Composite probiotics and their application in the preparation of fragrant braised soybeans

Abstract

The present invention discloses a composite probiotic and its application in the preparation of fragrant stewed soybeans. The composite probiotic includes Lactobacillus curvatus 5‑1 and Lactobacillus plantarum; the Lactobacillus curvatus 5‑1 is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No.12891. The Lactobacillus plantarum is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC 1.12934. The present invention provides a composite probiotic strain that converts daidzein into equol, and a soybean product having a significant effect in treating menopausal syndrome and estrogen secretion disorders such as breast hyperplasia and breast cancer.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a composite probiotic and application thereof in the preparation of fragrant stewed soybeans, which are used for treating women's menopausal syndrome. Background Art

[0002] Soy isoflavones, also known as "phytoestrogens", are mainly composed of daidzein and genistein. They have a wide range of biological activities. In addition to their estrogen-like functions, they also have antioxidant and cell cycle regulation functions. Equol is the intestinal bacterial metabolite of daidzein. Compared with daidzein and genistein, equol has higher estrogenic activity and antioxidant activity. Pathological studies have shown that equol can bind to estrogen receptors and has significant therapeutic effects in the treatment of menopausal syndrome, breast hyperplasia, breast cancer and other diseases with estrogen secretion disorders.

[0003] However, studies have shown that due to differences in intestinal flora and dietary structure, only about 30% to 45% of the population can produce equol in their bodies. That is to say, after humans ingest soy isoflavones from legumes, not all people can produce equol in their bodies. For non-equol producers, the health effects of ingesting soy isoflavones are relatively weaker than those of equol producers. It has been reported that equol, a metabolite of daidzein, cannot be detected in foods containing isoflavones, such as processed soy products, and it cannot be ingested from the regular diet.

[0004] Based on the above findings, the inventors conducted in-depth research and screened out strains that can produce equol, so as to further produce soybean products containing equol with high biological activity. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a composite probiotic and its application in the preparation of fragrant braised soybeans to solve the problems raised in the above technical background.

[0006] Chinese patent 2016110531735 (the applicant is Ningbo Institute of Technology, Zhejiang University) discloses a strain of Lactobacillus curvatus, which is classified and named as Lactobacillus curvatus 5-1, and is deposited in the China General Microbiological Culture Collection Administration Center. The deposit date is August 22, 2016, and the deposit number is CGMCC No.12891. The strain has the ability to efficiently degrade purine nucleosides. The strain can decompose the purine nucleosides in the ingested food in the intestine, thereby reducing the absorption of purine nucleosides by the small intestine. The Lactobacillus curvatus 5-1 used in the present invention was donated by researchers from Ningbo Institute of Technology, Zhejiang University. The Lactobacillus plantarum (deposit number: CGMCC 1.12934) used in the present invention was purchased from the China General Microbiological Culture Collection Administration Center. The two strains of bacteria in the present invention were obtained as gifts.

[0007] In order to screen out bacteria that can convert daidzein into equol, the research team of the applicant used the published bacteria in the list of bacteria that can be used in food (each bacteria is selected from a different source) individually and in combination and found that only the combined use of Lactobacillus curvatus 5-1 (CGMCC No.12891) and Lactobacillus plantarum (CGMCC 1.12934) has the effect of converting daidzein into equol, and the use of Lactobacillus curvatus 5-1 or Lactobacillus plantarum alone has no effect of converting daidzein into equol, and the use of other strains of Lactobacillus curvatus and Lactobacillus plantarum also has no such effect.

[0008] To achieve the above object, the present invention is implemented through the following technical solutions:

[0009] In one aspect, the present invention provides a composite probiotic comprising Lactobacillus curvatus 5-1 and Lactobacillus plantarum.

[0010] The Lactobacillus curvatus 5-1 is deposited in the China General Microbiological Culture Collection Center with the deposit number: CGMCC No.12891.

[0011] The Lactobacillus plantarum is deposited in the China General Microbiological Culture Collection Center with the deposit number: CGMCC 1.12934.

[0012] On the other hand, the present invention provides the use of composite probiotics in the preparation of fragrant braised soybeans.

[0013] As a further embodiment of the present invention, the present invention provides a method for preparing fragrant braised soybeans using composite probiotics, which specifically comprises the following steps:

[0014] (1) Activating separately: activating Lactobacillus curvatus 5-1 and Lactobacillus plantarum separately;

[0015] (2) steaming and stewing the soybeans;

[0016] (3) The stewed soybeans obtained in step (2) are placed in a fermentation tank, and the composite probiotics consisting of Lactobacillus curvatum 5-1 and Lactobacillus plantarum activated in step (1) are inoculated for fermentation to obtain fragrant stewed soybeans.

[0017] Furthermore, step (2) is specifically as follows: washing the soybeans and pouring them into a high pressure steamer for steaming with steam for 15 to 30 minutes, and then stewing for 10 to 20 minutes for later use;

[0018] Furthermore, in step (3), when inoculating Lactobacillus curvatus 5-1, the Lactobacillus curvatus 5-1 is added in the form of a Lactobacillus curvatus bacterial solution, and the concentration of Lactobacillus curvatus 5-1 in the Lactobacillus curvatus bacterial solution is 10 4 ~10 7 When inoculating Lactobacillus plantarum, the Lactobacillus plantarum is added in the form of a Lactobacillus plantarum bacterial solution, and the concentration of Lactobacillus plantarum in the Lactobacillus plantarum bacterial solution is 10 5 ~10 8 CFU / ml.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) Lactobacillus curvatus 5-1 and Lactobacillus plantarum are both highly safe food bacteria with guaranteed safety.

[0021] (2) providing a composite probiotic strain that converts daidzein into equol;

[0022] (3) The composite probiotics are applied to the preparation of stewed soybeans, providing a soybean product that has significant effects in treating menopausal syndrome and estrogen secretion disorders such as breast hyperplasia and breast cancer. DETAILED DESCRIPTION

[0023] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the following embodiments and features in the embodiments can be combined with each other without conflict.

[0024] Example 1

[0025] A composite probiotic is a bacterial strain used for metabolizing daidzein into equol, comprising Lactobacillus curvatus 5-1 and Lactobacillus plantarum.

[0026] The deposit number of the Lactobacillus curvatus 5-1 is CGMCC No.12891.

[0027] The deposit number of the Lactobacillus plantarum is CGMCC 1.12934.

[0028] The method for preparing fragrant braised soybeans using the composite probiotics specifically comprises the following steps:

[0029] (1) Separate activation: Lactobacillus curvatus 5-1 and Lactobacillus plantarum are activated separately; the activation culture can be carried out according to conventional methods in the art.

[0030] (2) steaming and stewing the soybeans; specifically, washing the soybeans and pouring them into a high-pressure steamer for steaming for 15 to 30 minutes, and then stewing for 10 to 20 minutes for later use;

[0031] (3) Put the braised soybeans obtained in step (2) into a fermentation tank, and inoculate the composite probiotics consisting of Lactobacillus curvatus 5-1 and Lactobacillus plantarum activated in step (1) for fermentation, the fermentation cultivation time is 36 to 72 hours, and the fermentation temperature is 30 to 37° C., to obtain fragrant braised soybeans. Further, when inoculating Lactobacillus curvatus 5-1, the Lactobacillus curvatus 5-1 is added in the form of a Lactobacillus curvatus bacterial liquid, and the concentration of Lactobacillus curvatus 5-1 in the Lactobacillus curvatus bacterial liquid is 104 ~10 7 CFU / ml, for example, the concentration is 10 6 When inoculating Lactobacillus plantarum, the Lactobacillus plantarum is added in the form of a Lactobacillus plantarum bacterial solution, and the concentration of Lactobacillus plantarum in the Lactobacillus plantarum bacterial solution is 10 5 ~10 8 CFU / ml, for example, the concentration is 10 6 CFU / ml. The inoculation amount of Lactobacillus curvatus 5-1 and Lactobacillus plantarum is 3~6ml / 100g, that is, 3~6ml of Lactobacillus curvatus liquid and Lactobacillus plantarum liquid are inoculated for every 100g of stewed soybeans (wherein, the ratio of Lactobacillus curvatus to Lactobacillus plantarum in the composite probiotics is 1~2:1). The preparation method of the composite probiotics is: after activating Lactobacillus curvatus 5-1 and Lactobacillus plantarum, mix them at a concentration of 1~2:1 in terms of the number of cells, and adjust the total concentration of the liquid to 10 6 ~10 7 CFU / ml.

[0032] Example 2

[0033] The method for preparing the fragrant braised soybeans in this embodiment is similar to that in the embodiment, but in step (3), Lactobacillus curvatus 5-1 is not added, and only Lactobacillus plantarum is added; the deposit number of the Lactobacillus plantarum is: CGMCC 1.12934.

[0034] Example 3

[0035] The method for preparing the fragrant braised soybeans in this embodiment is similar to that in the embodiment, but in step (3), Lactobacillus plantarum is not added, and only Lactobacillus curvatus 5-1 is added; the preservation number of the Lactobacillus curvatus 5-1 is: CGMCC No.12891.

[0036] Example 4

[0037] The method for preparing the fragrant braised soybeans in this embodiment is similar to that in the embodiment, but Lactobacillus plantarum (preservation number: CGMCC 1.12934) in step (3) is replaced by Lactobacillus plantarum (preservation number: CGMCC1.1856). Lactobacillus plantarum (preservation number: CGMCC 1.1856) was purchased from the China General Microbiological Culture Collection Center.

[0038] Example 5

[0039] The method for preparing the fragrant stewed soybeans in this embodiment is similar to that in the embodiment, but in step (2), the soybeans are not steamed or stewed but only soaked for 4 to 8 hours.

[0040] Example 6

[0041] The content of daidzein and equol in soybeans before fermentation (soybeans after stewing or soaking) and soybeans after fermentation (soybeans after fragrant stewing) was tested; the specific test method was to make soybean powder with the product to be tested (soybeans after stewing or soaking, soybeans after fragrant stewing), take 0.2g of soybean powder, add 25 times the weight of 90% methanol solution to the obtained soybean powder, ultrasonic treatment at 60-70℃, centrifuge for 4-6min, and the obtained supernatant was tested by HPLC, and the content of daidzein and equol in soybeans after stewing or soybeans after fragrant stewing was calculated, and each experiment was repeated 5 times to take the average value. The results are shown in Table 1.

[0042] Table 1 Contents of daidzein and equol in soybeans before and after fermentation in different examples

[0043]

[0044] As shown in Table 1, (1) equol was detected in soybeans after fermentation in Example 1, while equol was not detected in Examples 2-4, indicating that the use of Lactobacillus curvatus 5-1 or Lactobacillus plantarum alone had no effect on converting daidzein into equol, and the combined use of Lactobacillus curvatus 5-1 (CGMCC No. 12891) and Lactobacillus plantarum (CGMCC 1.1856) also had no effect on converting daidzein into equol; only the combined use of Lactobacillus curvatus 5-1 (CGMCC No. 12891) and Lactobacillus plantarum (CGMCC 1.12934) had the effect of converting daidzein into equol.

[0045] (2) Compared with Example 5, the content of daidzein in soybeans before fermentation and the content of equol in soybeans after fermentation in Example 1 are both lower than those in Example 1. This may be because daidzein is easier to extract and easier to be converted into equol after soybeans are steamed and stewed.

[0046] Example 7

[0047] 1. Experimental Animal Grouping

[0048] After adaptive feeding, female Wistar rats were monitored by vaginal exfoliative cytology, and rats with normal estrous cycle were selected as test rats. 35 rats weighing 280-300g were selected and divided into 7 groups. There was no statistical difference among the rats in each group.

[0049] Group 1: blank control group (normal rats);

[0050] Group 2: positive control group (Progynova group);

[0051] Group 3: Negative control group

[0052] Group 4: Braised soybean (Lactobacillus curvatus 5-1) treated group obtained in Example 2

[0053] Group 5: the group treated with the braised soybean (Lactobacillus plantarum CGMCC 1.12934) obtained in Example 3;

[0054] Group 6: Braised soybeans (Lactobacillus curvatus 5-1 + Lactobacillus plantarum CGMCC 1.12934) treated group obtained in Example 1

[0055] Group 7: Braised soybean (Lactobacillus curvatus 5-1 + Lactobacillus plantarum CGMCC 1.1856) treatment group obtained in Example 4. 5 rats in each group. Among them, the normal rat group was used as a blank control, and the positive control group, the negative control group, and the treatment group were all successfully modeled with premature ovarian failure (POF) rats. The Lactobacillus plantarum (preservation number is CGMCC 1.1856), the strain was purchased and preserved in the China General Microbiological Culture Collection Management Center.

[0056] 2. Method for modeling premature ovarian failure (POF) rats: 30 female Wistar rats were used for modeling. Each female Wistar rat was given Tripterygium wilfordii polyglycosides tablets (10 mg / tablet), 0.6 mg / (100 g.d) body weight per day, gavage once a day for 9 consecutive days to establish the POF rat model. Vaginal smears were taken daily to observe changes in the estrous cycle. When the estrous cycle of female rats in the model group was disordered, the estrus period was prolonged, there was no estrous cycle or estrus period, it indicated that the modeling of premature ovarian failure in rats was successful. The reserve function of the ovaries was evaluated by indicators such as the number of follicles, hormone levels, estrous cycle and fertility tests. The 30 rats with successful premature ovarian failure (POF) were given drugs according to groups 2-7. The other 5 rats from the same batch were not treated in any way and were treated according to group 1 as the normal group.

[0057] After the modeling was successful, the blank control group and the negative control group were not given any medication and were fed normally; the positive control group was gavaged with a suspension of Progynova, which was converted into a rat dosage according to the drug medication instructions. The concentration of Progynova in the suspension of Progynova was 0.1 mg / ml. The treatment group was gavaged with a suspension of fragrant stewed soybeans (the fragrant stewed soybeans were ground into powder and made into a suspension), and different groups were gavaged with corresponding suspensions of fragrant stewed soybeans. Each rat in the treatment group was gavaged with 0.1g of fragrant stewed soybeans / (100g.d) body weight, and the corresponding amount of suspension was gavaged according to the dosage; all groups were treated for 20 consecutive days, and serum and ovarian tissue data were collected on the 21st day.

[0058] 3. Serum test: After 20 days of treatment, blood was collected from the orbital vein of the rats on the 21st day, and the blood was collected in EP tubes. The serum was separated, and the upper serum was taken. The levels of estradiol (E2), follicle-stimulating hormone (FSH), and anti-Mullerian hormone (AMH) in the serum of the rats were tested according to the Elisa method described in the test kit. The test results are shown in Table 2.

[0059] Table 2 Serum test parameters of rats in different groups after treatment

[0060]

[0061] It can be found from Table 2 that compared with the negative control group (Group 3) and Groups 4, 5, and 7, the E2 and AMH of the stewed soybeans prepared in Example 1 in Group 6 were significantly increased, and the FSH was significantly decreased, which was close to the level of normal rats, indicating that the stewed soybeans obtained by the present invention can effectively improve the symptoms of premature ovarian failure.

[0062] 4. Follicle counting in rat ovarian tissue

[0063] The rats were anesthetized, and the left ovarian tissue of the rats was fixed in 4% paraformaldehyde. The fixed tissues were dehydrated with alcohol, transparentized with xylene, embedded in paraffin, and sliced ​​continuously. The slices were 5 μm thick, and HE staining was performed. Follicles of all levels in the ovarian tissue were observed under a microscope. It was found that compared with the negative control group (Group 3) and Groups 4, 5, and 7, the number of primary follicles, secondary follicles, and mature follicles in the ovarian tissue of the rats in Group 6 using the fragrant braised soybeans prepared in Example 1 were also significantly restored, and the number of atretic follicles was significantly reduced, close to the level of normal rats, indicating that the formula of the present invention can effectively improve the symptoms of premature ovarian failure.

[0064] In summary, the inventors have found in the process of research and practice that the combined use of Lactobacillus curvatus 5-1 (CGMCC No. 12891) and Lactobacillus plantarum (CGMCC 1.12934) as fermentation strains for soybean fermentation has the effect of converting daidzein into equol, which can effectively enhance the therapeutic effect on women's menopausal syndrome (premature ovarian failure). Lactobacillus curvatus 5-1 and Lactobacillus plantarum CGMCC 1.12934 are both highly safe food fungi with guaranteed safety. The braised soybeans prepared by them are very promising in the treatment of menopausal syndrome and diseases with estrogen secretion disorders such as breast hyperplasia and breast cancer. However, this effect was not observed when using Lactobacillus curvatus 5-1 and other Lactobacillus plantarum such as Lactobacillus plantarum CGMCC 1.1856 listed in Example 4. It is speculated that metabolites or secondary metabolites produced during the metabolism of these two strains just stimulated the expression of certain genes, thereby achieving this effect. The specific mechanism needs further study.

[0065] The above-mentioned embodiments only express the specific implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the protection scope of the present invention. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A composite probiotic, characterized in that: It is composed of Lactobacillus curvatus 5-1 and Lactobacillus plantarum; The Lactobacillus curvatus 5-1 is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No. 12891. The Lactobacillus plantarum is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC1.12934.

2. Use of the composite probiotics according to claim 1 in the preparation of fragrant braised soybeans.

3. The use according to claim 2, characterized in that Method for preparing fragrant soybeans using composite probiotics The body includes the following steps: (1) Activating respectively: activating Lactobacillus curvatus 5-1 and Lactobacillus plantarum respectively; (2) steaming and stewing the soybeans; (3) putting the braised soybeans obtained in step (2) into a fermentation tank and inoculating the activated Lactobacillus curvatus 5-1 and The composite probiotic composed of plant lactobacillus is fermented to obtain the fragrant braised soybeans.

4. The use according to claim 3, characterized in that Step (2) is as follows: wash the soybeans and pour them into the high pressure steamer. Use steam to cook in the pot for 15 to 30 minutes, then simmer for 10 to 20 minutes and set aside.

5. The use according to claim 3, characterized in that In step (3), when inoculating Lactobacillus curvatus 5-1, the Lactobacillus curvatum 5-1 was added in the form of a Lactobacillus curvatum bacterial solution, wherein the concentration of Lactobacillus curvatum 5-1 in the Lactobacillus curvatum bacterial solution was 10 4 ~10 7 When inoculating Lactobacillus plantarum, the Lactobacillus plantarum was added in the form of a Lactobacillus plantarum bacterial solution, the concentration of Lactobacillus plantarum in the Lactobacillus plantarum bacterial solution was 10 5 ~10 8 CFU / ml.

Images