Lactobacillus paracasei and malt extract composition for synergistically improving functional constipation

Abstract

The invention belongs to the technical field of microorganisms, and discloses a lactobacillus paracasei and malt extract composition for synergistically improving functional constipation, and the active ingredients of the composition comprise lactobacillus paracasei YRK-K5 and a malt extract; the lactobacillus paracasei YRK-K5 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO.28598, and the preservation date is October 9, 2023. The lactobacillus paracasei YRK-K5 can be used for preparing the lactobacillus paracasei YRK-K5. Experiments prove that the composition of the lactobacillus paracasei and the malt extract has a synergistic effect in treating and / or improving the functional constipation of animals, and the functional constipation is remarkably improved or treated by increasing the number of excrement grains and the water content of excrement, reducing the defecation time and improving the small intestine propulsion rate.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a combination of Lactobacillus paracasei and malt extract for synergistically improving functional constipation. Background Art

[0002] In today's society, with the accelerated pace of life, changes in eating habits and the increase in the aging population, constipation has become a global health problem. According to statistics, a considerable proportion of people of all ages are deeply troubled by constipation. Among adults, the incidence of chronic constipation can reach 10%~20%, and the elderly are as high as 20%~30%. Long-term constipation not only brings uncomfortable symptoms such as abdominal distension, abdominal pain, and loss of appetite to patients, but may also cause anorectal diseases such as hemorrhoids and anal fissures, seriously affecting the quality of life. More importantly, it is closely related to the disorder of the intestinal environment and the imbalance of intestinal flora, and is even regarded as a potential risk factor for some systemic diseases such as cardiovascular disease and diabetes. Therefore, in-depth exploration of the mechanism of constipation and the development of effective prevention and treatment methods are imminent.

[0003] In recent years, probiotic therapy has attracted much attention in the field of intestinal health. As a type of active microorganisms that are beneficial to the host, probiotics can regulate the structure of intestinal flora, inhibit the growth and reproduction of harmful bacteria, and strengthen the intestinal barrier function. Many studies have shown that supplementing specific probiotic strains can improve intestinal peristalsis and promote fecal excretion, opening up new ideas for the treatment of constipation. At the same time, medicinal and edible substances are gradually emerging. They are derived from natural ingredients, have both nutritional and medicinal effects, are highly safe, have few side effects, and are easily accepted by the public. For example, some food extracts rich in dietary fiber can increase the volume of stool, absorb water, and make the stool soft and easy to excrete. Combining probiotics with medicinal and edible substances is expected to give full play to the advantages of both, synergistically enhance the effect, not only treat the symptoms of constipation, but also treat the root cause and improve the intestinal ecology, providing constipation patients with a more comprehensive, long-term and gentle solution, and has broad application prospects in the future health industry. However, existing research lacks relevant research on the compounding of Lactobacillus paracasei and medicinal and edible substances. Summary of the invention

[0004] In order to overcome the problems of the prior art, the present invention proposes a combination of Lactobacillus paracasei and malt extract for synergistically improving functional constipation. The purpose of the invention is to provide a safe and effective functional constipation improvement solution. By rationally combining Lactobacillus paracasei and malt extract and utilizing the synergistic effect of the two, the functional constipation symptoms can be effectively alleviated, providing a new non-drug treatment option for a large number of functional constipation patients and improving their quality of life.

[0005] The object of the present invention is achieved in that: The first aspect of the present invention provides a combination of Lactobacillus paracasei and malt extract for synergistically improving functional constipation, wherein the active ingredients of the combination include Lactobacillus paracasei ( Lacticaseibacillus paracasei ) YRK-K5 and malt extract; The Lactobacillus paracasei YRK-K5 is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC NO. 28598 and a deposit date of October 9, 2023.

[0006] Furthermore, the malt extract is a water extract, and the extraction method includes: The malt is crushed into powder by a grinder, and 8 to 15 times the weight of water is added for decocting for 3 to 5 times, each time for 0.5 to 1.5 hours. The decocted malt is filtered, and the filtrate is combined and filtered. After filtering, the filtrate is rotary evaporated at 45 to 55° C. until the ratio of medicinal material to water is 1:1 to 2, and the malt extract is lyophilized into powder to obtain the malt extract.

[0007] Furthermore, in the combination, the final therapeutic dose of the Lactobacillus paracasei YRK-K5 is 1.0-3.0×10 9 CFU / d / kg body weight.

[0008] Furthermore, in the combination, the final therapeutic dose of the malt extract is 0.111-0.167 g / d / kg body weight.

[0009] Furthermore, the dosage form of the combination includes lyophilized powder, capsule, tablet or granule.

[0010] The second aspect of the present invention provides use of the combination of the first aspect in preparing a product for preventing and treating functional constipation.

[0011] Furthermore, the product is a medicine.

[0012] Furthermore, the application is one or more of the following: (1) The application can reduce the time of defecation; (2) The application can increase the number of fecal particles; (3) The application can increase the water content of feces; (4) The application can improve the small intestinal propulsion rate.

[0013] The third aspect of the present invention provides a probiotic combination, which comprises the combination of Lactobacillus paracasei and malt extract for synergistically improving functional constipation as described in the first aspect.

[0014] Furthermore, it also includes a medically acceptable carrier. Strain deposit information:

[0015] The strain of Lactobacillus paracasei was named YRK-K5, and the strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101; the strain was classified as Lactobacillus paracasei Lacticaseibacillus paracasei The deposit number is CGMCC NO. 28598, and the deposit date is October 9, 2023. The advantages and beneficial effects of the present invention are:

[0016] 1. This application screened and obtained a probiotic that can prevent and treat functional constipation. The strain was identified as Lactobacillus paracasei after preservation, with a preservation number of CGMCC NO. 28598 and a classification name of Lactobacillus paracasei. Lacticaseibacillus paracasei .

[0017] 2. The present application found that the use of Lactobacillus paracasei YRK-K5 can effectively increase the number of fecal particles and fecal water content, reduce defecation time, and improve the small intestinal propulsion rate to achieve the treatment of functional constipation. Its effect is better than that of the currently developed and marketed Lactobacillus paracasei LCS. Specifically, compared with the positive control bacteria Lactobacillus paracasei LCS, Lactobacillus paracasei YRK-K5 can significantly increase the number of fecal particles in the host in 2 hours by 10 particles.

[0018] 3. The combination of Lactobacillus paracasei and malt extract described in the present application has a significantly better intervention effect on improving constipation in mice than the intervention effect of a single substance of Lactobacillus paracasei YRK-K5 and malt extract, reaching the level of drug intervention, which has important practical significance for the development of efficient functional constipation drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0020] Figure 1 The figure shows the successful results of the functional constipation mouse model; A is the time of the first red pellet, B is the number of feces pellets in 2 hours, and C is the water content of feces; Figure 2 The results of treating functional constipation mice with different doses of malt extract are shown; A is the time of first red pellet excretion, B is the number of feces pellets in 2 hours, and C is the water content of feces; Figure 3 The results show that the expression of Lactobacillus paracasei YRK-K5 and malt extract was 1-3×10 9 Results of the treatment of functional constipation mice after the combination of CFU / d / kg and 0.111g / d / kg; where A is the time of first red pellet excretion, B is the number of fecal pellets in 2 hours, C is the fecal water content, and D is the intestinal propulsion rate; Figure 4The results show that the expression of Lactobacillus paracasei YRK-K5 and malt extract was 1-3×10 9 The synergistic enhancement results after the combination of 0.1 g / d / kg and 0.11 g / d / kg (combined normalized equivalent hologram); where A is the combination of Lactobacillus paracasei YRK-K5 and malt extract, and B is the combination of Lactobacillus paracasei LCS malt extract. DETAILED DESCRIPTION

[0021] The implementation of the present invention is not limited to the following embodiments, and any form of modification and / or change made to the present invention will fall within the scope of protection of the present invention. In the present invention, unless otherwise specified, all equipment and raw materials can be purchased from the market or are commonly used in the industry. Unless otherwise specified, the methods used in the embodiments are common techniques in the art.

[0022] For numerical ranges, the endpoints of each range, the endpoints of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be deemed to be specifically disclosed herein.

[0023] In the following examples, Lactobacillus paracasei YRK-K5 was provided by Shaanxi Yiruikang Biotechnology Co., Ltd., and the strain was deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration. The chemical reagents used were purchased from regular chemical reagent suppliers and were analytically pure.

[0024] In the following examples, the culture medium formulations involved include: The modified MRS liquid culture medium has a formula of: 10 g of peptone, 5 g of yeast extract powder, 20 g of glucose, 10 g of beef extract, 2 g of K2HPO4, 2 g of diammonium citrate, 0.58 g of MgSO4·7H2O, 0.25 g of MnSO4·4H2O, 5 g of anhydrous sodium acetate, 1 mL of Tween 80, 0.5 g of cysteine ​​hydrochloride, distilled water to 1 L, pH adjusted to 6.5, and sterilized at 121°C for 15 min. Example 1: Microbial culture

[0025] 1) Preparation of Lactobacillus paracasei YRK-K5 bacteria: A probiotic strain was isolated and identified as Lactobacillus paracasei Lacticaseibacillus paracasei , deposited in the General Microbiology Center of China Microbiological Culture Management Committee, with the deposit number CGMCCNO.28598 and the deposit date of October 9, 2023.

[0026] After Lactobacillus paracasei YRK-K5 was subcultured twice in the modified MRS liquid medium, it was inoculated in the modified MRS liquid medium at a 2% inoculum and incubated at 37±1°C for 12 h. The supernatant was removed by centrifugation (4000 rpm, 10 min), and the bacteria were washed twice with PBS. When gavage was performed, the bacteria were resuspended with 0.9% saline and the concentration of the bacterial suspension was adjusted to 1-3×10 9 CFU / g.

[0027] 2) Preparation of Lactobacillus paracasei LCS bacteria: After Lactobacillus paracasei LCS was subcultured twice in MRS liquid medium, it was inoculated in a modified MRS liquid medium at a 2% inoculum and cultured at 37 ± 1°C for 12 h. The supernatant was removed by centrifugation (4000 rpm, 10 min), and the bacteria were washed twice with PBS. When gavage was performed, the bacteria were resuspended in 0.9% saline and the concentration of the bacterial suspension was adjusted to 1-3×10 9 CFU / g. Example 2: Preparation of malt extract

[0028] This embodiment provides a malt extract, which is a water extract, and is prepared as follows: The malt was crushed into powder by a grinder, 10 times the weight of water was added and decocted for 3 times, each time for 1 hour, the filtrates were combined, filtered through 8 layers of gauze, and rotary evaporated at 50°C until the medicinal material: water = 1:1 (mass concentration of 1 g / mL), freeze-dried into powder, and weighed.

[0029] The concentration multiple between malt extract and malt was calculated, and the extract was resuspended with 0.9% saline and adjusted to a concentration of 0.022 g / mL and 0.033 g / mL during intragastric administration. Example 3: Preparation of animal model and animal experiment

[0030] SPF BALB / c (6-week-old, female) mice were used as experimental animals and purchased from Beijing Huafukang Biotechnology Co., Ltd. The animal experiment was divided into 11 groups, namely, blank group, model group, drug group, malt extract high-dose group, malt extract low-dose group, Lactobacillus paracasei YRK-K5 low-dose group, Lactobacillus paracasei YRK-K5 medium-dose group, Lactobacillus paracasei YRK-K5 high-dose group, Lactobacillus paracasei YRK-K5 + malt extract low-dose mixed group, Lactobacillus paracasei LCS, and Lactobacillus paracasei LCS + malt extract low-dose mixed group, 8 mice in each group, a total of 88 mice.

[0031] The mice were kept under the following conditions: temperature 24°C, 12h light, 12h dark, and bedding was changed every three days. After one week of adaptive feeding, each mouse was gavaged with 15 mg / kg bw of loperamide hydrochloride every day, and weighed once a week.

[0032] The blank group was gavaged with 0.9% saline until the end of the experiment; The model group and each intervention group were first gavaged with 0.1 mL of 15 mg / kg bw loperamide hydrochloride until the model was successful (7-14 days). After the model was successfully established, the model group and the intervention group were gavaged with 0.1 mL of 15 mg / kg bw loperamide hydrochloride every other day until the end of the experiment; at the same time, the drug group was gavaged with polyethylene glycol 4000 powder, and each intervention group was gavaged with low and high doses of malt extract (low dose 0.111 g / d / kg, high dose 0.167 g / d / kg), Lactobacillus paracasei YRK-K5 (low dose 1~3×10 7 CFU / d / kg, medium dose 1~3×10 8 CFU / d / kg, high dose 1~3×10 9 CFU / d / kg), 1~3×10 8 CFU / d / kg of Lactobacillus paracasei YRK-K5 and malt extract (0.111 g / d / kg) and 1-3×10 8 The mixed group of Lactobacillus paracasei LCS (CFU / d / kg) and malt extract (0.111g / d / kg) was given by intragastric administration with a volume of 0.1 mL for 2-3 weeks. Test Example 1: Testing of fecal water content, number of stool particles within 2 hours, and time of first red stool particle

[0033] This test example measures the feces water content, the number of stool pellets within 2 hours and the time of first red stool pellet in each group of experimental animals in Example 3. The specific method and results are as follows: Determination of fecal water content: On the 29th day, after gavage, mice were placed individually in a cage lined with absorbent paper to collect feces. The wet weight was obtained by weighing, and the dry weight was obtained after freeze-drying. The fecal water content was calculated according to formula (1):

[0034] Number of stool particles within 2 hours: On the 30th day, the blank group was given 0.1 mL of 0.9% saline, and the model group and intervention group were given 0.1 mL of loperamide hydrochloride solution (15 mg / kg bw). After 1 hour, each mouse was placed in a cage lined with absorbent paper, and feces were collected every 30 minutes for a total of 2 hours.

[0035] Time of first red stool: On the 30th day, the blank group was given 0.1 mL 0.9% saline, and the model group and intervention group were given 0.1 mL loperamide hydrochloride solution (15 mg / kg bw). One hour later, each group was gavaged with 0.1 mL 0.6% (m:v) carmine. Starting from the gavage of carmine, the time of the first red stool for each mouse was recorded.

[0036] like Figure 1 The results showed that 7 days after modeling, the time to first red pellet excretion, number of feces pellets and feces water content of mice in the model group were 3.7 times, 1.96 times and 1.81 times that of the blank group, respectively, and there were significant differences, indicating that the constipation model was successfully established.

[0037] The performance of the intervention group was compared with that of the model group, e.g. Figure 2 The results showed that after 14 days of intervention with different doses of malt, low dose of malt extract (0.111 g / d / kg), high dose of malt extract (0.167 g / d / kg), low dose of Lactobacillus paracasei YRK-K5 (1-3×10 7 CFU / d / kg), medium dose of Lactobacillus paracasei YRK-K5 (1~3×10 8 CFU / d / kg), high dose of Lactobacillus paracasei YRK-K5 (1~3×10 9 Compared with the model group, the first red grain excretion time of low-dose malt extract (0.111 g / d / kg) and high-dose malt extract (0.167 g / d / kg) were significantly reduced by 102.4 and 122.9 minutes, respectively ( Figure 2 Middle A); the number of fecal particles increased significantly by 8, 17 and 21 particles ( Figure 2 B) and increased fecal water content by 10% and 11% ( Figure 2 Compared with the model group, low doses (1-3×10 7 CFU / d / kg), medium dose of Lactobacillus paracasei YRK-K5 (1~3×10 8 CFU / d / kg and high doses of Lactobacillus paracasei YRK-K5 (1-3×10 9 CFU / d / kg) were significantly reduced by 55, 89.2 and 97.6 minutes, respectively ( Figure 2 Middle A); the number of fecal particles increased significantly by 12, 17 and 22 particles ( Figure 2 B) and significantly increased fecal water content by 2.11%, 4.77%, and 15% ( Figure 2C). There was no significant difference in the intervention effect between low-dose (0.11g / d / kg) and high-dose (0.167g / d / kg) malt extract. This indicates that Lactobacillus paracasei YRK-K5 has the effect of improving constipation in mice, and low-dose (0.111g / d / kg) and high-dose (0.167g / d / kg) malt extract have the best effect on improving constipation in mice.

[0038] like Figure 3 The results showed that the combined intervention of Lactobacillus paracasei YRK-K5 and malt extract (0.111 g / d / kg) for 14 days significantly improved the constipation symptoms of mice. Compared with the model group, the time to first red pill was significantly reduced by 156 minutes ( Figure 3 Middle A); the number of fecal particles increased significantly by 33 particles ( Figure 3 B) and significantly increased fecal water content by 23% ( Figure 3 C), and the effect was not significantly different from the drug intervention group. After 14 days of co-intervention of Lactobacillus paracasei LCS and malt extract (0.111g / d / kg) in constipated mice, the constipation symptoms of mice were also significantly improved. Compared with the model group, the time to first red pill was significantly reduced by 80 minutes ( Figure 3 Middle A); the number of fecal particles increased significantly by 15 ( Figure 3 B) and significantly increased fecal water content by 7% ( Figure 3 In the middle C, the intervention effect did not reach the intervention effect of the drug group. This shows that the combination of Lactobacillus paracasei YRK-K5 and malt extract (0.111g / d / kg) is better than the combination of Lactobacillus paracasei LCS and malt extract (0.11g / d / kg) in improving constipation in mice. Test Example 2: Determination of mouse small intestine propulsion rate experiment

[0039] This test example further measured the small intestinal propulsion rate of each group of experimental animals in Example 3. The specific method and results are as follows: On the 30th day, mice in each group were fasted overnight but not water-deprived. At 8:00 a.m. on the 31st day, the blank group was given 0.1 mL of 0.9% saline solution, and the model group and the bacterial injection group were given 0.1 mL of loperamide hydrochloride solution (15 mg / kg bw). 30 minutes later, the blank group and the model group were gavaged with 0.6% (m:v) carmine, and the bacterial injection group was gavaged with carmine containing the respective gavage contents. After 30 minutes, the mice were killed, the abdominal cavity was opened, and the upper end from the pylorus to the lower end to the cecum was cut off. The total length of the small intestine was measured as the "total length of the small intestine", and the length from the pylorus to the front of the carmine was the "carmine advancement length". The small intestine advancement rate was calculated according to formula (2):

[0040] like Figure 3The results showed that after 14 days of co-intervention of constipated mice with Lactobacillus paracasei YRK-K5 and malt extract (0.111 g / d / kg), the small intestinal propulsion rate of mice was significantly improved compared with the model group (increased by 2 times), and there was no difference with the blank group. The propulsion rate of the combination of Lactobacillus paracasei YRK-K5 and malt extract (0.111 g / d / kg) was no different from the propulsion rate of Lactobacillus paracasei YRK-K5 and malt extract alone. At the same time, the propulsion rate of the combination of Lactobacillus paracasei YRK-K5 and malt extract (0.111 g / d / kg) was significantly higher than the propulsion rate of the combination of Lactobacillus paracasei LCS and malt extract (0.111 g / d / kg).

[0041] Based on the Median-effect Principle (or Chou-Talalay combination index method), CompuSyn statistical software was used to draw the combination index curve under different effects, and the relationship between the combined effect of the two substances and the combination index was used to quantitatively evaluate whether there was a synergistic effect between the two substances.

[0042] Combination index CI = D1 / D X1 + D2 / D X2 ; D1 and D2 are the concentrations of the two substances when they produce the X effect when used alone, and D X1 , D X2 They are the concentrations of the two substances when the two substances produce the X effect when used together. When the CI is greater than 1, it indicates inhibition (i.e., the effect of the two substances when used together is antagonistic); CI=1 indicates that the effect of the two substances when used together is additive; when the CI is less than 1, it indicates promotion (i.e., the effect of the two substances when used together is synergistic), and the smaller the value, the stronger the synergistic effect.

[0043] like Figure 4 The results showed that CompuSyn software was used to analyze the red discharge time of Lactobacillus paracasei YRK-K5, Lactobacillus paracasei LCS and malt extracts in constipated mice alone and together. The CI greater than 1 indicated inhibition; CI = 1 indicated addition; CI less than 1 indicated promotion. 9 CFU / d / kg) and malt extract (0.111 g / d / kg) combined CI = 0.3207 ( Figure 4 A), while Lactobacillus paracasei LCS (1~3×10 9 CFU / d / kg) and malt extract (0.111 g / d / kg) combined CI = 1.8464 ( Figure 4The results showed that the combination of Lactobacillus paracasei YRK-K5 and malt extract (0.111 g / d / kg) had a promoting effect on the intervention of constipation in mice, while the combination of Lactobacillus paracasei LCS and malt extract (0.11 g / d / kg) had an inhibitory effect on the intervention of constipation in mice.

[0044] In summary, Lactobacillus paracasei YRK-K5, malt extract, and the combination of Lactobacillus paracasei YRK-K5 and malt extract can experimentally treat functional constipation by increasing the number of fecal particles and fecal water content, reducing defecation time, and improving small intestinal propulsion rate. The intervention effect of Lactobacillus paracasei YRK-K5 combined with malt extract in improving mouse constipation is better than the intervention effect of Lactobacillus paracasei YRK-K5 and malt extract alone, and has a synergistic effect.

[0045] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred arrangement scheme, a person skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A combination of Lactobacillus paracasei and malt extract for synergistically improving functional constipation, characterized in that: The active ingredients of the combination include Lactobacillus paracasei ( Lacticaseibacillus paracasei ) YRK-K5 and malt extract; The Lactobacillus paracasei YRK-K5 is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC NO. 28598 and a deposit date of October 9, 2023.

2. The combination according to claim 1, characterized in that The malt extract is a water extract, and the extraction method comprises the following steps: The malt is crushed into powder by a grinder, and 8 to 15 times the weight of water is added for decocting for 3 to 5 times, each time for 0.5 to 1.5 hours. The decocted malt is filtered, and the filtrate is combined and filtered. After filtering, the filtrate is rotary evaporated at 45 to 55° C. until the ratio of medicinal material to water is 1:1 to 2, and the malt extract is lyophilized into powder to obtain the malt extract.

3. The combination according to claim 1, characterized in that In the combination, the final therapeutic dose of the Lactobacillus paracasei YRK-K5 is 1.0-3.0×10 9 CFU / d / kg body weight.

4. The combination according to claim 1, characterized in that In the combination, the final therapeutic dose of the malt extract is 0.111-0.167 g / d / kg body weight.

5. The combination according to any one of claims 1 to 4, characterized in that: The dosage form of the combination includes lyophilized powder, capsule, tablet or granule.

6. Use of the combination according to any one of claims 1 to 5 in the preparation of a product for preventing and treating functional constipation.

7. The use according to claim 6, characterized in that The product described is a pharmaceutical product.

8. The use according to claim 6, characterized in that The application is one or more of the following: (1) The application can reduce the time of defecation; (2) The application can increase the number of fecal particles; (3) The application can increase the water content of feces; (4) The application can improve the small intestinal propulsion rate.

9. A probiotic combination, characterized in that: A combination of Lactobacillus paracasei and malt extract for synergistically improving functional constipation as described in any one of claims 1 to 5.

10. The probiotic combination according to claim 9, characterized in that: Medically acceptable carriers are also included.

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