Process for extracting alfalfa saponin through probiotic combined fermentation and ultrasonic optimization and application

Abstract

The invention belongs to the technical field of medicine and health care, and particularly relates to a process for extracting alfalfa saponin through probiotic combined fermentation and ultrasonic optimization and application of the alfalfa saponin to cholesterol reduction. According to the invention, three strains are used for fermenting alfalfa, and ultrasonic-assisted extraction of alfalfa saponin is carried out; a complete alfalfa saponin fermentation process (lactobacillus casei, bacillus subtilis and saccharomyces cerevisiae are combined for fermentation according to the ratio of 1: 1: 1, the fermentation temperature is 37 DEG C, the fermentation time is 50.32 h, the material-liquid ratio (alfalfa meal to distilled water) is 1: 1.27, the bacterium addition amount is 0.062 mL, and the glucose addition amount is 0.1 g) and an ultrasonic extraction process (lactobacillus casei, bacillus subtilis and saccharomyces cerevisiae are combined for fermentation according to the ratio of 1: 1: 1) are combined for fermentation according to the ratio of lactobacillus casei to bacillus subtilis to saccharomyces cerevisiae being 1: 1: 1). The ultrasonic time is 72.7 min, the ultrasonic temperature is 47.3 DEG C, and the material-to-liquid ratio (fermentation: 40% ethanol) is 1: 223. 1). According to the probiotic combined fermentation and ultrasonic optimization extraction process provided by the invention, the extraction rate of alfalfa saponin is improved by 93.8%, and the probiotic combined fermentation and ultrasonic optimization extraction process is high in active ingredient, green and environment-friendly, and easy for large-scale production.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine and health care, and specifically relates to a process for extracting alfalfa saponins by combined fermentation of probiotics and coordinated ultrasonic optimization, and an application of the process in lowering cholesterol. Background Art

[0002] Natural products and their derivatives have a multi-link and multi-target comprehensive effect in the prevention and treatment of hyperlipidemia, and exert unique therapeutic effects. Plant extracts, especially active ingredients that lower cholesterol, have the advantages of wide sources, low toxicity and side effects, and obvious lipid-lowering effects. They have become the focus and hotspot of lipid-lowering drug research and development in recent years. Among the active substances in plants, saponins have the most obvious lipid-lowering effect and are the most concerned. They are a new research direction for the development of new lipid-lowering drugs, food and feed additives.

[0003] Alfalfa (Medicago sativa L.), known as the "king of forage grass", is the world's largest cultivated and most widely used perennial leguminous forage grass. It is rich in nutritional value and contains a variety of biologically active ingredients. Alfalfa saponins are an important class of biologically active substances in alfalfa. As a pure natural plant secondary metabolite, they are distributed in all tissues of alfalfa. The average total saponin content is about 2.2%, and its structure is a pentacyclic triterpene compound. At present, the traditional extraction methods commonly used for alfalfa saponins include solvent extraction, water extraction and alcohol precipitation, supercritical CO2 extraction, microwave-assisted extraction, etc. The solvent extraction process is simple and easy to operate, but it takes a long time, has low efficiency and high energy consumption; the water extraction and alcohol precipitation method can extract various plant saponins, but the extraction efficiency is low and high temperature affects the activity; the supercritical CO2 extraction method has no solvent residue and is environmentally friendly, but the extraction rate of polar saponins is low and the cost is high; microwave-assisted extraction is fast and green and environmentally friendly, but the equipment is expensive, and it is easy to degrade at high temperature, which is harmful to the human body. Alfalfa saponins are wrapped in the lignin of the cell wall. The complex structure of lignin is difficult to degrade, which greatly affects the extraction of saponins. How to fully and effectively realize large-scale industrial extraction and solve the problems of low enrichment rate, high cost, poor stability and low biological activity of traditional extraction methods are currently urgent problems to be solved. Summary of the invention

[0004] The present invention mainly aims at the problems of low enrichment rate, low extraction amount, low biological activity, poor stability, and difficulty in industrialization in the extraction of alfalfa saponins by traditional methods. It has developed a suitable probiotic fermentation combination process and an ultrasonic-assisted extraction process, and provided an optimal extraction process for the combined fermentation of probiotics and ultrasonic-assisted extraction of alfalfa saponins, which increases the release of effective saponin components, improves the extraction efficiency and extraction amount of alfalfa saponins, is green and environmentally friendly, and is easy to scale up production. At the same time, it has high biological activity, which can reduce obesity and liver fat deposition, lower cholesterol, and improve dyslipidemia. The obtained alfalfa saponins have broad application value and economic value in the fields of medicine, food, feed, etc.

[0005] In a first aspect, the present invention provides a method for the combined fermentation of probiotics and ultrasonic-assisted extraction of alfalfa saponins, comprising:

[0006] Step (1), preparing a fermentation broth with fermentation bacteria, with the viable count being 1×10 8 cfu / mL - 8×10 8 cfu / mL, and storing it in a 4°C refrigerator for later use;

[0007] Step (2), cutting alfalfa during the budding stage to the early flowering stage, naturally drying it, and then crushing and sieving it to make alfalfa grass powder;

[0008] Step (3), mixing the alfalfa grass powder with distilled water, adding the fermentation broth and glucose, and performing fermentation to obtain a fermentation product;

[0009] Step (4), adding 40% ethanol to the fermentation product, performing ultrasonic-assisted extraction, and obtaining an alfalfa saponin extract after post-treatment.

[0010] In some embodiments, in step (1), the fermentation bacteria are selected from Saccharomyces cerevisiae, Bacillus subtilis, Lactobacillus casei, and combinations thereof.

[0011] In some embodiments, in step (1), the fermentation bacteria are activated before use. Use an inoculation loop to pick a single colony of the fermentation bacteria from the slant medium, place it in a test tube containing 5 mL of liquid medium, culture it at 37°C with shaking at 200 r / min for 12 h, inoculate it into the liquid medium at a volume ratio of 2%, continue to culture it on a shaker for 12 h, measure the turbidity value and adjust it to a viable count of 1×10 8 cfu / mL - 8×10 8 cfu / mL, collect the fermentation broth, and store it in a 4°C refrigerator for later use.

[0012] In some embodiments, in step (1), YPD medium is used to prepare the Saccharomyces cerevisiae solution. The YPD medium contains 2 parts by weight of peptone, 2 parts by weight of glucose, and 1 part by weight of yeast extract. LB medium is used to prepare the Bacillus subtilis solution. The LB medium contains 1 part by weight of NaCl, 1 part by weight of peptone, and 0.5 part by weight of yeast extract. MRS medium is used to prepare the Lactobacillus casei solution. The MRS medium contains 2 parts by weight of glucose, 0.75 part by weight of sodium acetate, 1.5 parts by weight of peptone, 0.03 part by weight of magnesium sulfate, 1.5 parts by weight of beef extract, 0.2 part by weight of ammonium citrate, 0.75 part by weight of yeast extract, and 0.2 part by weight of dipotassium hydrogen phosphate.

[0013] In some embodiments, in step (2), the alfalfa meal is in the form of green or light green powder, has a grassy smell, and has no impurities, fermentation, mildew, or caking, meeting the quality grading standard of alfalfa meal above grade two (T / CAAA 086-2022).

[0014] In some embodiments, in step (3), the fermentation time is 24 - 96 h, preferably 24 - 72 h.

[0015] In some embodiments, in step (3), the material-liquid ratio of alfalfa meal to distilled water is 1 (g): 0.8 (mL) to 1 (g): 2.0 (mL), preferably 1 (g): 0.8 (mL) to 1 (g): 1.6 (mL).

[0016] In some embodiments, in step (3), compared with 1 g of alfalfa meal, the addition amount of the fermentation broth is 0.04 - 0.1 mL, preferably 0.04 - 0.08 mL.

[0017] In some embodiments, in step (3), compared with 1 g of alfalfa meal, the addition amount of glucose is 0.05 - 0.2 g, preferably 0.1 g.

[0018] In some embodiments, in step (3), the fermentation broth is Saccharomyces cerevisiae solution, Bacillus subtilis solution, Lactobacillus casei solution, and their combinations. Preferably, the Saccharomyces cerevisiae solution, Bacillus subtilis solution, and Lactobacillus casei solution are combined in a volume ratio of 1:1:1.

[0019] In some embodiments, in step (3), 1 g of alfalfa meal is added to 1.27 mL of distilled water, 0.062 mL of the fermentation broth and 0.1 g of glucose are added thereto, the fermentation temperature is 37 °C, and the fermentation time is 50.32 h to obtain the fermentation product. The fermentation broth is a mixture of Saccharomyces cerevisiae solution, Bacillus subtilis solution, and Lactobacillus casei solution in a volume ratio of 1:1:1.

[0020] In some embodiments, in step (4), the ultrasonic time is 45 - 90 min, preferably 60 - 90 min.

[0021] In some embodiments, in step (4), the ultrasonic temperature is 25 - 61 °C, preferably 37 - 61 °C.

[0022] In some embodiments, in step (4), the material ratio of fermented product to 40% ethanol is 1 (g): 10 (mL) to 1 (g): 40 (mL), preferably 1 (g): 10 (mL) to 1 (g): 30 (mL).

[0023] In some embodiments, in step (4), 1 g of the fermentation product is added to 23.1 mL of 40% ethanol, and ultrasonic treatment is carried out at 47.3 °C for 72.7 min, and after post-treatment, alfalfa saponin extract is obtained.

[0024] In the second aspect, the present invention provides an alfalfa saponin extract obtained by the above method. Among them, the extraction amount of alfalfa saponin obtained only by the fermentation process in the present invention is increased by 63% compared with the traditional extraction method. With the combined fermentation of the probiotic combination in the present invention and the ultrasonic-assisted extraction process, the extraction rate of alfalfa saponin is increased by 93.8% compared with the conventional extraction method. At the same time, oleanane-type triterpenoid saponins and lupane-type triterpenoid compounds are also detected in the alfalfa saponin extracted by the present invention, indicating that the alfalfa saponin extracted by the present invention has the effects of antibacterial, anti-inflammatory, antioxidant, cholesterol-lowering and immune-enhancing.

[0025] In the third aspect, the present invention provides the use of the alfalfa saponin extract in the preparation of a drug for reducing fat or cholesterol.

[0026] In some embodiments, the alfalfa saponin extract reduces the body weight of high-fat rats by 7.4%, reduces the levels of serum TC, TG and LDL-C, increases the level of HDL-C, and the hepatic steatosis changes from extremely severe to moderate, and the effects of reducing fat and cholesterol are significant.

[0027] The beneficial effects of the present invention are:

[0028] The present invention uses three strains of bacteria to ferment alfalfa, and synergistically uses ultrasonic wave-assisted extraction of alfalfa saponins, forming a complete set of alfalfa saponin fermentation processes (Lactobacillus casei + Bacillus subtilis + Saccharomyces cerevisiae in a 1:1:1 combination for fermentation, fermentation temperature of 37 °C, fermentation time of 50.32 h, solid-liquid ratio (alfalfa powder: distilled water) of 1 (g): 1.27 (mL), bacterial addition amount of 0.062 mL, glucose addition amount of 0.1 g) and ultrasonic extraction processes (after fermentation with Lactobacillus casei, Bacillus subtilis and Saccharomyces cerevisiae in a 1:1:1 combination, ultrasonic time of 72.7 min, ultrasonic temperature of 47.3 °C, solid-liquid ratio (fermented product: 40% ethanol) of 1 (g): 23.1 (mL)). The probiotic combination fermentation and synergistic ultrasonic optimization extraction process provided by the present invention increases the extraction rate of alfalfa saponins by 93.8%, has high active ingredients, is green and environmentally friendly, and is easy to scale up production.

[0029] Meanwhile, the alfalfa saponins extracted by the present invention reduce the body weight of hyperlipidemic rats by 7.4%. Compared with the alfalfa saponins extracted by conventional methods, the body weight of rats is reduced by 2.4%; the contents of serum total cholesterol, triglyceride and low-density lipoprotein cholesterol are significantly reduced, and the content of high-density lipoprotein cholesterol is significantly increased; the hepatic steatosis changes from extremely severe to moderate, the fatty degeneration of hepatocytes decreases, the number of lipid droplets in the liver decreases by 24.4 times, and the total area of lipid droplets decreases by 30.7 times, showing a significant effect on improving the accumulation of liver fat. It can be seen that the alfalfa saponins extracted by the process of the present invention have significant effects on reducing fat and cholesterol, and have broad application value and economic value in the fields of medicine, food, feed, etc. Brief Description of the Drawings

[0030] Figure 1 Shows the effect of fermentation time on the extraction amount of alfalfa saponins in Example 2;

[0031] Figure 2 Shows the effect of solid-liquid ratio (alfalfa powder: distilled water) on the extraction amount of alfalfa saponins in Example 2;

[0032] Figure 3 Shows the effect of bacterial addition amount on the extraction amount of alfalfa saponins in Example 2;

[0033] Figure 4 Shows the effect of glucose addition amount on the extraction amount of alfalfa saponins in Example 2;

[0034] Figure 5 Shows the effect of the interaction of three fermentation factors on the total yield of alfalfa saponins in Example 2;

[0035] Figure 6 Shows the effect of ultrasonic time on the extraction amount of alfalfa saponins in Example 3;

[0036] Figure 7Shows the effect of ultrasonic temperature on the extraction amount of alfalfa saponins in Example 3;

[0037] Figure 8 Shows the effect of the solid-liquid ratio (ferment: 40% ethanol) on the extraction amount of alfalfa saponins in Example 3;

[0038] Figure 9 Shows the effect of the interaction of three ultrasonic factors on the total yield of alfalfa saponins in Example 3;

[0039] Figure 10 Shows the effect of alfalfa saponins on the body weight of high-fat rats in Example 4;

[0040] Figure 11 Shows the effect of alfalfa saponins on the serum biochemical indexes of high-fat rats in Example 4;

[0041] Figure 12 Shows the effect of alfalfa saponins on the liver of high-fat rats in Example 4.

[0042] Detailed embodiments

[0043] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to specific embodiments.

[0044] Example 1: Process for extracting alfalfa saponins by combined fermentation of probiotics and synergistic optimization by ultrasound

[0045] Step 1,

[0046] Preparation of culture medium:

[0047] Preparation of YPD (Yeast Extract Peptone Dextrose Medium) culture medium: 2 g of peptone, 2 g of glucose, 1 g of yeast extract, 2 g of agar is added to the solid culture medium, and no agar is added to the liquid culture medium. Sterilize and reserve for culturing yeast strains.

[0048] Preparation of LB (Luria-Bertani Medium) culture medium: 1 g of NaCl, 1 g of peptone, 0.5 g of yeast extract, 2 g of agar powder is added to the solid culture medium, and no agar is added to the liquid culture medium. Sterilize and reserve for culturing Bacillus subtilis strains.

[0049] Preparation of MRS (De Man, Rogosa and Sharpe Medium): 2 g of glucose, 0.75 g of sodium acetate, 1.5 g of peptone, 0.03 g of magnesium sulfate, 1.5 g of beef extract, 0.2 g of ammonium citrate, 0.75 g of yeast extract, 0.2 g of dipotassium hydrogen phosphate. For solid medium, add 2 g of agar powder; for liquid medium, do not add agar. Sterilize and reserve for culturing Lactobacillus casei strains.

[0050] Preparation of fermented liquid strains:

[0051] Strains: Saccharomyces cerevisiae (S. cerevisiae, S117 isolate), Bacillus subtilis (B. subtilis, B117 isolate), and Lactobacillus casei (L. casei, L117 isolate) are all strains isolated and preserved by the Key Laboratory of Forage Resources Innovation and Utilization in Henan Province, and are activated before use.

[0052] Preparation of Saccharomyces cerevisiae liquid: Use an inoculation loop to pick a single colony of Saccharomyces cerevisiae (S. cerevisiae, S117 isolate) from the slant medium, place it in a test tube containing 5 mL of liquid medium, culture it at 37 °C with shaking at 200 r / min for 12 h, inoculate it into a triangular flask liquid medium with a liquid filling volume of 20% according to a volume ratio of 2%, continue to culture it on a shaker for 12 h, measure the turbidity value and adjust it to a viable cell count of 1×10 8 cfu / mL - 8×10 8 cfu / mL. Collect the Saccharomyces cerevisiae liquid and store it in a 4 °C refrigerator for later use.

[0053] Preparation of Bacillus subtilis liquid: Use an inoculation loop to pick a single colony of Bacillus subtilis (B. subtilis, B117 isolate) from the slant medium, place it in a test tube containing 5 mL of liquid medium, culture it at 37 °C with shaking at 200 r / min for 12 h, inoculate it into a triangular flask liquid medium with a liquid filling volume of 20% according to a ratio of 2%, continue to culture it on a shaker for 12 h, measure the turbidity value and adjust it to a viable cell count of 1×10 8 cfu / mL - 8×10 8 cfu / mL. Collect the Bacillus subtilis liquid and store it in a 4 °C refrigerator for later use.

[0054] Preparation of Lactobacillus casei liquid: Use an inoculation loop to pick a single colony of Lactobacillus casei (L. casei, L117 isolate) from the slant medium, place it in a test tube containing 5 mL of liquid medium, culture it at 37 °C with shaking at 200 r / min for 12 h, inoculate it into a triangular flask liquid medium with a liquid filling volume of 90% according to a ratio of 2%, continue to culture it on a shaker for 12 h, measure the turbidity value and adjust it to a viable cell count of 1×10 8cfu / mL - 8×10 8 cfu / mL, collect the Lactobacillus casei bacterial liquid and store it in a 4°C refrigerator for later use.

[0055] Step 2,

[0056] Preparation of alfalfa powder: Alfalfa (Henan Agricultural University strain) was collected from the Yuanyang Science and Education Park of Henan Agricultural University, which is a self-cultivated variety of the Key Laboratory of Forage Resource Innovation and Utilization in Henan Province. Alfalfa was cut during the budding to early flowering stage, naturally air-dried, and then mechanically crushed and processed into a powdery substance with a certain fineness, and passed through a sieve for later use. The alfalfa grass powder is green or light green in powder form, has a grassy fragrance, and has no impurities, fermentation, mildew, or caking, meeting the standards above the second level of the alfalfa grass powder quality grading (T / CAAA 086 - 2022).

[0057] Step 3,

[0058] Add 1 g of alfalfa grass powder to 1.27 mL of distilled water, add 0.062 mL of fermentation bacterial liquid and 0.1 g of glucose thereto, ferment at 37°C for 50.32 h to obtain a fermentation product. The fermentation bacterial liquid is a mixture of Saccharomyces cerevisiae bacterial liquid, Bacillus subtilis bacterial liquid, and Lactobacillus casei bacterial liquid in a volume ratio of 1:1:1.

[0059] Step 4,

[0060] Add 1 g of the fermentation product to 23.1 mL of 40% ethanol, ultrasonically treat at 47.3°C for 72.7 min, and obtain alfalfa saponin extract after post-treatment.

[0061] Example 2: Fermentation process optimization

[0062] (1) Construct a standard curve for alfalfa saponins

[0063] Accurately weigh 10 mg of oleanolic acid standard product with an analytical balance, dissolve it with ethanol and make up the volume to 50 mL in a volumetric flask to prepare an oleanolic acid standard solution. Respectively transfer 0.10, 0.20, 0.30, 0.40, 0.50 mL of the oleanolic acid standard solution into 10 mL stoppered test tubes, volatilize the solvent in a 70°C water bath, then add 0.20 mL of 5% vanillin solution and 0.80 mL of perchloric acid, shake in a 70°C water bath for 15 min, cool to 0°C in ice water, add 5.0 mL of glacial acetic acid to dilute and mix evenly, let stand for 15 min, and then measure the absorbance value at a wavelength of 560 nm with a UV-visible spectrophotometer. Taking the absorbance as the ordinate (Y) and the different concentrations of oleanolic acid as the abscissa (X), construct a standard curve for alfalfa saponins: y = 26.176x + 0.035, R 2 = 0.9991.

[0064] (2) Screen the strain combination and fermentation parameters

[0065] Four key parameters, namely fermentation time, glucose addition amount, material-liquid ratio (alfalfa meal: distilled water), and bacterial liquid addition amount, were set, and each parameter had 4 levels: fermentation time (24 h, 48 h, 72 h, and 96 h), glucose addition amount (0.05 g, 0.1 g, 0.15 g, and 0.2 g), material-liquid ratio (1:0.8, 1:1.2, 1:1.6, and 1:2 (unit, g:mL)), and bacterial liquid addition amount (0.04 mL, 0.06 mL, 0.08 mL, and 0.1 mL). By measuring the effects of 3 strain combinations and 4 parameters on the yield of alfalfa saponins, the strain combination and fermentation parameters were screened. The results are as Figure 1 - 4 shown.

[0066] It can be Figure 1 seen that the strains and fermentation time had a significant effect on the extraction amount of alfalfa saponins (P < 0.001), and there was also an obvious interaction between the strains and fermentation time (P < 0.001). Compared with the non-fermented control group, whether it was single-strain fermentation or combined fermentation, the extraction amount of alfalfa saponins increased significantly, and the effect of combined fermentation was the best (P < 0.05). With the extension of fermentation time, the extraction amount of alfalfa saponins showed a trend of first increasing and then decreasing, and the fermentation effect was the best at 48 h (P < 0.05). Three strain combinations were continued to be fermented, and response surface experiments were carried out at three times of 24 h, 48 h, and 72 h.

[0067] Similar to the results of fermentation time (see Figure 2 ), the strains and material-liquid ratio also had a significant effect on the extraction amount of alfalfa saponins (P < 0.001), and there was an obvious interaction between the strains and material-liquid ratio (P < 0.001). Compared with the control group, after single-strain fermentation and combined fermentation treatments, the extraction amount of alfalfa saponins increased significantly, and the effect of combined fermentation with 3 strains was the best (P < 0.05). With the increase of the material-liquid ratio, the extraction amount of alfalfa saponins showed a trend of first increasing and then decreasing, and the fermentation effect was the best at the material-liquid ratio of 1:1.2 (P < 0.05). Therefore, combined fermentation with 3 strains was selected, and response surface experiments were carried out at material-liquid ratios of 1:0.8, 1:1.2, and 1:1.6.

[0068] The effect of the addition amount of bacteria on the yield of alfalfa saponins is shown in Figure 3The strain and the amount of added bacteria had a significant effect on the extraction yield of alfalfa saponins (P<0.001), and there was an obvious interaction between the strain and the amount of added bacteria (P<0.001). When the amount of added bacteria was between 0.04 - 0.06 mL, insufficient amount of added bacteria might lead to slow fermentation start and low fermentation efficiency. When the amount of added bacteria exceeded 0.06 mL, it could cause competition for nutrients or accumulation of metabolites, inhibiting the growth of bacteria and reducing the extraction efficiency, hindering the dissolution of alfalfa saponin components. When fermented with a combination of 3 strains and the amount of added bacteria was 0.06 mL, the yield of alfalfa saponins was the highest (P<0.05). Therefore, a response surface experiment was carried out by selecting fermentation with a combination of 3 strains and three levels of the amount of added bacteria, namely 0.04, 0.06, and 0.08 mL.

[0069] It can be seen from Figure 4 that the strain and the amount of added glucose had a significant effect on the extraction yield of alfalfa saponins (P<0.01), and there was an interaction between the two (P<0.05). The extraction yields of alfalfa saponins after single-strain fermentation and combined fermentation were both significantly higher than those of the control group (P<0.05), but there was no significant difference between the fermentation treatment groups (P>0.05). The optimal amount of added glucose was 0.1 g, but this factor was not included in the response surface optimization experiment.

[0070] (3) Based on Box - Bhenken to optimize the fermentation parameters to obtain the optimal fermentation process

[0071] Taking the screened fermentation time, solid - liquid ratio (alfalfa meal: distilled water), and the amount of added bacterial liquid as the main factors, with the total saponin yield as the index, Design - Expert 13 software was used for response surface analysis. The regression model (Y = 11.67 + 0.4303A + 0.3721B + 0.121C - 0.1418AB + 0.3864AC - 0.2259BC - 0.5923A 2 - 0.204B 2 - 0.4436C 2 ) was used to predict the fermentation conditions, and further verified the reliability by variance analysis to obtain the optimal fermentation process. The factor and level design of the Box - Bhenken test is shown in Table 1, the results of variance analysis are shown in Table 2, the contour plot (a) and response surface plot (b) of the interaction of the three fermentation factors are shown in Figure 5 , and the results of the optimal fermentation process are shown in Table 5.

[0072] Table 1 Factor and level design of Box - Bhenken fermentation test

[0073]

[0074]

[0075] Table 2 Variance analysis of the regression model of fermentation parameters

[0076] Source of Variance Sum of Squares Degree of Freedom Mean Square F Value P Value Significance Model 24.72 9 2.75 48.67 <0.0001 ** A - Liquid - to - Material Ratio 1.48 1 1.48 26.25 0.0014 ** B - Fermentation Time 1.11 1 1.11 19.62 0.0030 ** C - Bacterial Addition 0.1171 1 0.1171 2.07 0.1929 NS AB 0.0804 1 0.0804 1.42 0.2716 NS AC 0.5974 1 0.5974 10.58 0.0140 * BC 0.2041 1 0.2041 3.62 0.0990 NS <![CDATA[A 2 > 1.48 1 1.48 26.17 0.0014 ** <![CDATA[B 2 > 17.59 1 17.59 311.65 <0.0001 ** <![CDATA[C 2 > 0.8287 1 0.8287 14.68 0.0064 ** Residual 0.3951 7 0.0564 Lack - of - Fit 0.3069 3 0.1023 4.64 0.0862 NS Pure Error 0.0882 4 0.0220 Total Deviation 25.12 16 <![CDATA[R 2 > 0.9843 <![CDATA[R 2 Adj > 0.9640

[0077] Note: * indicates significant difference (P < 0.05); ** indicates extremely significant difference (P < 0.01); NS indicates no significant difference (P > 0.05).

[0078] The results of the two - factor interactions of liquid - to - material ratio (A), fermentation time (B), and inoculum amount (C) on the yield of alfalfa saponins are as Figure 5 shown. The interaction surface between the liquid - to - material ratio (A) and the inoculum amount (C) has a maximum value and a relatively steep slope, indicating that the interaction between these two groups has a significant effect on the total yield of alfalfa saponins (P < 0.05). While the slopes of the interaction surfaces between the liquid - to - material ratio (A) and fermentation time (B), and between fermentation time (B) and inoculum amount (C) are relatively gentle, indicating that the interaction of this group has no significant effect on the total yield of alfalfa saponins (P > 0.05). This is consistent with the analysis results of the P - value of the interaction terms in Table 2.

[0079] Conclusion: The optimal fermentation process for extracting alfalfa saponins is as follows: a combination of Lactobacillus casei, Bacillus subtilis, and Saccharomyces cerevisiae in a ratio of 1:1:1, fermentation temperature of 37°C, fermentation time of 50.32 h, liquid - to - material ratio (alfalfa meal: distilled water) of 1:1.27, inoculum amount of 0.062 mL, and glucose addition amount of 0.1 g. Compared with the conventional extraction method, the extraction rate of alfalfa saponins can be increased by 63% only by using the probiotic combination fermentation process of the present invention.

[0080] Example 3: Optimization of ultrasonic - assisted extraction process

[0081] Continue to screen the strain combination and determine the ultrasonic parameters to obtain the optimal ultrasonic extraction process. Use the optimal fermentation process in Example 2 to obtain the fermentation product, and cooperate with the ultrasonic - assisted extraction process to finally extract alfalfa saponins with high yield and high activity.

[0082] (1) Construct the standard curve of alfalfa saponins

[0083] The construction method and the final standard curve of alfalfa saponins are the same as those in Example 2.

[0084] (2) Screen the strain combination and ultrasonic parameters

[0085] Set three key parameters: ultrasonic time, ultrasonic temperature, and liquid - to - material ratio (fermentation product: ethanol). Each parameter has 4 levels: ultrasonic temperature (25°C, 37°C, 49°C, and 61°C), ultrasonic time (45 min, 60 min, 75 min, and 90 min), and liquid - to - material ratio (1:10, 1:20, 1:30, and 1:40 (unit: g:mL)). By measuring the effects of 3 strain combinations and 3 ultrasonic parameters on the yield of alfalfa saponins, screen the strain combination and ultrasonic parameters. The results are as Figures 6 - 8as shown

[0086] It can be seen from Figure 6 that the strain and ultrasonic time had a significant effect on the extraction amount of alfalfa saponins (P<0.001), and there was also an obvious interaction between the strain and ultrasonic time (P<0.001). Compared with the unfermented control group, whether it was single-strain fermentation or combined fermentation, the extraction amount of alfalfa saponins increased significantly, and the effect of combined post-fermentation was the best (P<0.05). With the extension of ultrasonic time, the extraction amount of alfalfa saponins showed a trend of first increasing and then decreasing, and the fermentation effect was the best at 75 min (P<0.05). Therefore, three strains were selected for combination, and response surface experiments were carried out at three times of 60 min, 75 min, and 90 min.

[0087] Similar to the ultrasonic time results (see Figure 7 ), the strain and ultrasonic temperature also had a significant effect on the extraction amount of alfalfa saponins (P<0.001), and there was an obvious interaction between the two (P<0.001). Compared with the control group, after single-strain ultrasonic treatment and composite-strain ultrasonic treatment, the extraction amount of alfalfa saponins increased significantly, and the effect of ultrasonic extraction with three-strain combination was the best (P<0.05). Compared with the control group and other treatment groups, the extraction effect was the best at an ultrasonic temperature of 49 °C (P<0.05). Therefore, three strains were continuously selected for combination, and response surface experiments were carried out at three levels of 37 °C, 49 °C, and 61 °C.

[0088] As Figure 8 shown, the strain and solid-liquid ratio (fermented product: 40% ethanol) also had a significant effect on the ultrasonic extraction amount of alfalfa saponins (P<0.001), and there was an obvious interaction between the strain and solid-liquid ratio (P<0.001). Compared with the control group, after single-strain ultrasonic treatment and composite-strain ultrasonic treatment, the extraction amount of alfalfa saponins increased significantly, and the effect of ultrasonic extraction with three-strain combination was the best (P<0.05). From the results, the saponin extraction amount was the highest at a solid-liquid ratio of 1:20 (P<0.05). Therefore, three strains were selected for combination, and response surface experiments were continued at three levels of solid-liquid ratios of 1:10, 1:20, and 1:30.

[0089] (3) Optimize the ultrasonic parameters based on Box-Bhenken to obtain the best ultrasonic-assisted extraction process

[0090] The screened ultrasonic time, ultrasonic temperature, and solid-liquid ratio (fermented product: 40% ethanol) were used as the main factors, and the total saponin yield was used as the index. Response surface analysis was carried out using Design-Expert 13 software, and the regression model (Y = 13.83 + 0.4320A - 0.3460B - 0.0727C + 0.0836AB - 0.04224AC - 0.5471BC - 0.4945A 2 - 0.7308B 2-0.1.06C 2 )Predict the ultrasonic conditions, further verify the reliability by analysis of variance, and obtain the optimal ultrasonic-assisted extraction process. The factors and levels of the Box-Bhenken test are shown in Table 3, the analysis of variance results of the ultrasonic parameter regression model are shown in Table 4, and the contour plots (a) and response surface plots (b) of the interaction of the three ultrasonic factors are shown in Figure 9 , and the results of the optimal ultrasonic-assisted extraction process are shown in Table 5.

[0091] Table 3 Factors and levels of Box-Bhenken ultrasonic test design

[0092]

[0093] Table 4 Analysis of variance of ultrasonic parameter regression model

[0094] Source of Variance Sum of Squares Degree of Freedom Mean Square F Value P Value Significance Model 13.23 9 1.47 19.44 0.0004 ** A - Liquid - to - Material Ratio 1.49 1 1.49 19.74 0.0030 ** B - Ultrasonic Time 0.9579 1 0.9579 12.66 0.0092 ** C - Ultrasonic Temperature 0.0422 1 0.0422 0.5584 0.4793 NS AB 0.0280 1 0.0280 0.3696 0.5624 NS AC 0.7137 1 0.7137 9.43 0.0180 * BC 1.20 1 1.20 15.83 0.0053 ** <![CDATA[A 2 > 1.03 1 1.03 13.61 0.0078 ** <![CDATA[B 2 > 2.25 1 2.25 29.73 0.0010 ** <![CDATA[C 2 > 4.70 1 4.70 62.09 0.0001 ** Residual 0.5295 7 0.0756 Lack - of - Fit 0.3983 3 0.1328 4.05 0.1051 NS Pure Error 0.1312 4 0.0328 Total Deviation 13.76 16 <![CDATA[R 2 > 0.9615 <![CDATA[R 2 Adj]]> 0.9121

[0095] Note: * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01); NS indicates no significant difference (P>0.05).

[0096] The effects of the pairwise interactions of the solid-liquid ratio (A), ultrasonic time (B), and ultrasonic temperature (C) on the yield of alfalfa saponins are as Figure 9 shown. For the interactions between the solid-liquid ratio (A) and ultrasonic temperature (C), and between ultrasonic time (B) and ultrasonic temperature (C), the surfaces of the interactions both have maximum values and the slopes are relatively steep, indicating that the AC and BC interactions have a significant effect on the total yield of alfalfa saponins (P<0.05). The slope of the interaction between the solid-liquid ratio (A) and ultrasonic time (B) is relatively gentle, indicating that the AB interaction has no significant effect on the total yield of alfalfa saponins (P>0.05). This is consistent with the analysis results of the P values of the interaction terms in Table 4.

[0097] Conclusion: The optimal ultrasonic-assisted extraction process for alfalfa saponins is as follows: a combination of Lactobacillus casei, Bacillus subtilis, and Saccharomyces cerevisiae in a ratio of 1:1:1, ultrasonic time of 72.7 min, solid-liquid ratio (ferment: 40% ethanol) of 1:23.1, and ultrasonic temperature of 47.3 °C. Compared with the conventional extraction method, the extraction rate of alfalfa saponins is increased by 93.8% using the probiotic combination fermentation and ultrasonic-assisted extraction process of the present invention.

[0098] Table 5 Results of the optimal fermentation process + ultrasonic-assisted extraction process

[0099]

[0100] Example 4: Application of alfalfa saponins in reducing cholesterol

[0101] This experiment was approved by the Animal Welfare and Ethics Committee of Henan Agricultural University. Thirty-six SPF-grade male healthy SD rats, weighing (180 ± 20) g, were randomly divided into 4 groups (n = 9):

[0102] ① Control group, fed with basal diet, intragastrically administered with 2 mL / d of normal saline from week 5 to 8;

[0103] ② High-fat group, fed with high-fat diet, intragastrically administered with 2 mL / d of normal saline from week 5 to 8;

[0104] ③ Saponin treatment group Ⅰ, fed with high-fat diet, intragastrically administered with 2 mL / d of alfalfa saponin from week 5 to 8. The alfalfa saponin was a pure product extracted by a conventional process (without fermentation and ultrasonic assistance, directly extracting saponin from alfalfa meal with ethanol), with a concentration of 240 mg·kg -1 ;

[0105] ④ Saponin treatment group Ⅱ, fed with high-fat diet, intragastrically administered with 2 mL / d of alfalfa saponin from week 5 to 8. The alfalfa saponin was a pure product purified from the alfalfa saponin extract obtained in Example 1 of the present invention, with a concentration of 240 mg·kg -1 .

[0106] Each group of rats was housed individually in a cage. The pre-trial period was 1 week and the trial period was 8 weeks. The nutritional levels of the high-fat diet were: crude protein 20.96%, crude fat 28.72%, carbohydrate 37.19%, total energy 20.55%, calcium 0.95%, and phosphorus 0.48%. The experimental management conditions for each group were the same, with a temperature of 20 - 22 °C, humidity of 45% ± 5%, a 12 h light / dark cycle, and free access to water.

[0107] At a fixed time every week (09:00 - 10:00), the rats were weighed on an empty stomach (accurate to 0.1 g), and the health status of the rats was observed every day. On the last day of the 4th week and the 8th week respectively, blood was taken from the ophthalmic veins of the rats. The rats were fasted but not water-deprived for 12 h before blood collection. The blood samples were allowed to stand at room temperature for 0.5 h and then centrifuged at 12000 rpm for 15 min. According to the requirements of the kit (Nanjing Jiancheng Bioengineering Institute), the contents of TC, TG, HDL-C, and LDL-C in the serum were measured using an automatic biochemical analyzer (Hitachi-7020, Hitachi, Japan). After the end of the 8th week, the rats were anesthetized by intraperitoneal injection of 0.1 mL / 100 g of 3% sodium pentobarbital, the liver was removed, the hepatic lobules were rinsed with normal saline, and fixed in 4% paraformaldehyde solution for histological examination of the liver and determination of other indexes.

[0108] Data statistics:

[0109] The data was sorted using Excel 2021. One-way ANOVA of the SPSS 26.0 software was adopted, and multiple comparisons were carried out using the Duncan method. P < 0.05 indicates significant differences, and P > 0.05 indicates no significant differences. Significant differences from the control group were marked as: *P < 0.05, **P < 0.01; significant differences between the saponin treatment group I and the high-fat group were marked as: # P < 0.05, ## P < 0.01; significant differences between the saponin treatment group II and the high-fat group were marked as: & P < 0.05, && P < 0.01; significant differences between the two saponin treatment groups were marked as: △ P < 0.05, △△ P < 0.01.

[0110] The results are as Figure 10 shown. After 4 weeks of high-fat diet, the body weight of the rats was significantly higher than that of the normal rats in the control group (P < 0.05). From the 4th week to the 8th week, the high-fat group and the two treatment groups continued the high-fat diet, and at the same time, the rats in the two treatment groups were intragastrically administered pure alfalfa saponins with different extraction processes. The results showed that the body weight of the rats in the high-fat group was still significantly higher than that of the normal rats (P < 0.05); while the body weights of the rats in the saponin treatment group I and the saponin treatment group II decreased significantly and were significantly lower than those of the high-fat rats (P < 0.05), but there was no difference from the normal rats (P > 0.05).

[0111] The effects of different treatments on the serum biochemical indexes of the rats are shown in Figure 11 . Compared with the control group, after 4 weeks of high-fat diet, the contents of total cholesterol (TC) and triglyceride (TG) in the serum of the high-fat group increased significantly (P < 0.01), and the content of high-density lipoprotein cholesterol (HDL-C) decreased significantly (P < 0.01). After saponin treatment, the contents of serum TC, TG and LDL-C in the two treatment groups were significantly lower than those in the high-fat group (P < 0.05), and the content of HDL-C increased significantly (P < 0.05). However, the effect of reducing the serum TG content in the saponin II group was better than that in the saponin I group (P < 0.05). It shows that the alfalfa saponins extracted by the present invention can better reduce the serum TC, TG and LDL-C of high-fat rats, increase HDL-C and reduce the body cholesterol level.

[0112] From the results of liver pathological morphology examination (Table 6), the liver of the rats in the high-fat group showed extremely severe fatty degeneration; after treatment with the saponin I group, the liver changed from extremely severe fatty degeneration to severe level; while after treatment with the saponin II group, the liver recovered from extremely severe fatty degeneration to moderate level, with better effect.

[0113] Table 6 Pathological morphology examination

[0114] Item Steatosis Normal Control Group 1 High - Fat Model Group 4 Saponin Treatment Group Ⅰ 3 Saponin Treatment Group Ⅱ 2

[0115] Note: The lesions were evaluated according to the degree from mild to severe, and semi-quantitatively as follows: extremely small amount or no lesion was negative "-", recorded as 0; mild or small amount was "+", recorded as 1; moderate or medium amount was "++", recorded as 2; severe or large amount was "+++", recorded as 3; extremely severe or massive amount was "++++", recorded as 4.

[0116] The results of HE staining of the liver showed ( Figure 12 , Figure 12 a: control group (100X); Figure 12 b: high-fat group (100X); Figure 12 c: saponin treatment group I (100X); Figure 12 d: saponin treatment group II (100X)). The hepatic lobule structure of normal rats was intact, the hepatocytes were arranged closely, the hepatic cord structure was clear, the cytoplasm was abundant, and the cell morphology was normal. In the high-fat group, extensive fatty degeneration of hepatocytes was visible, with round vacuoles of different sizes appearing in the cytoplasm, and small focal infiltration of inflammatory cells around the portal area and within the lobules. After treatment with alfalfa saponin, the fatty degeneration of hepatocytes decreased, and a varying number of tiny round vacuoles could be seen in the cytoplasm, and no other obvious abnormalities were observed. In particular, the effect of reducing inflammation in saponin treatment group II was obvious, and the effect of improving liver fat accumulation was significant.

[0117] In addition, compared with the control group (Table 7), the number of lipid droplets, the total area of lipid droplets, and the average area of lipid droplets in the liver of high-fat rats increased significantly (P<0.05); after treatment with alfalfa saponin, the number of lipid droplets, the total area of lipid droplets, and the average area of lipid droplets in the liver of rats were all reduced compared with the high-fat group (P<0.05), and among them, the effect of saponin treatment group II was significantly better than that of saponin treatment group I (P<0.05).

[0118] Table 7 Digital pathological image analysis

[0119] Item Normal Control Group High - Fat Model Group Saponin Treatment Group Ⅰ Saponin Treatment Group Ⅱ Number of Lipid Droplets 19476±11259d 809777±96909a 70496.5±9673b 33129±27820c Total Area of Lipid Droplets 61043±48074d 3968065±1426581a 1224920±253810b 129275±62855c Average Area of Lipid Droplets 2.19±0.79b 3.85±1.05a 2.30±0.18b 1.51±0.25c

[0120] Conclusion: After treatment with alfalfa saponin group II, the body weight of rats was reduced by 7.4% compared with the high-fat group (P<0.05), and by 2.4% compared with saponin treatment group I; the contents of serum total cholesterol, triglyceride, and low-density lipoprotein cholesterol decreased significantly (P<0.05), and the content of high-density lipoprotein cholesterol increased significantly (P<0.05); the hepatic steatosis changed from extremely severe to moderate, the fatty degeneration of hepatocytes decreased, the number of liver lipid droplets decreased by 24.4 times, the total area of lipid droplets decreased by 30.7 times, and the effect of improving liver fat accumulation was significant. It can be seen that the alfalfa saponin extracted by the process of the present invention has a significant effect in reducing fat and cholesterol.

[0121] The above are only embodiments of the present invention, and do not thus limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the content of the specification and drawings of the present invention, or directly or indirectly applied in other related technical fields, shall similarly be included within the patent protection scope of the present invention.

Claims

1. A method for extracting alfalfa saponins by combined fermentation of probiotics and ultrasound-assisted extraction, comprising: Step (1), using fermentation bacteria to prepare a fermentation solution, the number of viable bacteria is 1×10 8 cfu / mL-8×10 8 cfu / mL, stored in a 4°C refrigerator for later use; Step (2), alfalfa is cut during the budding stage to the early flowering stage, dried naturally, and then crushed and sieved to produce alfalfa powder; Step (3), mixing alfalfa powder with distilled water, adding fermentation liquid and glucose, and fermenting to obtain a fermentation product; Step (4), adding 40% ethanol to the fermentation product, performing ultrasound-assisted extraction, and obtaining alfalfa saponin extract through post-treatment.

2. The method according to claim 1, wherein: In step (1), the fermentation bacteria is selected from Saccharomyces cerevisiae, Bacillus subtilis, Lactobacillus casei and a combination thereof.

3. The method according to claim 1, wherein: In step (2), the alfalfa powder is green or light green in color, has a grass aroma, is free of impurities, fermentation, mildew, or agglomeration, and meets the alfalfa powder quality grading standard of level 2 or above.

4. The method according to claim 1, wherein: In step (3), the fermentation time is 24-96 h; the material-liquid ratio of alfalfa powder: distilled water is 1 (g): 0.8 (mL) to 1 (g): 2.0 (mL).

5. The method according to claim 1, wherein: In step (3), compared with 1 g of alfalfa powder, the amount of fermentation liquid added is 0.04-0.1 mL, and the amount of glucose added is 0.05-0.2 g.

6. The method according to claim 1, wherein: In step (3), the fermentation broth is a combination of Saccharomyces cerevisiae broth, Bacillus subtilis broth, Lactobacillus casei broth, and the three bacteria in a volume ratio of 1:1:

1.

7. The method according to claim 1, wherein: In step (4), the ultrasonic time is 45-90 min; and the ultrasonic temperature is 25-61°C.

8. The method according to claim 1, wherein: In step (4), the material-liquid ratio of fermentation product: 40% ethanol is 1 (g): 10 (mL) to 1 (g): 40 (mL).

9. Alfalfa saponin extract obtained by the method according to any one of claims 1-9.

10. Use of the alfalfa saponin extract obtained by the method according to any one of claims 1 to 9 in the preparation of a drug for reducing fat or lowering cholesterol.

Images