Bacillus licheniformis KZQ-5 and application thereof in fermentation of American ginseng

Abstract

The invention discloses a bacillus licheniformis KZQ-5 with the preservation number of CGMCC No.27417, and relates to the technical field of microbial fermentation of American ginseng and screening of related strains. The bacillus licheniformis KZQ-5 is good in safety, is used for fermentation of American ginseng, can remarkably improve the content of active ingredients and enhance the antioxidant activity of American ginseng, and has certain repairing capacity for oxidative damage cells. The fermentation process is simple, no allogenic material is added, large-scale production is easy to realize, and the fermentation product can meet the application field requirements of cosmetics and health care products.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation of American ginseng and related strain screening, and more specifically to a strain of Bacillus licheniformis KZQ-5 and application thereof in fermenting American ginseng. Background Art

[0002] American ginseng, also known as American ginseng, is a perennial herbaceous plant of the Araliaceae family and the genus Panax. It is a traditional precious Chinese medicinal material and a very commonly used agricultural product ingredient. It can be used as both medicine and food and has a wide range of uses. American ginseng is cool in nature, sweet and slightly bitter in taste, and has the effects of replenishing qi and nourishing yin, clearing away heat and promoting fluid production. It is mainly used for qi deficiency and yin deficiency syndrome. American ginseng was first recorded as a medicinal material in "Supplement to Compendium of Materia Medica". "Medical Records of Chinese and Western Medicine" said that it "is cool in nature and replenishing. Anyone who wants to use ginseng but cannot tolerate the warming effect of ginseng can use this instead". It has the replenishing nature of ginseng but not the dryness of ginseng. It is a cold tonic product with a wide range of clinical applications. The main components of American ginseng, such as saponins and polysaccharides, have been proven to have a variety of pharmacological activities, such as improving cardiovascular system diseases, especially in atherosclerotic diseases, and have anti-inflammatory effects, enhancing antioxidant enzyme activity, protecting endothelial cell function, and inhibiting vascular smooth muscle cell migration and proliferation.

[0003] Traditionally, the extraction of active ingredients of American ginseng adopts water extraction or alcohol extraction, and the extraction efficiency is not high. Later studies introduced microbial fermentation extraction, which improved the extraction effect of active ingredients of American ginseng. For example, CN 107550961 B discloses a method for preparing ginseng / American ginseng extract by using microbial rapid fermentation, wherein ginseng / American ginseng is fermented with probiotics and yeast to obtain enzyme liquid. The method extracts ginseng / American ginseng extract with high content of active ingredients, short production cycle, and can realize automated production; CN 108969430 B discloses a mixed fermentation of American ginseng with Lactobacillus plantarum, Leuconostoc mesenteroides, and Lactobacillus acidophilus, and uses it as a skin care product or skin care product additive. The obtained skin care product can stimulate collagen formation and reduce collagen degradation, and has an improvement effect on red blood streaks, wrinkles, acne, and pigmentation; CN 107375374B discloses a fermentation extraction method of American ginseng extract with the effect of assisting in improving sleep, wherein American ginseng is subjected to secondary fermentation by Aspergillus oryzae to produce functional food containing GABA, thereby assisting in improving sleep. The above patents ferment American ginseng by single or multiple mixed fermentation of yeast, lactobacillus, mold, etc. to obtain a fermentation product with a higher content of active ingredients. There is no report on the fermentation of American ginseng by Bacillus licheniformis. Summary of the invention

[0004] In view of this, the present invention provides a strain of Bacillus licheniformis KZQ-5 and application thereof in fermenting American ginseng.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A strain of Bacillus licheniformis KZQ-5, the strain preservation number of which is CGMCC No. 27417, was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on May 24, 2023, and was classified and named Bacillus licheniformis.

[0007] Another object of the present invention is to provide a microbial agent comprising the above-mentioned Bacillus licheniformis KZQ-5.

[0008] Another object of the present invention is to provide the use of the above-mentioned Bacillus licheniformis KZQ-5 or the above-mentioned microbial agent.

[0009] As a preferred solution, the application is any one of the following:

[0010] A. The amount of active ingredients extracted from ginseng plants;

[0011] B. Improve the antioxidant activity of active ingredients in ginseng plants;

[0012] C. Improve the ability of ginseng plant active ingredients to repair oxidatively damaged cells.

[0013] More preferably, the ginseng plant is American ginseng.

[0014] Another object of the present invention is to provide a method for extracting active ingredients from ginseng plants, using the above-mentioned Bacillus licheniformis KZQ-5 or the above-mentioned microbial agent to ferment ginseng plants, taking the supernatant of the fermentation liquid, freeze-drying, and obtaining the active ingredients.

[0015] As a preferred solution, in the method for extracting active ingredients from ginseng plants, the fermentation temperature is 28° C., the fermentation time is 48-168 hours, and the pH value of the fermentation liquid is 6-9.

[0016] As a preferred solution, in the method for extracting active ingredients from ginseng plants, the Bacillus licheniformis KZQ-5 or the microbial agent is first activated for two generations before preparing the seed liquid for fermentation. ;

[0017] In the method for extracting active ingredients from ginseng plants, the ginseng plants are prepared into powder, and then prepared into a 2-10% suspension with water, and after ultrasonic mixing, the pH is adjusted to 6-9 and sterilized;

[0018] Moreover, the volume ratio of the seed solution to the ginseng plant suspension is 1:10-1:50.

[0019] As a preferred embodiment, in the method for extracting active ingredients from ginseng plants, the ginseng plant is American ginseng.

[0020] Another object of the present invention is to provide the use of the product extracted by the above extraction method in the preparation of cosmetics and health products.

[0021] Through the above technical scheme, it can be known that compared with the prior art, the licheniformis KZQ-5 isolated and purified from the root tissue of wild button seven in Qinling Mountains in the present invention has good safety and can be used for American ginseng fermentation to significantly increase the content of active ingredients, enhance its antioxidant activity, and have a certain ability to repair oxidatively damaged cells. The fermentation process of the present invention is simple, without any exogenous substances added, and it is easy to achieve large-scale production. The fermentation product can meet the application needs of cosmetics and health products. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0023] Figure 1 The colony morphology characteristics of strain KZQ-5;

[0024] Figure 2 is the Gram staining characteristics of strain KZQ-5;

[0025] Figure 3 is the phylogenetic tree of strain KZQ-5;

[0026] Figure 4 is the hemolytic activity of strain KZQ-5;

[0027] Figure 5 It is the product of American ginseng fermented by strain KZQ-5;

[0028] Figure 6 The content of active ingredients in the fermentation broth of American ginseng fermented by strain KZQ-5 at different time points; * indicates p<0.05 compared with unfermented American ginseng.

[0029] Figure 7 The antioxidant activity of the fermentation products of American ginseng fermented by strain KZQ-5 at different time points; * indicates p < 0.05 compared with unfermented American ginseng.

[0030] Figure 8 This is the repair effect of the American ginseng fermented by strain KZQ-5 on cell oxidative damage. * indicates p<0.05 compared with the normal group, # indicates p<0.05 compared with the model, and Δ indicates p<0.05 compared with the same concentration of unfermented American ginseng. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example 1

[0033] Isolation and identification of strain KZQ-5

[0034] (1) Isolation and purification of strains: The root tissue of wild Psoralea corylifolia collected from Qinling Mountains was cut into 1 cm pieces, and the surface was disinfected with 75% ethanol for 1 min, 2.5% sodium hypochlorite for 2 min, and 75% ethanol for 1 min, respectively. Finally, it was washed with sterile water for 3-5 times and placed in a pre-sterilized culture dish. The tissue block was added to LB liquid culture medium with sterile tweezers, and cultured at 28°C and 180 rpm for 48 h. After gradient dilution, it was spread on solid LB culture medium, and a single colony was picked for purification, Gram staining and observation under a microscope; Gram-positive strains with a rod-shaped microscopic morphology were selected and frozen at -80°C with 25% glycerol for future use (for colony characteristics, see Appendix Figure 1 , Gram staining characteristics see Appendix Figure 2 ).

[0035] (2) Strain identification: The DNA of strain KZQ-5 was extracted using a bacterial genome extraction kit (TaKaRaMiniBEST Bacterial GenomicDNA Extraction kit ver2.0), and PCR amplification was performed using bacterial 16S rDNA universal primers: upstream primer 27F: AGAG TTTG ATCM TGGC TCAG; downstream primer 1492R: GGTT ACCT TGTT ACGACTT. PCR amplification reaction system (50 μL): 5.0 μL 10× Taq enzyme buffer, 0.4 μmol / L primer, 4 μL 200 mmol / L dNTP, 40 ng DNA template, 1U Taq enzyme (TaKaRa), 34 μL ddH 2O. PCR reaction program: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 55℃ annealing for 1min, 72℃ extension for 1min, 35 cycles; finally 72℃ extension for 5min. The reaction was carried out on a Bio-RadPCR instrument, and the 16S rDNA amplified fragment was detected by 1% agarose gel electrophoresis and sent to Shenzhen BGI Genomics Co., Ltd. for sequencing. The sequencing results were compared with the sequences in GenBank by Blast. The results showed that the 16SrDNA gene sequence of the strain of the present invention (as shown in SEQ ID NO.1) had the highest similarity with the gene sequence of Bacillus licheniformis strain ATCC 14580, which was 99%. The phylogenetic tree was constructed using MEGA-X software (see Appendix). Figure 3 ), it can be known that the strain is Bacillus licheniformis.

[0036]

[0037] The strain KZQ-5 was deposited on May 24, 2023 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101, with the deposit number CGMCC No. 27417 and the classification name Bacillus licheniformis.

[0038] Example 2

[0039] Hemolytic activity assay of strain KZQ-5

[0040] KZQ-5 was activated in YPD medium, LGG was activated in MRS medium, and Staphylococcus aureus (CGMCC 1.0089) was activated in LB medium. All the above strains were activated to the second generation before the experiment.

[0041] Place the blood plate on a clean bench and sterilize it under ultraviolet light for 15 to 30 minutes. Use a sterilized bamboo stick to streak the second-generation KZQ-5, LGG, and Staphylococcus aureus on the blood plate. Place KZQ-5 upside down in a 28°C incubator, and place LGG and Staphylococcus aureus in a 37°C incubator. Cultivate for 24 hours, observe the results, and take photos for record.

[0042] Result judgment basis: β-hemolytic activity: a transparent circle appears around the colony, and no red blood cells remain around it.

[0043] The results show that (see Appendix Figure 4 ), the control Staphylococcus aureus produced a transparent circle on the blood plate, which had β-hemolytic activity, while the strain KZQ-5 and the control strain LGG had no transparent circle around their bodies, so they did not have β-hemolytic activity and were safe.

[0044] Example 3

[0045] Fermentation of American ginseng by strain KZQ-5

[0046] (1) Seed solution preparation: The strain KZQ-5 stored at -80°C was activated for two generations, inoculated into YPD medium, and cultured in a shaking incubator at 28°C and 180 rpm for 24 h to obtain seed solution;

[0047] (2) Take 10 g of American ginseng powder, add ultrapure water to 200 mL, prepare an American ginseng suspension with a mass concentration of 5%, mix well and adjust the solution pH to 7.0, sterilize at 121°C for 20 min, place at room temperature to cool, add KZQ-5 seed solution at a volume ratio of 1:24, and culture at 28°C, 180 rpm on a shaker for 72 h;

[0048] (3) After the fermentation is completed, the fermentation liquid is collected, treated in a 90°C water bath for 30 min, and freeze-dried at -80°C to obtain the fermentation product (see Appendix Figure 5 ).

[0049] Example 4

[0050] Determination of active ingredients in American ginseng fermented by strain KZQ-5

[0051] In order to compare the changes in the content of effective ingredients and antioxidant activity in the products of American ginseng before and after fermentation, the following experiments were performed to demonstrate the ability of strain KZQ-5 to ferment American ginseng.

[0052] Prepare 5% American ginseng culture medium, adjust the pH of the mixture to 7.0, sterilize with high pressure steam at 121°C for 20 min, and then add the strain KZQ-5 seed solution at a volume ratio of 4%. Cultivate in a shaking table at 28°C and 180 rpm for 7 days, and take samples on days 1, 3, 4, 5, 6, and 7 for subsequent testing.

[0053] 1. Determination of total phenol, total flavonoids and total saponin content in fermentation broth

[0054] Total phenol determination: Take 50 μL of each sample and add 1 mL of 2% Na 2 CO 3 , shake and mix, let stand for 3 minutes, then add 50 μL of 50% Folin phenol reagent, let stand at 25°C for 30 minutes, add 200 μL of reaction solution to each well of the 96-well plate, detect the absorbance at a wavelength of 750 nm, and calculate the total phenol content in the sample according to the standard curve.

[0055] Total flavonoids determination: Take 100 μL of each sample and add 20 μL 5% NaNO 2 , 800 μL 60% ethanol, mixed and incubated at room temperature for 6 minutes, then added 20 μL 10% AlCl 3 , 60 μL 4% NaOH, incubated at 25°C for 30 minutes, added 200 μL of reaction solution to each well of the 96-well plate, detected the absorbance at a wavelength of 405 nm, and calculated the total flavonoid content in the sample according to the standard curve.

[0056] Determination of total saponins: Take 500 μL of each sample, add 0.525 mL of 5% vanillin-glacial acetic acid solution and 2.5 mL of perchloric acid solution, add 200 μL of reaction solution to each well of a 96-well plate, detect the absorbance at a wavelength of 540 nm, and calculate the total saponin content in the sample according to the standard curve.

[0057] The results are attached Figure 6As shown, it can be seen that compared with unfermented American ginseng, the total flavonoid content did not change significantly after 1 day of fermentation, but on the third day of culture, the total flavonoid content increased significantly, from 0.013 mg / mL at the initial stage to 0.043 mg / mL. Similarly, the total phenol content of unfermented American ginseng was 0.024 mg / mL, and the content reached 0.131 mg / mL after 3 days of fermentation. The total saponin content in unfermented American ginseng was 0.646 mg / mL at the initial stage, and the saponin content reached 2.349 mg / mL after 3 days of fermentation. It can be seen that KZQ-5 fermentation can significantly increase the content of active substances in American ginseng.

[0058] 2. Determination of antioxidant activity of fermentation broth

[0059] Sample preparation: 4 mL of fermented liquid and unfermented liquid were taken and divided into two equal parts for freeze drying. After freeze drying, 2 mL of ultrapure water was added to each part and 2 mL of methanol was added to the other part. Ultrasonic extraction was performed at 60°C for 4 h, and then centrifuged at 5000 rpm for 10 min at room temperature. The supernatant was taken and filtered with a 0.22 μm filter membrane to obtain the water extract (W) and alcohol extract (M) of the sample.

[0060] ABTS free radical scavenging test: Take 7mM ABTS stock solution and 2.45mM potassium persulfate solution, mix them and react, then keep them away from light for 14 hours to obtain ABTS+ mother solution, which should be stored away from light. Add PBS to the ABTS+ mother solution to dilute it to an absorbance of 0.7 to obtain ABTS+ working solution. Add 50μL sample solution to a 96-well plate, then add 150μL ABTS+ working solution, and react at room temperature away from light for 5 minutes. Detect the absorbance at a wavelength of 734nm to calculate the ABTS free radical scavenging rate.

[0061] DPPH free radical scavenging test: Weigh 0.004g DPPH and dissolve it in anhydrous ethanol and mix well. Then dilute it to 100mL with anhydrous ethanol and store it in the dark. Add 50μL of fermentation sample alcohol extract to a 96-well plate, then add 150μL DPPH solution and react at room temperature in the dark for 30min. Detect the absorbance at a wavelength of 517nm and calculate the DPPH free radical scavenging rate.

[0062] The results are attached Figure 7As shown, it can be seen that the antioxidant activity of the methanol extract of American ginseng (M) is low. After fermentation, its antioxidant activity increases significantly. The scavenging ability of the American ginseng extract for 3 days on DPPH free radicals reaches more than 60%, and the ABTS free radical scavenging activity of the American ginseng methanol extract (M) after 3 days of fermentation is also increased from about 20% of the unfermented American ginseng to more than 85%. The water extract of unfermented American ginseng (W) has a high ABTS free radical scavenging activity (>50%), which is still significantly improved after 3 days of fermentation, and the antioxidant activity can also reach more than 90%. It can be seen that the product of American ginseng fermented by Bacillus licheniformis KZQ-5 has good in vitro antioxidant activity.

[0063] 3. Repair effect of fermentation products on oxidatively damaged cells

[0064] After the fermentation liquid was freeze-dried, a certain mass of freeze-dried powder was weighed, 100 mL of deionized water was added, and after ultrasonic extraction for 2 h, the supernatant was centrifuged and concentrated to 100 mg / mL sample mother liquor by rotary evaporation and stored at 4°C for later use. The sample liquid of unfermented American ginseng that had been treated in the same way was used as a control.

[0065] NIH-3T3 cells were added to 96-well plates at a rate of 5000 cells / well. After the cells adhered to the wall, the supernatant was discarded. Except for the normal group, the other groups were added with 0.98 mM H 2 O 2 The cells were pretreated with culture medium for 1 hour, and then the supernatant was removed and rinsed with PBS. DMEM complete culture medium (containing 10% FBS and 1% penicillin-streptomycin mixture) was added to both the normal group and the model group, and different concentrations of fermented and unfermented American ginseng dilutions (0.01 mg / mL, 0.05 mg / mL, and 0.1 mg / mL) were added to the experimental group. After culturing for 24 hours, MTT was used to detect cell activity.

[0066] The results are attached Figure 8 As shown, H 2 O 2 The cell activity of the model group and the experimental group after treatment was significantly decreased compared with the normal group, down to about 40%. The cell activity of the non-fermented American ginseng (NF) treatment did not increase significantly compared with the model group, while the cell activity of the KZQ-5 fermented American ginseng product was significantly improved after treatment, indicating that the Bacillus licheniformis KZQ-5-American ginseng fermentation product has an important effect on H 2 O 2 The oxidative damage to fibroblasts caused by stimulation has a certain degree of repair effect.

[0067] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0068] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of Bacillus licheniformis KZQ-5, characterized in that: The strain is deposited with the CGMCC No. 27417 on May 24, 2023 at the General Microbiology Center of China National Microbiological Culture Collection Administration, and is classified and named Bacillus licheniformis.

2. A microbial agent, characterized in that: It comprises the Bacillus licheniformis KZQ-5 according to claim 1.

3. Use of the Bacillus licheniformis KZQ-5 according to claim 1 or the microbial agent according to claim 2.

4. The use according to claim 3, characterized in that: The application is any of the following: A. Increase the extraction amount of active ingredients from ginseng plants; B. Improve the antioxidant activity of active ingredients in ginseng plants; C. Improve the ability of ginseng plant active ingredients to repair oxidatively damaged cells.

5. The use according to claim 4, characterized in that: The ginseng plant is American ginseng.

6. A method for extracting active ingredients from ginseng plants, characterized in that: The ginseng plant is fermented by using the Bacillus licheniformis KZQ-5 described in claim 1 or the microbial agent described in claim 2, and the supernatant of the fermentation liquid is taken and freeze-dried to obtain the active ingredient.

7. The method for extracting active ingredients from ginseng plants according to claim 6, characterized in that: The fermentation temperature is 28°C, the fermentation time is 48-168h, and the pH value of the fermentation liquid is 6-9.

8. The method for extracting active ingredients from ginseng plants according to claim 6, characterized in that: The Bacillus licheniformis KZQ-5 or the microbial agent is activated for two generations before preparing a seed liquid for fermentation; The ginseng plant is prepared into powder, and then mixed with water to form a 2-10% suspension, after ultrasonic mixing, the pH is adjusted to 6-9, and sterilized; Moreover, the volume ratio of the seed liquid to the ginseng plant suspension is 1:10-1:

50.

9. The method for extracting active ingredients from ginseng plants according to claim 6, characterized in that: The ginseng plant is American ginseng.

10. Use of the product extracted by any one of the extraction methods of claims 6 to 9 in the preparation of cosmetics and health products.

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