CN113481243A

CN113481243A - Angelica sinensis fermentation liquor with effects of resisting oxidation, resisting aging and regulating intestinal flora, and preparation method and application thereof

Info

Publication number: CN113481243A
Application number: CN202110938802.7A
Authority: CN
Inventors: 张春江; 卢雪蕊; 李君翔
Original assignee: Lanzhou University
Current assignee: Lanzhou University
Priority date: 2021-08-16
Filing date: 2021-08-16
Publication date: 2021-10-08
Anticipated expiration: 2041-08-16
Also published as: CN113481243B

Abstract

The invention belongs to the technical field of traditional Chinese medicine fermentation, and provides angelica sinensis fermentation liquor with functions of resisting oxidation and aging and regulating intestinal flora, and a preparation method and application thereof, wherein the preparation method comprises the following steps: soaking radix Angelicae sinensis in water, decocting, filtering, collecting filtrate, adding water into residue, decocting, filtering, collecting filtrate, mixing filtrates, concentrating, centrifuging to obtain water extractive solution; fermenting the angelica water extract: mixing Lactobacillus plantarum LZU-J-TSL6 and LZU-S-ZCJ, inoculating into radix Angelicae sinensis water extractive solution, and fermenting to obtain Angelica sinensis fermentation liquid. The angelica sinensis fermentation liquor obtained by the preparation method has the effects of resisting oxidation and aging, well removing free radicals, regulating intestinal flora and having good health care effect.

Description

Angelica sinensis fermentation liquor with effects of resisting oxidation, resisting aging and regulating intestinal flora, and preparation method and application thereof

Technical Field

The invention relates to the technical field of traditional Chinese medicine fermentation, in particular to angelica sinensis fermentation liquor with functions of resisting oxidation and aging and regulating intestinal flora, and a preparation method and application thereof.

Background

Aerobic organisms in the world are gradually exposed to environments generated by oxidants or electrophiles, which are affected by the processes of enzymatic reactions in the body and by external harmful substances. These oxidizing poisons have serious effects on the body, and in order to avoid these adverse effects, the body has evolved a complex yet stable antioxidant defense system to sustain the redox reactions in the body. ROS (reactive oxygen species) excessively generated by cells in a body and ROS generated by an electrophile can induce free radical chain reaction, so that biomacromolecules in the cells are subjected to cross-linking polymerization to generate Malondialdehyde (MDA), and the survival of the cells is greatly threatened. An increase in oxidative stress is an important cause of aging.

The angelica contains various chemical components, and researches show that angelica polysaccharide, organic acid, flavone and volatile oil have the effects of resisting oxidation and removing free radicals. In the field of traditional Chinese medicines, the traditional Chinese medicine has a good curative effect on treatment of chronic diseases, but is subject to scaling due to slow treatment process, bitter taste, long decoction time and the like. Most of the effective components of the traditional Chinese medicine are wrapped in compact cell walls and are difficult to dissolve. The traditional Chinese medicine fermentation technology is characterized in that traditional Chinese medicines containing active ingredients are used as a culture medium, fermented strains are added into the culture medium, and the traditional Chinese medicines interact with the strains to influence each other. On one hand, the strain can grow by itself by utilizing the nutrients provided by the traditional Chinese medicine culture medium, and on the other hand, the enzyme generated in the growth process of the strain can accelerate the dissolution of the effective components of the traditional Chinese medicine and improve the efficacy of the traditional Chinese medicine.

Disclosure of Invention

The invention aims to provide a preparation method of angelica sinensis fermentation liquor, which has the effects of resisting oxidation and aging, regulating intestinal flora and having good health care effect.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a preparation method of angelica sinensis fermentation liquor, which comprises the following steps:

(1) soaking radix Angelicae sinensis in water, decocting, filtering, collecting filtrate, adding water into residue, decocting, filtering, collecting filtrate, mixing filtrates, concentrating, centrifuging to obtain water extractive solution;

(2) fermenting the angelica water extract:

mixing Lactobacillus plantarum LZU-J-TSL6 and LZU-S-ZCJ, inoculating into radix Angelicae sinensis water extractive solution, and fermenting to obtain Angelica sinensis fermentation liquid.

Preferably, the soaking time in the step (1) is 8-16 h, the volume ratio of the first decocted angelica to water is 1: 8-12, and the decocting time is 0.5-2.5 h; the volume ratio of the filter residue obtained in the second decoction to water is 1: 6-10, the decoction time is 0.5-2.5 h, and the water is used for soaking.

Preferably, the lactobacillus plantarum LUZ-J-TSL6 is preserved in Guangdong provincial microorganism strain collection center in 2020, 10 and 23 days, with the preservation numbers as follows: GDMCC No: 61242 Lactobacillus plantarum LZU-S-ZCJ, deposited in the Guangdong province collection of microorganisms at 31/12/2020 with the deposit numbers: GDMCC No: 61402 of; the nucleic acid sequence of the Lactobacillus plantarum LZU-S-ZCJ is shown in SEQ NO. 1.

Preferably, in the step (1), each 1mL of the angelica water extract is prepared from 0.4-1.0 g of angelica.

Preferably, the volume ratio of the Lactobacillus plantarum LZU-J-TSL6 to the LZU-S-ZCJ in the step (2) is (1-3): (1-3), the concentration of the two strains is (1-9) x10¹²CFU/mL, the inoculation amount of the mixed strain is 3-5%.

Preferably, the fermentation temperature in the step (2) is 36-38 ℃, and the fermentation time is 60-100 h.

The invention also provides angelica sinensis fermentation liquor.

The invention also provides application of the angelica sinensis fermentation liquor in preparation of a medicine for improving intestinal beneficial bacteria, and the prepared medicine of the angelica sinensis fermentation liquor can improve the abundance of beneficial bacteria of lactobacillus.

The invention also provides application of the angelica sinensis fermentation liquor in preparing in-vitro antioxidant products, wherein the angelica sinensis fermentation liquor can remove DPPH, hydroxyl free radicals, superoxide anions and ABTS free radicals.

The angelica sinensis fermentation liquor obtained by the preparation method has the effects of resisting oxidation and aging, better removing free radicals, regulating intestinal flora and having good health care effect. The cleaning agent has good cleaning effect on DPPH free radicals, superoxide anions and hydroxyl free radicals; can reduce oxidation products of aged mice liver tissue induced by D-galactose, improve the content of antioxidant enzyme of liver tissue, and activate the expression of antioxidant pathway Nrf2 and HO-1 gene; the abundance of beneficial bacterium lactobacillus in intestinal tracts of aged mice induced by D-galactose can be improved; can accelerate the metabolism passage related to the D-galactose-induced feces metabolites of the aged mice and relieve the aging process.

Drawings

FIG. 1 shows the results of the antioxidant activity assay of the fermentation broth of Angelica sinensis (FQ for Angelica sinensis extract group, FH for Angelica sinensis fermentation broth group, J for Lactobacillus plantarum group, P for L-ascorbic acid group)

FIG. 2 shows the change of the oxidation index and inflammation index in liver tissue of each mouse group (C is normal control group, M is model control group, P is L-ascorbic acid group, F is angelica fermentation broth group, S is angelica water extract group, J is Lactobacillus plantarum group, JS is angelica water extract + Lactobacillus plantarum group)

FIG. 3 shows the effect of treatment with fermentation and aqueous extracts of Angelica sinensis on histopathological analysis of liver (H & E staining, magnification x 40) (blank control group A, D-gal model group B, L-ascorbic acid group C, fermentation and aqueous extracts of Angelica sinensis E)

FIG. 4 shows the effect on the expression of Nrf2 signal pathway genes Nrf2(4-A) and HO-1(4-B) (C is normal control group, M is model control group, S is radix Angelicae sinensis water extract group, F is radix Angelicae sinensis fermentation broth group, #: represents that P is less than 0.05 compared with M group)

FIG. 5-A shows the effect of Men's horizontal fermentation broth of Angelica gigas nakai on regulating intestinal flora structure of mice

FIG. 5-B shows the effect of horizontal fermentation broth of Angelica sinensis on the regulation of intestinal flora structure of mice in each group

FIG. 5-C shows the prediction of intestinal differential flora function (CH for normal control group, MH for aging model group, PH for L-ascorbic acid group, SH for water extract of radix Angelicae sinensis, FH for fermentation broth of radix Angelicae sinensis) for each group of mice

FIG. 6-A is a PCA principal component analysis chart of mouse fecal metabolism FH group to FQ group

FIG. 6-B is an OPLS-DA score plot of the FH group versus the FQ group of the fecal metabolism of mice

FIG. 6-C is a graph showing the results of the replacement test of the OPLS-DA model of the FH group versus the FQ group in the feces metabolism of mice

FIG. 6-D is a volcano plot of mouse differential metabolite screening by FH groups to FQ group

FIG. 6-E is a hierarchical clustering analysis thermodynamic diagram of mouse fecal metabolism FH groups versus FQ groups

FIG. 6-F is a diagram of the metabolic pathway involved in the mouse metabolic defect

FIG. 6-G is a diagram showing the metabolic pathways involved in mouse metabolic errors (ascorbic acid and alduronate metabolic pathways, purine metabolic pathways, glycerophospholipid metabolic pathways)

FIG. 7 shows the selection of strains and ratios

FIG. 8 shows the effect of single factor on the fermentation broth of Angelica sinensis (A is the concentration of Angelica sinensis aqueous extract, B is the inoculum size, C is the fermentation temperature, D is the fermentation time)

Deposit description

Lactobacillus plantarum LZU-J-TSL6, Latin is Lactobacillus plantarum, which is deposited in the Guangdong province collection of microorganisms with the following addresses: the preservation date of the No. 59 building 5 of the No. 100 college of the Xieli Zhonglu city is 10 months and 23 days of 2020, and the preservation numbers are as follows: GDMCC No: 61242.

lactobacillus plantarum LZU-S-ZCJ, Latin is Lactobacillus plantarum, which is deposited in the Guangdong province collection of microorganisms with the addresses: the preservation date of the No. 59 building 5 building of the No. 100 college of the Xieli Zhonglu City is 12 months and 31 days of 2020, and the preservation numbers are as follows: GDMCC No: 61402.

Detailed Description

(2) fermenting the angelica water extract:

In the step (1) of the present invention, the soaking time is preferably 8 to 16 hours, more preferably 10 to 14 hours, and even more preferably 12 hours.

In the step (1) of the present invention, the volume ratio of the first angelica decoction to the water is preferably 1:8 to 12, more preferably 1:8 to 10, and even more preferably 1:10, and the decoction time is preferably 0.5 to 2.5, more preferably 1 to 2 hours, and even more preferably 1 hour. The Chinese medicinal herbs are directly decocted after being soaked, and water does not need to be changed.

In the step (1) of the present invention, the volume ratio of the second decoction residue to water is preferably 1: 6-10, more preferably 1: 6-8, even more preferably 1:8, and the decoction time is preferably 0.5-2.5 hours, even more preferably 1-2 hours, even more preferably 1 hour.

As a preferable scheme of the invention, the Lactobacillus plantarum LUZ-J-TSL6 is preserved in Guangdong provincial microorganism strain collection center in 2020, 10 and 23 days, and the preservation numbers are as follows: GDMCC No: 61242, respectively; lactobacillus plantarum LZU-S-ZCJ is preserved in Guangdong province microorganism strain preservation center in 12-31 of 2020, with the preservation number being: GDMCC No: 61402 of; the nucleic acid sequence of the Lactobacillus plantarum LZU-S-ZCJ is shown in SEQ NO. 1.

In the step (1), the water extract of angelica is prepared from 0.4 to 1.0g of angelica per 1mL, more preferably 0.5 to 0.7g, and still more preferably 0.6 g.

In the step (2) of the invention, the volume ratio of the Lactobacillus plantarum LZU-J-TSL6 to the LZU-S-ZCJ is preferably (1-3): (1-3), more preferably 1 (1-3), and still more preferably 1: 3.

In the step (2) of the present invention, the concentration of the Lactobacillus plantarum LZU-J-TSL6 and LZU-S-ZCJ is preferably (1-9). times.10¹²CFU/mL, more preferablyIs (3-7) × 10¹²CFU/mL, more preferably 5X 10¹²CFU/mL, the inoculation amount of the mixed strain is preferably 3-5%, more preferably 3.5-4.5%, and still more preferably 4%.

In the step (2), the fermentation temperature is preferably 36-38 ℃, more preferably 36-37 ℃, even more preferably 36 ℃, and the fermentation time is preferably 60-100 h, more preferably 70-90 h, even more preferably 72 h.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Adding 10 times of water into radix Angelicae sinensis, soaking for 12h, decocting for 1h, filtering, collecting filtrate, adding 8 times of water into residue, decocting for 1h, filtering, collecting filtrate, mixing filtrates, concentrating filtrate to obtain concentrated solution 0.6g/mL, centrifuging at 4 deg.C and 3000rmp, and collecting supernatant. Mixing Lactobacillus plantarum LZU-J-TSL6 and LZU-S-ZCJ at a volume ratio of 1:3, wherein the concentration of the two strains is 5 × 10¹²CFU/mL was inoculated at 4% inoculum size to an aqueous extract of Angelica sinensis prepared as described above at 1mL of extract per 0.6g of Angelica sinensis. Fermenting for 72h at the fermentation temperature of 36 ℃ to obtain the angelica fermentation liquor.

Example 2

Adding 8 times of water to radix Angelicae sinensis, soaking for 8 hr, decocting for 0.5 hr, filtering, collecting filtrate, adding 6 times of water to the residue, decocting for 0.5 hr, filtering, collecting filtrate, mixing the filtrates, concentrating the filtrate to obtain concentrated solution 0.4g/mL, centrifuging at 4 deg.C and 3000rmp, and collecting supernatant. Mixing Lactobacillus plantarum LZU-J-TSL6 and LZU-S-ZCJ at a volume ratio of 3:1, wherein the concentration of the two strains is 1 × 10¹²CFU/mL was inoculated at 3% inoculum size to an aqueous extract of Angelica sinensis prepared as described above at 1mL per 0.4g of Angelica sinensis. Fermenting for 100h at 37 deg.C to obtain the final product.

Example 3

Adding 12 times of water to radix Angelicae sinensis, soaking for 16 hr, decocting for 2.5 hr, filtering, collecting filtrate, adding 10 times of water to the residue, decocting for 2.5 hr, filtering, collecting filtrate, mixing the filtratesThen, the filtrate was concentrated to 1.0g/mL, centrifuged at 4 ℃ and 3000rmp, and the supernatant was collected for use. Mixing Lactobacillus plantarum LZU-J-TSL6 and LZU-S-ZCJ at a volume ratio of 1.5:1.5, wherein the concentration of the two strains is 9 × 10¹²CFU/mL, 5% inoculum size was inoculated into 1mL of the above aqueous extract of Angelica sinensis per 1g of Angelica sinensis. Fermenting for 60h at the fermentation temperature of 38 ℃ to obtain the angelica fermentation liquor.

Example 4

Adding 11 times of water to radix Angelicae sinensis, soaking for 14h, decocting for 1.5h, filtering, collecting filtrate, adding 9 times of water to the residue, decocting for 1.5h, filtering, collecting filtrate, mixing the filtrates, concentrating the filtrate to obtain concentrated solution 0.7g/mL, centrifuging at 4 deg.C and 3000rmp, and collecting supernatant. Mixing Lactobacillus plantarum LZU-J-TSL6 and LZU-S-ZCJ at a volume ratio of 1.5:1, wherein the concentration of the two strains is 7 × 10¹²CFU/mL was inoculated at 4.5% inoculum size to an aqueous extract of Angelica sinensis prepared as described above at 1mL per 0.7g of Angelica sinensis. Fermenting for 90 hours at the fermentation temperature of 37.5 ℃ to obtain the angelica fermentation liquor.

Example 5

Adding 9 times of water into radix Angelicae sinensis, soaking for 10h, decocting for 1h, filtering, collecting filtrate, adding 7 times of water into residue, decocting for 1h, filtering, collecting filtrate, mixing filtrates, concentrating filtrate to obtain concentrated solution 0.6g/mL, centrifuging at 4 deg.C and 3000rmp, and collecting supernatant. Mixing Lactobacillus plantarum LZU-J-TSL6 and LZU-S-ZCJ at a volume ratio of 1.5:3, wherein the concentration of the two strains is 3 × 10¹²CFU/mL, 3.5% inoculum size was inoculated into 1mL of the above aqueous extract of Angelica sinensis per 0.6g of Angelica sinensis. Fermenting for 70h at the fermentation temperature of 36 ℃ to obtain the angelica fermentation liquor.

Comparative example 1

Adding 10 times of water into radix Angelicae sinensis, soaking for 12h, decocting for 1h, filtering, collecting filtrate, adding 8 times of water into residue, decocting for 1h, filtering, collecting filtrate, mixing filtrates, concentrating filtrate to obtain concentrated solution 0.2g/mL, centrifuging at 4 deg.C and 3000rmp, and collecting supernatant. Mixing Lactobacillus plantarum LZU-J-TSL6 and LZU-S-ZCJ at a volume ratio of 1:3, wherein the concentration of the two strains is 5 × 10¹²CFU/mL, 4% inoculation amount of radix Angelicae sinensis in which 1mL of the above extract solution per 0.2g of radix Angelicae sinensis is preparedExtracting with water. Fermenting for 72h at the fermentation temperature of 36 ℃ to obtain the angelica fermentation liquor.

Comparative example 2

Adding 10 times of water into radix Angelicae sinensis, soaking for 12h, decocting for 1h, filtering, collecting filtrate, adding 8 times of water into residue, decocting for 1h, filtering, collecting filtrate, mixing filtrates, concentrating filtrate to obtain concentrated solution 0.6g/mL, centrifuging at 4 deg.C and 3000rmp, and collecting supernatant. Mixing Lactobacillus plantarum LZU-J-TSL6 and LZU-S-ZCJ at a volume ratio of 1:3, wherein the concentration of the two strains is 5 × 10¹²CFU/mL was inoculated at an inoculum size of 2% to an aqueous extract of Angelica sinensis prepared as described above at 1mL per 0.6g of Angelica sinensis. Fermenting for 72h at the fermentation temperature of 36 ℃ to obtain the angelica fermentation liquor.

Comparative example 3

Adding 10 times of water into radix Angelicae sinensis, soaking for 12h, decocting for 1h, filtering, collecting filtrate, adding 8 times of water into residue, decocting for 1h, filtering, collecting filtrate, mixing filtrates, concentrating filtrate to obtain concentrated solution 0.6g/mL, centrifuging at 4 deg.C and 3000rmp, and collecting supernatant. Mixing Lactobacillus plantarum LZU-J-TSL6 and LZU-S-ZCJ at a volume ratio of 1:3, wherein the concentration of the two strains is 5 × 10¹²CFU/mL was inoculated at an inoculation amount of 1% to the aqueous extract of Angelica sinensis prepared as described above, wherein 1mL of the extract was prepared per 0.6g of Angelica sinensis. Fermenting for 72h at the fermentation temperature of 36 ℃ to obtain the angelica fermentation liquor.

Comparative example 4

Adding 10 times of water into radix Angelicae sinensis, soaking for 12h, decocting for 1h, filtering, collecting filtrate, adding 8 times of water into residue, decocting for 1h, filtering, collecting filtrate, mixing filtrates, concentrating filtrate to obtain concentrated solution 0.6g/mL, centrifuging at 4 deg.C and 3000rmp, and collecting supernatant. Mixing Lactobacillus plantarum LZU-J-TSL6 and LZU-S-ZCJ at a volume ratio of 1:3, wherein the concentration of the two strains is 5 × 10¹²CFU/mL was inoculated at 4% inoculum size to an aqueous extract of Angelica sinensis prepared as described above at 1mL of extract per 0.6g of Angelica sinensis. Fermenting for 72 hours at the fermentation temperature of 35 ℃ to obtain the angelica fermentation liquor.

Comparative example 5

Adding 10 times of water into radix Angelicae sinensis, soaking for 12 hr, decocting for 1 hr, filtering, collecting filtrate, adding 8 times of water into residue, decocting for 1 hr,filtering and collecting filtrate, combining the two filtrates, concentrating the filtrate into 0.6g/mL, centrifuging at 4 ℃ and 3000rmp, and taking supernatant for later use. Mixing Lactobacillus plantarum LZU-J-TSL6 and LZU-S-ZCJ at a volume ratio of 1:3, wherein the concentration of the two strains is 5 × 10¹²CFU/mL was inoculated at 4% inoculum size to an aqueous extract of Angelica sinensis prepared as described above at 1mL of extract per 0.6g of Angelica sinensis. Fermenting for 72 hours at the fermentation temperature of 39 ℃ to obtain the angelica fermentation liquor.

Comparative example 6

Adding 10 times of water into radix Angelicae sinensis, soaking for 12h, decocting for 1h, filtering, collecting filtrate, adding 8 times of water into residue, decocting for 1h, filtering, collecting filtrate, mixing filtrates, concentrating filtrate to obtain concentrated solution 0.6g/mL, centrifuging at 4 deg.C and 3000rmp, and collecting supernatant. Mixing Lactobacillus plantarum LZU-J-TSL6 and LZU-S-ZCJ at a volume ratio of 1:3, wherein the concentration of the two strains is 5 × 10¹²CFU/mL was inoculated at 4% inoculum size to an aqueous extract of Angelica sinensis prepared as described above at 1mL of extract per 0.6g of Angelica sinensis. Fermenting for 40h at the fermentation temperature of 36 ℃ to obtain the angelica fermentation liquor.

Comparative example 7

Adding 10 times of water into radix Angelicae sinensis, soaking for 12h, decocting for 1h, filtering, collecting filtrate, adding 8 times of water into residue, decocting for 1h, filtering, collecting filtrate, mixing filtrates, concentrating filtrate to obtain concentrated solution 0.6g/mL, centrifuging at 4 deg.C and 3000rmp, and collecting supernatant. Mixing Lactobacillus plantarum LZU-J-TSL6 and LZU-S-ZCJ at a volume ratio of 1:3, wherein the concentration of the two strains is 5 × 10¹²CFU/mL was inoculated at 4% inoculum size to an aqueous extract of Angelica sinensis prepared as described above at 1mL of extract per 0.6g of Angelica sinensis. Fermenting for 20h at the fermentation temperature of 36 ℃ to obtain the angelica fermentation liquor.

Comparative example 8

Adding 10 times of water into radix Angelicae sinensis, soaking for 12h, decocting for 1h, filtering, collecting filtrate, adding 8 times of water into residue, decocting for 1h, filtering, collecting filtrate, mixing filtrates, concentrating filtrate to obtain concentrated solution 0.6g/mL, centrifuging at 4 deg.C and 3000rmp, and collecting supernatant. MRS agar was inoculated in an amount of 4% to the aqueous extract of Angelica sinensis prepared as described above to prepare 1mL of the extract per 0.6g of Angelica sinensis. Fermenting for 72h at the fermentation temperature of 36 ℃ to obtain the angelica fermentation liquor.

Comparative example 9

Adding 10 times of water into radix Angelicae sinensis, soaking for 12h, decocting for 1h, filtering, collecting filtrate, adding 8 times of water into residue, decocting for 1h, filtering, collecting filtrate, mixing filtrates, concentrating filtrate to obtain concentrated solution 0.6g/mL, centrifuging at 4 deg.C and 3000rmp, and collecting supernatant. The LZU-J-TSL6 and LZU-S-ZCJ dead bacteria (SJ group) were inoculated at a volume ratio of 1:1 in an inoculum size of 4% to an aqueous extract of Angelica sinensis prepared in an amount of 1mL per 0.6g of the above extract. Fermenting for 72h at the fermentation temperature of 36 ℃ to obtain the angelica fermentation liquor.

Comparative example 10

Adding 10 times of water into radix Angelicae sinensis, soaking for 12h, decocting for 1h, filtering, collecting filtrate, adding 8 times of water into residue, decocting for 1h, filtering, collecting filtrate, mixing filtrates, concentrating filtrate to obtain concentrated solution 0.6g/mL, centrifuging at 4 deg.C and 3000rmp, and collecting supernatant. Lactobacillus plantarum LZU-J-TSL6 at a concentration of 5X 10¹²CFU/mL was inoculated at 4% inoculum size to an aqueous extract of Angelica sinensis prepared as described above at 1mL of extract per 0.6g of Angelica sinensis. Fermenting for 72h at the fermentation temperature of 36 ℃ to obtain the angelica fermentation liquor.

Comparative example 11

Adding 10 times of water into radix Angelicae sinensis, soaking for 12h, decocting for 1h, filtering, collecting filtrate, adding 8 times of water into residue, decocting for 1h, filtering, collecting filtrate, mixing filtrates, concentrating filtrate to obtain concentrated solution 0.6g/mL, centrifuging at 4 deg.C and 3000rmp, and collecting supernatant. Lactobacillus plantarum LZU-S-ZCJ at a concentration of 5X 10¹²CFU/mL was inoculated at 4% inoculum size to an aqueous extract of Angelica sinensis prepared as described above at 1mL of extract per 0.6g of Angelica sinensis. Fermenting for 72h at the fermentation temperature of 36 ℃ to obtain the angelica fermentation liquor.

The results of exploring the fermentation strains and the ratios thereof using example 1 as an experimental group and comparative examples 1 to 11 as a control group are shown in FIG. 7, in which the suitable ranges of the volume ratios of Lactobacillus plantarum LZU-J-TSL6 and LZU-S-ZCJ are 1 to 3:1 to 3, and the optimal volume ratios of Lactobacillus plantarum LZU-J-TSL6 and LZU-S-ZCJ are 1: 3; the influence of single factors on the angelica sinensis fermentation broth is explored, and the result is shown in fig. 8, wherein the concentration of the angelica sinensis water extract is 0.6g/mL, the inoculum size is 4%, the fermentation temperature is 36 ℃, and the fermentation time is 72 hours, which is the best angelica sinensis fermentation broth preparation parameter.

Determination of antioxidant activity of angelica sinensis fermentation liquor

The total antioxidant capacity (ABTS method), total antioxidant capacity (FRAP method), superoxide anion radical scavenging activity, hydroxyl radical scavenging activity, DPPH radical scavenging activity of the angelica sinensis fermentation broth obtained in example 1 was measured by using the angelica sinensis fermentation broth as an experimental group according to the methods and procedures of the kit instructions. The kits used above were all purchased from Shanghai Haosheng practice Co., Ltd.

As shown in FIG. 1, Angelica sinensis has a stronger antioxidant activity after fermentation than before fermentation. Wherein, the clearance rate of angelica for eliminating hydroxyl radical, superoxide anion and DPPH radical is obviously changed after fermentation (P is less than 0.05, P is less than 0.01), and the total antioxidant capacity is not obviously changed after fermentation (P is more than 0.05).

The angelica sinensis fermentation liquor provided by the invention has strong antioxidant activity of removing free radicals.

Effect of Angelica sinensis fermentation broth on D-galactose-induced aging mice

Grouping, modeling and administration of test animals (the fermentation broth of Angelica sinensis obtained in example 1 was set F)

Grouping: 70 animals for the test were acclimatized for 7 days and divided into 7 groups of 10 animals each. The method comprises the following steps: normal control group (C), model control group (M), L-ascorbic acid group (P), radix Angelicae sinensis fermentation liquid group (F), radix Angelicae sinensis water extract group (S), Lactobacillus plantarum group (J), and radix Angelicae sinensis water extract + bacterium group (JS).

Molding: apart from normal control group, 2.5g/kg of D-galactose solution was administered by intraperitoneal injection to each group in an amount of 0.1mL/10g, respectively.

Administration: and converting the dose into the mouse dose according to the pre-experimental result and the human dose range of the Chinese angelica in the reference pharmacopoeia. After 14 days of molding, the other groups except the normal control group were subjected to intragastric administration while injecting D-galactose for a period of 30 days. The dosage is 0.1mL/10g, and the normal control group and the model group are filled with the same amount of normal saline. The L-ascorbic acid group is administered with 120mg/kg L-ascorbic acid solution. Lactobacillus plantarum group 10⁹CFU/mL, the radix Angelicae sinensis water extractive solution group is given radix Angelicae sinensis water extractive solution of 1mL extractive solution per 0.6g radix Angelicae sinensis, and the radix Angelicae sinensis fermentation liquid group is given radix Angelicae sinensis fermentation liquid of 0.6 g/mL.

Sample collection and biochemical index detection

After the administration period is finished, fasting is carried out for 24 hours without water prohibition, eyeballs of mice in each group are picked, blood is taken, blood samples are placed in a test tube rack for standing for 2 hours, and then supernatant is obtained by centrifugation and refrigerated for later use. After blood is taken, liver tissues are quickly separated, fatty tissues around the liver tissues are removed, residual blood is washed, water is sucked dry, weighing is carried out, visceral organ indexes are calculated, and the liver tissues are homogenized and frozen for later use.

The MDA content, SOD, CAT, GSH-Px, T-AOC activity, IL-1 beta, IL-6, TNF-alpha level and other cell factor expressions in serum and liver tissue are measured according to the kit instructions.

As shown in FIG. 2, administration of D-Gal resulted in significantly decreased T-AOC levels and significantly increased MDA levels (P < 0.05) in liver tissues of mice in the aging group, as well as SOD, CAT and GSH-PX. Treatment with L-ascorbic acid, angelica broth and angelica water extract reversed these changes, respectively.

HE staining of liver tissue

Liver tissue from each mouse was fixed in 10% neutral formalin for 48H, dehydrated, waxed, embedded, sectioned, and stained with hematoxylin-eosin (H & E). Histological images were taken under a fluorescence microscope at 40-fold magnification.

As shown in FIG. 3, the structure of normal liver is clearly characterized in the blank control group mice, the cells are arranged in order, the damage of the model group liver tissue caused by the induction of D-galactose is obvious, and the liver cell edema, the cytoplasm is loosened, and some cytoplasm is vacuolated to be balloon-like. The treatment of the angelica fermentation liquor and the angelica water extract can prevent liver injury, so that cells lose balloon degeneration and vacuole of cytoplasm is reduced, and the improvement of the angelica fermentation liquor group is more obvious and is similar to that of the angelica group P.

Determination of liver tissue Nrf2, HO-1mRNA expression

Extracting RNA of liver tissue, detecting concentration and purity, and performing PCR amplification after reverse transcription of RNA. The primers are as follows:

Nrf2-F TAGATGACCATGAGTCGCTTGC

Nrf2-R GCCAAACTTGCTCCATGTCC

HO1-F GATAGAGCGCAACAAGCAGAA

HO1-R CAGTGAGGCCCATACCAGAAG

a PCR amplification system;

the total system is as follows: mu.l, cDNA template, 1. mu.l (Note: cDNA template diluted 5-fold used.) primer (F + R) 1. mu.l, 2. x. qPCRMix 10. mu.l, sterile water 8. mu.l.

And (3) PCR reaction conditions:

pre-denaturation: at 95 ℃ for 3 min; denaturation at 95 ℃ for 5 s; renaturation at 60 ℃ for 5 s; extension at 72 ℃ for 15s for 40 cycles;

the results are shown in FIG. 4: compared with the blank group, the expression levels of Nrf2 mRNA and HO-1mRNA in the liver of P, M, F mice are increased, and the difference has statistical significance (P <0.05 and P < 0.01); compared with the aging group, the expression levels of Nrf2 mRNA and HO-1mRNA in the liver of P, M, F mice are increased, and the difference is statistically significant (P <0.05 and P < 0.01).

Influence of Angelica sinensis fermentation broth on D-galactose-induced intestinal flora and fecal metabolites thereof in aging mice

Influence of Angelica sinensis fermentation broth on intestinal flora of D-galactose-induced aging mice

The angelica sinensis fermentation liquid obtained in example 1 is used as an experimental group, about 0.1g of fresh excrement of mice of each group after administration is collected in a sterilized test tube, and is frozen and stored at the temperature of minus 80 ℃ for later use. Analyzing the result through the steps of sample preparation → DNA extraction and detection → PCR amplification → product purification → library preparation and library inspection → NovaSeq on-machine sequencing and the like.

The primers are as follows:

bacterium 16SrRNA (V3+ V4)5'-ACTCCTACGGGAGGCAGCA-3'

5'-GGACTACHVGGGTWTCTAAT-3'

Fungal ITS1 region 5'-CTTGGTCATTTAGAGGAAGTAA-3'

5'-GCTGCGTTCTTCATCGATGC-3'

PCR amplification System: genomic DNA: 40-60ng/5 ul; VnF (10 μ M): 1.5 mul; VnR (10 μ M): 1.5 mul; KODFXneo (TOYOBO): 1 mul; KODFXNeoBuf (2X): 25 mul; 2 MmdNTP: 10 mu l of the mixture; ddH 2O: make up to 50 μ l total.

And (3) PCR reaction conditions: after electrophoresis at 95 ℃ for 5min, 95 ℃ for 1min, 50 ℃ for 1min, 72 ℃ for 7min, → 15cycles, 1.8% agarose gel, 120V for 40min, the target fragment was cut and recovered.

The results are shown in FIGS. 5-A to C: the angelica sinensis fermentation liquor can regulate the mouse intestinal flora damaged by oxidation, so that the abundance of the scleritis and Lactobacillus is obviously increased. Lactic acid produced by lactobacilli can also promote the production of butyrate, which can be transferred from the intestinal tract to the liver through the portal vein, further activates Nrf2, and influences the host metabolic pathways through the axicon-intestinal axis, which have correlation with changes in intestinal flora.

Effect of Angelica sinensis fermentation broth on D-galactose-induced fecal metabolites of aging mice

The angelica sinensis fermentation broth obtained in example 1 was used as an experimental group, and about 0.1g of fresh feces of mice in the angelica sinensis fermentation broth group was collected after 14 days of molding and 30 days of administration, and the feces were stored in a sterilized test tube and frozen at-80 ℃ for testing. Weighing 50mg of sample to be detected, and adding 1000 mu L of extracting solution (acetonitrile: methanol: water: 2: 1(V/V), isotope labeling internal standard mixture); grinding at 35Hz for 4min, and performing ultrasonic treatment in ice water bath for 5 min; repeating the steps for 2-3 times; standing at-40 ℃ for 1 h; the sample was centrifuged at 12000rpm (centrifugal force 13800 (Xg), radius 8.6cm) for 15min at 4 ℃; and (4) injecting the centrifuged supernatant into a sample injection bottle, and performing sample injection detection on the sample injection bottle.

As shown in fig. 6-a to G, the glycerophospholipids compounds such as glycerophospholipids and glycerolipids, the fatty acid compounds such as ethyl myristate, nervonic acid and erucic acid, and the compounds such as uric acid and inositol may become potential biomarkers for aging, and these differential metabolites mainly participate in metabolic pathways such as metabolism of ascorbic acid and aldehydic acid, biosynthesis of unsaturated fatty acid, ABC transporter, phosphatidylinositol signal system and glycerophospholipids metabolism.

The embodiment of the invention proves that the angelica sinensis fermentation liquor prepared by the preparation method has the effects of resisting oxidation and aging, better removing free radicals, regulating intestinal flora and having good health care effect. The cleaning agent has good cleaning effect on DPPH free radicals, superoxide anions and hydroxyl free radicals; can reduce oxidation products of aged mice liver tissue induced by D-galactose, improve the content of antioxidant enzyme of liver tissue, and activate the expression of antioxidant pathway Nrf2 and HO-1 gene; the abundance of beneficial bacterium lactobacillus in intestinal tracts of aged mice induced by D-galactose can be improved; can accelerate the metabolism passage related to the D-galactose-induced feces metabolites of the aged mice and relieve the aging process.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Lanzhou university

<120> angelica sinensis fermentation liquor with functions of oxidation resistance, aging resistance and intestinal flora regulation, and preparation method and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1433

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ttaggcgctg gttctaaagg ttaccccacc gactttgggt gttacaaact ctcatggtgt 60

gacgggcggt gtgtacaagg cccgggaacg tattcaccgc ggcatgctga tccgcgatta 120

ctagcgattc cgacttcatg taggcgagtt gcagcctaca atccgaactg agaatggctt 180

taagagatta gcttactctc gcgagttcgc aactcgttgt accatccatt gtagcacgtg 240

tgtagcccag gtcataaggg gcatgatgat ttgacgtcat ccccaccttc ctccggtttg 300

tcaccggcag tctcaccaga gtgcccaact taatgctggc aactgataat aagggttgcg 360

ctcgttgcgg gacttaaccc aacatctcac gacacgagct gacgacaacc atgcaccacc 420

tgtatccatg tccccgaagg gaacgtctaa tctcttagat ttgcatagta tgtcaagacc 480

tggtaaggtt cttcgcgtag cttcgaatta aaccacatgc tccaccgctt gtgcgggccc 540

ccgtcaattc ctttgagttt cagccttgcg gccgtactcc ccaggcggaa tgcttaatgc 600

gttagctgca gcactgaagg gcggaaaccc tccaacactt agcattcatc gtttacggta 660

tggactacca gggtatctaa tcctgtttgc tacccatact ttcgagcctc agcgtcagtt 720

acagaccaga cagccgcctt cgccactggt gttcttccat atatctacgc atttcaccgc 780

tacacatgga gttccactgt cctcttctgc actcaagttt cccagtttcc gatgcacttc 840

ttcggttgag ccgaaggctt tcacatcaga cttaaaaaac cgcctgcgct cgctttacgc 900

ccaataaatc cggacaacgc ttgccaccta cgtattaccg cggctgctgg cacgtagtta 960

gccgtggctt tctggttaaa taccgtcaat acctgaacag ttactctcag atatgttctt 1020

ctttaacaac agagttttac gagccgaaac ccttcttcac tcacgcggcg ttgctccatc 1080

agactttcgt ccattgtgga agattcccta ctgctgcctc ccgtaggagt ttgggccgtg 1140

tctcagtccc aatgtggccg attaccctct caggtcggct acgtatcatt gccatggtga 1200

gccgttacct caccatctag ctaatacgcc gcgggaccat ccaaaagtga tagccgaagc 1260

catctttcaa actcggacca tgcggtccaa gttgttatgc ggtattagca tctgtttcca 1320

ggtgttatcc cccgcttctg ggcaggtttc ccacgtgtta ctcaccagtt cgccactcac 1380

tcaaatgtaa atcatgatgc aagcaccaat caataccaga gttcgtcgac tgc 1433

Claims

1. The preparation method of the angelica sinensis fermentation liquor is characterized by comprising the following steps:

(2) fermenting the angelica water extract:

2. The method for preparing the angelica sinensis fermentation broth according to claim 1, wherein the soaking time in the step (1) is 8-16 h, the volume ratio of the first angelica sinensis decoction to water is 1: 8-12, and the decoction time is 0.5-2.5 h; the volume ratio of the filter residue obtained in the second decoction to water is 1: 6-10, and the decoction time is 0.5-2.5 h.

3. The method for preparing the angelica sinensis fermentation broth according to claim 1, wherein the lactobacillus plantarum LUZ-J-TSL6 is deposited in Guangdong province collection of microorganisms at 23/10/2020 with the deposit number: GDMCC No: 61242 Lactobacillus plantarum LZU-S-ZCJ, deposited in the Guangdong province collection of microorganisms at 31/12/2020 with the deposit numbers: GDMCC No: 61402 of; the nucleic acid sequence of the Lactobacillus plantarum LZU-S-ZCJ is shown in SEQ NO. 1.

4. The method of preparing the fermentation broth of Dang Gui according to claim 1, wherein the water extract of Dang Gui in step (1) is prepared from Dang Gui in an amount of 0.4 to 1.0g per 1 mL.

5. The method for preparing the fermentation broth of Angelica sinensis according to claim 3 or 4, wherein the plant straw is used in step (2)The volume ratio of the bacteria LZU-J-TSL6 to the bacteria LZU-S-ZCJ is (1-3): (1-3), the concentration of the two strains is (1-9) x10¹²CFU/mL, the inoculation amount of the mixed strain is 3-5%.

6. The method for preparing angelica sinensis fermentation broth according to claim 5, wherein the fermentation temperature in step (2) is 36-38 ℃ and the fermentation time is 60-100 h.

7. An Angelica sinensis fermentation broth obtained by the production method according to any one of claims 1 to 6.

8. The use of the angelica sinensis fermentation broth in the preparation of a medicament for increasing beneficial bacteria in intestinal tract, according to claim 7, wherein the angelica sinensis fermentation broth is used for preparing a medicament capable of increasing the abundance of beneficial bacteria in lactobacillus.

9. The use of the angelica sinensis fermentation broth of claim 7 for the preparation of an in vitro antioxidant product, wherein said angelica sinensis fermentation broth is capable of scavenging DPPH, hydroxyl radicals, superoxide anions, ABTS radicals.