Probiotic fermentation method for improving total saponins of astragalus membranaceus based on response surface optimization

Abstract

The invention discloses a probiotic fermentation method for improving total saponins of astragalus membranaceus based on response surface optimization, which comprises the following steps: inoculating a fermentation seed solution into a fermentation substrate containing astragalus membranaceus, and carrying out shake culture for 48 hours under the conditions of 40-45 DEG C and 150-200r / min; the inoculation amount is 1-5%; the fermentation seed liquid is obtained by culturing lactobacillus delbrueckii subsp. Bulgaricus CICC 20247; the fermentation substrate containing astragalus membranaceus comprises astragalus membranaceus powder, skim milk powder, white granulated sugar, yeast extract and sterile water. After the lactobacillus delbrueckii subsp. Bulgaricus CICC20247 liquid is used for fermenting the astragalus membranaceus, the content of total saponins in the astragalus membranaceus can be greatly increased, and the medicine effect of the astragalus membranaceus can be improved.

Description

Technical fields:

[0001] This invention relates to the field of technology, and specifically to a probiotic fermentation method for increasing total saponins in astragalus based on response surface methodology. Background technology:

[0002] Astragalus membranaceus belongs to the legume family. The medicinal part is the dried root of Astragalus membranaceus or Astragalus mongholicus. It is mild in nature and sweet in taste. In traditional Chinese medicine, it is considered a traditional tonic, possessing the effects of tonifying qi and raising yang, consolidating the exterior and stopping sweating, promoting diuresis and reducing swelling, and promoting tissue regeneration. Modern research shows that astragalus can enhance heart function, improve immunity, and regulate blood pressure and blood sugar. Modern chemical composition studies show that astragalus is rich in saponins, polysaccharides, and flavonoids. Among these, total astragalus saponins are important physiologically active components of astragalus, and therefore are often used as qualitative and quantitative indicators for the medicinal properties of astragalus.

[0003] In my country, the resources of Astragalus membranaceus (Huang Qi) mainly rely on artificial cultivation, with Mongolian Astragalus membranaceus being the mainstream commercial product. However, due to multiple factors, such as variations in cultivation methods and growth years affecting the yield and quality of Astragalus membranaceus, the supply of medicinal Astragalus membranaceus falls far short of market demand. Currently, researchers are combining traditional Chinese medicine fermentation techniques with modern fermentation technologies to improve the extraction rate of effective components from Chinese medicines, thereby further enhancing their medicinal value. This research has a very broad development prospect. Furthermore, in recent years, with the accelerated pace of life, anxiety and depression have become hot topics, and the antidepressant and anti-anxiety effects of fermented Astragalus membranaceus have also become a research focus in Chinese medicinal materials.

[0004] To date, reports on the extraction of active ingredients from Astragalus membranaceus have mostly focused on the extraction of polysaccharides and saponins, while research on the fermentation process of total saponins from Astragalus membranaceus is relatively limited. Summary of the Invention:

[0005] The purpose of this invention is to provide a probiotic fermentation method for increasing the total saponin content of astragalus based on response surface methodology, which can increase the total saponin content of astragalus through probiotic fermentation.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A probiotic fermentation method for increasing total saponins of Astragalus membranaceus based on response surface methodology includes the following steps: inoculating the fermentation seed liquid into a fermentation substrate containing Astragalus membranaceus, and shaking and culturing it at 40–45℃ and 150–200 r / min for 48 h; the inoculation amount is 1–5%;

[0008] The fermentation substrate containing Astragalus includes the following components: 2-20% Astragalus powder, 10-20% skim milk powder, 1-5% white sugar, 0-0.5% yeast extract, and the remainder is sterile water.

[0009] Furthermore, the method for preparing the fermentation seed liquid is as follows: Lactobacillus delbrueckii subsp. bulgaricus is inoculated into MRS liquid medium and cultured with shaking at 40-45℃ and 150-200 r / min for 48 h to obtain the fermentation seed liquid; the preservation number of Lactobacillus delbrueckii subsp. bulgaricus is CICC No. 20247.

[0010] Beneficial effects: Liquid fermentation of Astragalus membranaceus using Lactobacillus delbrueckii subsp. bulgaricus CICC20247 can significantly increase the total saponin content of Astragalus membranaceus, which is beneficial for enhancing its medicinal efficacy. Attached image description:

[0011] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0012] Figure 1 A schematic diagram of the steps in a probiotic fermentation method for increasing total saponins in astragalus based on response surface methodology.

[0013] Figure 2 This is a diagram showing the factors influencing the effect of skim milk powder on the response value during the optimization using response surface methodology in Example 4.

[0014] Figure 3 This is a diagram showing the influence of white sugar on the response value in the optimization using response surface methodology in Example 4.

[0015] Figure 4 This is a diagram showing the factors influencing the response value of yeast extract in the optimization using response surface methodology in Example 4.

[0016] Figure 5 This is a diagram showing the cross-effects of white sugar and yeast extract in the optimization using response surface methodology in Example 4.

[0017] Figure 6 This is a diagram showing the cross-effects of white sugar and skim milk powder in the optimization using response surface methodology in Example 4.

[0018] Figure 7 This is a diagram showing the cross-effects of yeast extract and skim milk powder in the optimization using response surface methodology in Example 4. Detailed implementation method:

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] In the following examples and comparative examples, all medicinal materials and reagents were commercially available.

[0021] Example 1

[0022] A probiotic fermentation method for increasing total saponins in astragalus based on response surface methodology, such as... Figure 1 As shown, the steps include:

[0023] (1) Lactobacillus delbrueckii subsp. bulgaricus was inoculated into MRS liquid medium and cultured with shaking at 45℃ and 200r / min for 48h to obtain fermentation seed liquid; Lactobacillus delbrueckii subsp. bulgaricus was purchased from China Industrial Microbial Culture Collection Center with the accession number CICC No. 20247.

[0024] (2) The fermentation seed liquid was inoculated into the fermentation substrate containing Astragalus membranaceus and cultured with shaking at 45℃ and 150r / min for 48h; the inoculation amount was 5%.

[0025] The fermentation substrate containing Astragalus membranaceus comprises the following components: 2-20% Astragalus membranaceus powder (all by weight), 10-20% skim milk powder, 1-5% white sugar, 0-0.5% yeast extract, and the remainder being sterile water. The Astragalus membranaceus powder is obtained by grinding Astragalus membranaceus slices into coarse powder and passing it through a 24-mesh sieve. The Astragalus membranaceus powder is in a suspension state within the fermentation substrate containing Astragalus membranaceus.

[0026] Example 2

[0027] A probiotic fermentation method for increasing total saponins from astragalus based on response surface methodology includes the following steps:

[0028] (1) Lactobacillus delbrueckii subsp. bulgaricus was inoculated into MRS liquid medium and cultured with shaking at 42℃ and 180r / min for 48h to obtain fermentation seed liquid; Lactobacillus delbrueckii subsp. bulgaricus was purchased from China Industrial Microbial Culture Collection Center with the accession number CICC No. 20247.

[0029] (2) The fermentation seed liquid was inoculated into the fermentation substrate containing Astragalus membranaceus and cultured with shaking at 42℃ and 180r / min for 48h; the inoculation amount was 2%.

[0030] The fermentation substrate containing Astragalus includes the following components: 10% Astragalus powder, 10% skim milk powder, 1.5% white sugar, and the remainder is sterile water.

[0031] Example 3

[0032] Different experimental groups were set up to study the effect of substrate concentration on the changes in saponin content in the Astragalus fermentation medium.

[0033] Accurately weigh 3.0g, 5.0g, and 8.0g of Astragalus membranaceus powder into 250mL Erlenmeyer flasks. Extract the effective components of the Astragalus membranaceus powder twice using ultrasonic extraction. For the first extraction, add 10 times the volume of water; for the second, add 7 times the volume of water. Extract each time with ultrasound for 45min. Filter, combine the two filtrates, and finally concentrate both to 50mL under reduced pressure. Adjust the pH to 6.20, autoclave at 120℃ for 30min, and then cool to room temperature to obtain the Astragalus membranaceus extract. Inoculate the Astragalus membranaceus extract with a 2% inoculum of fermentation seed liquid (using Lactobacillus delbrueckii subsp. bulgaricus CICC 20247 preserved on an slant, inoculated into MRS liquid medium, and cultured on a shaker at 42℃ and 180r / min for 48h). Then, incubate on a dual-function air bath at 42℃ and 180r / min for 48h. Perform three parallel experiments for each experimental group; the blank group was not inoculated with fermentation seed liquid, and all other conditions were the same as the experimental groups. The total saponin content in the samples of each experimental group was measured after inoculation and after 48 hours of culture. The results are shown in Table 1.

[0034] Table 1: Effect of different substrate concentrations on total saponin content

[0035]

[0036] Based on a comprehensive comparison of the above data, the following conclusions can be drawn: (1) The total saponin content of Astragalus membranaceus was increased after fermentation with Lactobacillus delbrueckii subsp. bulgaricus; (3) Under the same conditions, different substrate concentrations will have a certain impact on the saponin increase rate. Moreover, the increase in substrate concentration and the increase in total saponin content are not always positively correlated. At a certain concentration, the total saponin content increases with the increase in substrate concentration. When a certain range is reached, increasing the substrate concentration will actually decrease the total saponin content.

[0037] Example 4

[0038] Seventeen experimental groups were set up to investigate three factors in the Astragalus fermentation substrate: carbon source (skim milk powder, white sugar), nitrogen source (skim milk powder, yeast extract), and total saponins content (mg / g) of Astragalus after fermentation. Each factor was set at three levels, and the response value was the total saponin content of Astragalus after fermentation. The factor level table and experimental design table are shown in Tables 2 and 3 below.

[0039] Table 2. Levels of experimental factors in response surface methodology.

[0040]

[0041]

[0042] Preparation method of Astragalus-containing fermentation substrate for response surface methodology: 5g of Astragalus powder was placed in a 250mL Erlenmeyer flask. Skim milk powder, white sugar, and yeast extract were added according to Table 3. Then, 50mL of distilled water was added. The fermentation seed culture (Lactobacillus delbrueckii subsp. bulgaricus CICC 20247 preserved on a slant agar was inoculated into MRS liquid medium and cultured on a shaker at 42℃ and 180r / min for 48h to obtain the fermentation seed culture) was obtained. The pH was adjusted to 6.20, and the culture was carried out at 42℃ and 180r / min in a dual-function air bath constant temperature shaker for 48h. The total saponin content of Astragalus was then determined. The results are as follows: Figure 2-7 As shown.

[0043] Table 3. Response Surface Experiment Design and Results

[0044]

[0045]

[0046] Based on the experimental results, a multiple regression analysis was performed. The influence of the main factors on the response value is expressed by the following formula:

[0047] Y=0.9491+0.0203A-0.1851B-0.0397C-0.0169AB-0.0616AC-0.0218BC-0.0569A 2 +0.1257B 2 +0.2081C 2 .

[0048] Therefore, it can be concluded that the optimal combination of 5g of astragalus powder, 4.52g of skim milk powder, 0.70g of white sugar, and 0.011g of yeast extract, with sterile water added to a final volume of 50mL, resulted in the best enhancement of the total astragalus saponin content after fermentation. This experiment demonstrates that the total astragalus saponin content significantly increased after fermentation with *Lactobacillus delbrueckii*.

[0049] Example 5

[0050] The fermentation seed liquid (CCICC 20247 of Lactobacillus delbrueckii subsp. bulgaricus preserved on a slant was inoculated into MRS liquid medium and cultured on a shaker at 42℃ and 180 r / min for 48 h to obtain the fermentation seed liquid) was inoculated at a rate of 2% into four different culture media and cultured on a dual-function air bath constant temperature shaker at 42℃ and 180 r / min for 48 h.

[0051] Culture medium ①: 5g of Astragalus powder was extracted twice with 10 times and 7 times the amount of purified water by ultrasonic extraction, each extraction lasting 45min. The extracts were combined and concentrated to 50mL. The pH was adjusted to 6.20 to obtain Astragalus extract. The extract was then sterilized by autoclaving at 120℃ for 30min. After that, it was taken out and cooled to room temperature for later use.

[0052] Culture medium ②: Weigh 5g of Astragalus powder and 0.5g of NaCl, and put them into a 250mL Erlenmeyer flask. Then add distilled water to 50mL, adjust the pH to 6.20, sterilize at 121℃ for 30min, and then remove and cool to room temperature for later use.

[0053] Culture medium ③: 5g Astragalus powder, 5g skim milk powder, 1.5g white sugar, 0.025g yeast extract, add sterile water to 50mL.

[0054] Culture medium ④: 5g Astragalus powder, 5g skim milk powder, 1.5g white sugar, add sterile water to 50mL.

[0055] The results of the determination of total saponin content of Astragalus membranaceus after culturing in different culture media for 48 hours are shown in Table 4 below. Among them, the increase rate of total saponin content of Astragalus membranaceus after fermentation is A% = (W-W0) / W0, where: A - increase rate of total saponin content of Astragalus membranaceus, W - total saponin content of Astragalus membranaceus after fermentation, and W0 - total saponin content of unfermented Astragalus membranaceus.

[0056] The results showed that after fermentation in culture medium ④, the total saponin content of Astragalus membranaceus increased from 0.5565 mg / g to 1.4894 mg / g, an increase of 167.63%, significantly improving the total saponin content of Astragalus membranaceus. Therefore, the Astragalus membranaceus-containing fermentation substrate provided by this invention is beneficial for increasing the total saponin content of Astragalus membranaceus.

[0057] Table 4. Data on the influence of different culture media on total saponin content after fermentation.

[0058]

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

Claims

1. A probiotic fermentation method for increasing total saponins in astragalus based on response surface methodology, characterized in that, The steps are as follows: inoculate the fermentation seed liquid into the fermentation substrate containing Astragalus membranaceus, and culture it with shaking at 40-45℃ and 150-200 r / min for 48 h; the inoculation amount is 1-5%; The fermentation substrate containing Astragalus includes the following components: 2-20% Astragalus powder, 10-20% skim milk powder, 1-5% white sugar, 0-0.5% yeast extract, and the remainder is sterile water.

2. The probiotic fermentation method for increasing total saponins of astragalus based on response surface methodology according to claim 1, characterized in that, The method for preparing the fermentation seed liquid is as follows: Lactobacillus delbrueckii subsp. bulgaricus is inoculated into MRS liquid medium and cultured with shaking at 40-45℃ and 150-200 r / min for 48 h to obtain the fermentation seed liquid; the preservation number of Lactobacillus delbrueckii subsp. bulgaricus is CICC No. 20247.

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