lactobacillus plantarum and Lactobacillus bulgaricus

[0075] Take thermophilic Streptococcus 17140, Lactobacillus plantarum and Lactobacillus bulgaricus stored at -80℃ and streak them on MRS solid medium respectively, incubate in a constant temperature incubator at 37℃ for 16h, pick a single colony, place it in 3mL MRS liquid medium, shake and culture it at 37℃, 200rpm overnight. Inoculate 1% of the inoculum into 5mL MRS liquid medium, shake and culture for 5-6h until OD 600 =0.6.

[0076] Centrifuge the activated bacterial suspensions at 4°C, 2900 × g for 5 min, discard the supernatant, and resuspend the cells in appropriate amount of sterile water to an OD of 600＝ A composite bacterial solution was prepared according to the volume ratio of thermophilic Streptococcus 17140: Lactobacillus plantarum: Lactobacillus bulgaricus of 2:1:1, and added to 50% pasteurized fresh milk at an inoculum amount of 5% (volume percentage), and the red ginseng water extract obtained in Example 5 above was added until the final concentration of ginsenosides was 5 mg·mL -1 , 37°C, 200rpm aerobic shaking culture for 144h. The fermentation liquid was added to pasteurized fresh milk at a 1% inoculum, mixed evenly, and then fermented at 40°C for 12h to obtain ginseng fermented milk (containing 50μg / ml ginsenosides).

[0077] The content of rare ginsenosides in fermented milk was determined by HPLC, and the content of rare ginsenoside F2 was 0.75 μg g -1 ; The content of Rg3 is 4.92μg·g -1 ; Rh2 content is 0.35μg·g -1 ; CK content is 0.63μg·g -1 The total amount of rare ginsenosides increased by 333% after fermentation. The number of lactic acid bacteria in fermented milk was detected to be 3×10 7 CFU·mL -1 .

Claims

1. A thermophilic Streptococcus capable of transforming rare ginsenosides, characterized in that: The strain is Streptococcus thermophilus 17140, which was deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on September 23, 2024, with the deposit number: GDMCC NO.65173.

2. An application of the thermophilic Streptococcus having rare ginsenoside conversion function as claimed in claim 1, characterized in that: The application of the thermophilic streptococcus in transforming rare ginsenosides.

3. A thermophilic streptococcus agent having rare ginsenoside conversion effect, characterized in that: The bacterial agent contains the thermophilic streptococcus described in claim 1.

4. The bacterial agent according to claim 3, characterized in that: The bacterial agent is a fermentation liquid, a crude enzyme liquid, a culture supernatant or a subculture liquid containing the strain.

5. The bacterial agent according to claim 3, characterized in that: The fermentation broth is obtained by inoculating a single bacterial colony into a fermentation medium for fermentation and culturing; The obtained fermentation liquid is separated and the obtained bacteria are resuspended, and then crushed and centrifuged to remove the debris to obtain the crude enzyme solution; The fermentation liquid is centrifuged and the supernatant is collected to obtain a culture supernatant; The obtained fermentation liquid is further subcultured to obtain the subculture liquid.

6. An application of the bacterial agent according to claim 3, characterized in that: The use of the bacterial agent in the transformation and preparation of rare ginsenosides; or, the use of the bacterial agent in the preparation of dairy products containing rare ginsenosides.

7. A method for preparing rare ginsenosides, characterized in that: The strain described in claim 1 or the bacterial agent described in claim 3 is inoculated into a culture system to obtain a culture solution, and then ginseng extract is added to the culture solution for further cultivation, thereby realizing the conversion of ginsenosides in ginseng extract to obtain rare ginsenosides.

8. The method for preparing rare ginsenosides according to claim 7, characterized in that: The strain or bacterial agent is inoculated into a culture system and cultured at 32°C to 47°C and a pH of 5.0 to 6.

5.

9. A method for preparing a dairy product containing rare ginsenosides, characterized in that: The strain described in claim 1 or the bacterial agent described in claim 3 is activated respectively, and after activation, ginseng extract is added to the system for mixing, and after mixing, milk is added thereto for culturing, so as to obtain a dairy product containing rare ginsenosides.

10. A method for preparing a dairy product containing rare ginsenosides, characterized in that: The strain described in claim 1 or the bacterial agent described in claim 3 is activated separately, mixed with probiotics, inoculated into a culture system containing milk, and ginseng extract is added for fermentation culture to obtain a dairy product containing rare ginsenosides.

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