TWI744028B - Prebiotic composition for promoting growth of clostridium butyricum and uses thereof - Google Patents

Abstract

The present invention provides use of a composition including aAbelmoschus esculentus ferment for manufacture of a prebiotic composition that promotes the growth ofClostridium butyricum , and a prebiotic composition including theAbelmoschus esculentus ferment. The prebiotic composition promotes the growth of the gut bacteriumClostridium butyricum , reduces waist circumference, and improves bowel movement in users.

Description

Probiotic composition for promoting butyric acid bacteria growth and its use

The present invention relates to a probiotic composition, in particular to a probiotic composition that promotes the growth of butyric acid bacteria.

Many medical studies have shown that the intestinal environment is closely related to the maintenance of normal body functions and disease prevention. The intestine is not only a digestive organ, but also hosts a large number of immune cells to resist pathogens from invading the body, and densely distributed nerve branches to collect physiological information and feed back to the central nervous system. Therefore, intestinal health has a huge impact on human health. In recent decades, studies have found that more than a thousand intestinal bacteria live in the intestinal mucosa. They communicate with body organs and give appropriate responses, such as supplying nutrients needed by the body, regulating the development of intestinal cells, and inducing the development of the immune system Therefore, intestinal bacteria can be described as an essential organ of the body.

The composition of intestinal bacteria Bacteria have specific functions. When their composition is out of balance due to bad habits, diseases, antibiotics and other reasons, the body may lose health. For example, if an individual consumes a high-fat diet, the fat-eating Gram-negative bacteria in the large intestine will proliferate in large numbers and produce lipopolysaccharides (LPS), which can cause chronic inflammation throughout the body through blood circulation, and eventually may Lead to chronic diseases such as type 2 diabetes, cardiovascular disease, and even neurodegenerative diseases and cancer. Studies have also shown that mice whose intestinal bacterial composition is destroyed Memory decline in stressful situations. Therefore, maintaining a constant composition of intestinal bacteria helps maintain physical and mental health.

Now known intestinal bacteria beneficial to human health in addition to Bifidobacterium (Bifidobacterium) and germ Lactobacillus (Lactobacillus) bacteria, butyric acid bacteria (Clostridium butyricum) is also a great potential of probiotics. This bacteria accounts for 1% to 4% of adult intestinal bacteria. It is an anaerobic gram-positive bacteria that grows in the large intestine. Butyric acid bacteria can produce butyric acid, which can help defecation and promote the growth of lactic acid bacteria. In mouse animal experiments, when the mice ingested butyric acid bacteria under a high-fat diet, the weight gain of the mice was reduced.

In view of the importance of butyric acid bacteria in maintaining the integrity of the intestinal tract, it is necessary to develop a probiotic composition that promotes the growth of butyric acid bacteria in the intestinal tract.

For this reason, one object of the present invention is to provide a composition containing fermented okra for the preparation of a probiotic composition for promoting the growth of butyric acid bacteria.

In an embodiment of the present invention, the okra fermentation product is obtained by simultaneously fermenting an okra pulp and an air culture solution with a yeast (Saccharomyces cerevisiae) and a Lactobacillus plantarum (Lactobacillus plantarum).

In an embodiment of the present invention, the air culture solution is obtained by mixing okra pulp and water in a weight ratio of 1:1 to 1:20, and the inoculation amounts of the yeast and the embryonic Lactobacillus are 0.1% to 0.4%(w/v) and 0.2% to 0.5%(w/v).

In a preferred embodiment of the present invention, the composition containing the fermented okra further includes an oligosaccharide selected from the group consisting of xylo-oligosaccharides, fructo-oligosaccharides, and combinations thereof.

In some embodiments, the composition comprising okra fermented product further comprises a sugar alcohol.

In some embodiments, the oligosaccharide is xylo-oligosaccharide, and the sugar alcohol is lactitol.

In some embodiments, the weight ratio of the fermented okra, the xylo-oligosaccharide, and the lactitol ranges from 3 to 5: 2 to 4: 2 to 4. In a preferred embodiment, the weight ratio of the fermented okra, the xylo-oligosaccharide, and the lactitol is in the range of 4:3:3.

Another object of the present invention is to provide a probiotic composition for promoting the growth of butyric acid bacteria. Sugar, and combinations thereof.

Another object of the present invention is to provide a use of the aforementioned probiotic composition for preparing a pharmaceutical composition for reducing waist circumference.

In one embodiment of the present invention, the probiotic composition increases the number of butyric acid bacteria in the intestine of obese subjects, and reduces the weight, body fat rate and waist circumference of obese subjects.

The present invention discloses multiple probiotics and their combinations that are beneficial to the growth of butyric acid bacteria. The probiotic combination can be used to prepare a probiotic composition that effectively promotes the growth of butyric acid bacteria, for example, as a medium supplement for in vitro bacterial culture, or as a probiotic product that promotes the proliferation of butyric acid bacteria in the intestine of a living body (such as a human body) . The probiotic composition can have a dosage form of powder, granule, solution, or capsule, and can be made into food, drink, medicine, or nutritional supplement, and administered to a body by oral administration.

The following will further illustrate the embodiments of the present invention in conjunction with the drawings. The following examples are used to illustrate the inventive features and applications of the present invention, rather than to limit the scope of the present invention. Anyone familiar with the art will not depart from Within the spirit and scope of the present invention, some changes can be made Movement and modification, therefore, the scope of protection of the present invention shall be subject to the scope of the attached patent application.

Figure 1 shows the relative bacterial count of butyric acid bacteria in a medium with or without probiotics after anaerobic culture for 24 hours.

Figure 2A shows the statistical results of the average number of defecation days before the subjects take the probiotic composition according to an embodiment of the present invention.

Figure 2B shows the statistical results of the average number of defecation days after the subjects took the probiotic composition according to an embodiment of the present invention for four weeks.

Figure 3A shows the statistical results of the frequency of unclean defecation experienced by subjects before taking the probiotic composition of an embodiment of the present invention.

Figure 3B shows the statistical results of the frequency of unclean defecation experienced by subjects after taking the probiotic composition of an embodiment of the present invention for four weeks.

Fig. 4A shows the statistical results of the stool status of subjects before taking the probiotic composition according to an embodiment of the present invention.

Figure 4B shows the statistical results of the stool status of subjects after taking the probiotic composition of an embodiment of the present invention for four weeks.

FIG. 5A shows the statistical results of the subject's intention to defecate and discharge before taking the probiotic composition of an embodiment of the present invention.

FIG. 5B shows the statistical results of the subject's intentional defecation after taking the probiotic composition of an embodiment of the present invention for four weeks.

Figure 6 shows the average waist circumference changes of subjects after taking the probiotic composition of an embodiment of the present invention for four weeks.

Figure 7 shows the average weight change of subjects after taking the probiotic composition of an embodiment of the present invention for four weeks.

Figure 8 shows the change in body fat percentage of subjects after taking the probiotic composition of an embodiment of the present invention for four weeks.

The present invention provides a use of a composition containing a fermented okra to prepare a probiotic composition for promoting the growth of butyric acid bacteria. In addition to the fermented okra, the probiotic composition may further include an oligosaccharide selected from the group consisting of xylo-oligosaccharides, fructooligosaccharides, and combinations thereof, and a sugar alcohol, such as sorbitol, mannitol, and erythrose Alcohol, maltitol, lactitol, and xylitol. The probiotic composition preferably includes fermented okra, xylo-oligosaccharides, and lactitol, and the weight ratio is preferably in the range of 3~5:2~4:2~4. The following examples show the growth-promoting effects of the components and combinations in the probiotic composition on butyric acid bacteria, and also show that the probiotic composition can reduce the body weight, overall body fat percentage, and waist circumference of obese subjects.

definition

The numerical values used herein are approximate values, and all experimental data are expressed in the range of 20%, preferably in the range of 10%, and most preferably in the range of 5%.

The so-called "okra" in this article refers to an annual or perennial herbaceous plant in the genus Abelmoschus of the Malvaceae family (Malvaceae), also known as hollyhock, carob, ashwagandha, Apingtian, Ukra, Okra , Vietnamese sesame, goat carob and other aliases.

The term "xylo-oligosaccharides" as used herein refers to oligosaccharides containing 3 to 10 xylose units, which can be derived from plants, or produced by microbial fermentation, enzyme transformation, or chemical synthesis.

According to the present invention, a food product can be used as a food additive, which can be added during the preparation of raw materials by a conventional method, or added during the production process of the food, and formulated with any edible material for supply Food products consumed by humans and non-human animals.

According to the present invention, the types of food products include, but are not limited to: beverages, fermented foods, bakery products, health foods, and dietary supplements.

As used herein, the terms " Saccharomyces cerevisiae " and " Lactobacillus plantarum " are intended to cover those brewer's yeast and Lactobacillus plantarum that are readily available to those skilled in the art (for example, It can be purchased from domestic or foreign depository institutions), or strains of Saccharomyces cerevisiae and Lactobacillus embryonicum isolated and purified from natural sources using the usual microbial isolation methods used in the art.

Materials and Methods Preparation of fermented okra

Okra (Abelmoschus esculentus) is an annual or perennial herbaceous plant in the genus Abelmoschus of the Malvaceae family (Malvaceae ), also known as hollyhock, carob, hair eggplant, Apingtian, Ukra, Okla, Vietnam Sesame, croissant and other aliases. It is native to Abyssini in northeastern Africa, Egypt and the Caribbean or tropical Asia. It was introduced to Taiwan in 1901. The production area is distributed in Changhua, Yunlin, Chiayi and other counties. The season of production is from April to September each year. The okra plant is erect, about one meter high, the whole plant is covered with hairs, the stem is lignified, and the flowers are fully flowered and light yellow. The tender fruits can be harvested about three to four days after the flowers die. The fruit of the sunflower is a capsule, about ten centimeters long, slender like a lamb's horn or a chili, with a thin and leathery skin, and a pointed tip. The fruit color ranges from light green to dark green, and there are also purple-red ones. The okra fruit contains special slimy substances and has a special aroma or flavor. Fermentation of okra in the present invention will obtain a fermented product with more abundant constituent molecules.

The preparation method of the fermented okra used in Examples 1-3 herein is briefly described as follows. First, the okra pulp and water are mixed in a weight ratio of 1:1 to 1:20, preferably 1:1 to 1:5, to serve as an air culture solution. After the air culture solution is processed by the pasteurization method, it is cooled to room temperature for subsequent fermentation. Then, 0.01-0.5% (w/w) of yeast (Saccharomyces cerevisiae , purchased from the Biological Resources Conservation and Research Center, Taiwan, numbered BCRC20271) and 0.01-0.25% (w/w) were implanted in the air culture solution at the same time w) Lactobacillus plantarum (Lactobacillus plantarum, purchased from the Biological Resources Conservation and Research Center, Taiwan, numbered BCRC910760), fermented at 25-35°C for 1-7 days, the actual time varies depending on the fermentation state. Finally, without removing the two kinds of bacteria, use the set sugar content range of 2-4°, pH<4, alcohol>5% and other specifications. If the test meets the specifications, the fermentation is determined to be completed and the fermented product is obtained. It is also an experimental sample of the fermented okra used in Examples 1-3. Next, the fermentation broth was concentrated under reduced pressure at 45-70°C, filtered through a 200-400 mesh sieve, and 40-70% isomalt-oligosaccharides were added to adjust the specifications, and then heated at 95-105°C for 70 -90 minutes for sterilization. The okra fermentation broth can be made into a powdered product by drying methods such as spray drying.

Oligosaccharides, polysaccharides and sugar alcohols

The oligosaccharides used in the examples herein include xylo-oligosaccharides (purchased from Shandong Longli Biotechnology Co., Ltd.) and fructo-oligosaccharides (purchased from MEIJI CO., Ltd.). The polysaccharides used include inulin (purchased from Cosucra Groupe Warcoing SA). The sugar alcohol used includes lactitol (purchased from Hongwei Biotechnology Co., Ltd.), sorbitol, mannitol, erythritol, maltitol, and xylitol.

Source of butyric acid bacteria and sample preparation

The following examples used Clostridium butyricum (BCRC 81048) purchased from the Bioresource Collection and Research Center (BCRC) of the Food Industry Development Institute.

Carry out an activation procedure to activate butyric acid bacteria. First, the butyric acid bacteria frozen bacteria were cultured in a liquid Lactobacilli MRS medium (BD Difco ^TM Lactobacilli MRS Broth, product model DF0881-17-5, purchased from Thermo Fischer Scientific) at a bacterial mass of 10% by volume, and the content was 37 Cultivate for 16 hours to activate butyric acid bacteria. After the bacterium is thawed and activated, it can be used for probiotics test.

Example 1 Probiotic test of butyric acid bacteria

In order to identify the nutrients that are beneficial to the growth of butyric acid bacteria, the pre-cultured butyric acid bacteria were added to the MRS medium containing 1% (w/w) candidate probiotics at an inoculum of 3%, and cultured at 37°C under anaerobic conditions After 24 hours, the obtained bacterial liquid was spread on the MRS medium and cultured for 72 hours to estimate the bacterial count. The candidate probiotic system is xylo-oligosaccharide, lactitol, inulin, fructooligosaccharide, or okra fermented product. As a control, the butyric acid bacteria were additionally cultured in MRS medium (blank group) that did not contain candidate probiotics.

Figure 1 shows the effects of the aforementioned various candidate probiotics on the growth of butyric acid bacteria. In this experiment, a spectrophotometer was used to measure the turbidity of the MRS medium after 72 hours, and the wavelength was 600nm to measure the concentration of viable butyric acid bacteria. According to Figure 1, the OD ₆₀₀ of the blank group is 1.1. Compared with the blank group, the candidate probiotics that significantly increased the number of bacteria include oligosaccharides, inulin, fructo-oligosaccharides, and okra fermented products, and their OD ₆₀₀ increased to about 2.2, 1.8, 1.7, and 2.9, respectively. This result indicates that any of okra fermentation product, xylo-oligosaccharides, fructooligosaccharides, and lactitol can be used as probiotics for butyric acid bacteria.

Example 2 Probiotic composition for promoting growth of butyric acid bacteria

In order to evaluate the effect of different combinations of the aforementioned butyric acid bacteria probiotics on the growth of butyric acid bacteria, the pre-cultured butyric acid bacteria were added to the MRS medium containing 1% (w/w) probiotic composition at an inoculum of 3%. Cultivate for 24 hours under anaerobic conditions at 37°C, and then spread the obtained bacterial solution on MRS agar medium and cultivate for 72 hours to estimate the number of bacteria. The probiotic composition in the MRS medium has the following composition: 0.4% okra fermentation product, 0.3% xylo-oligosaccharide, and 0.3% lactitol. As a control, the butyric acid bacteria were additionally cultured in an MRS medium without probiotics (blank group).

Figure 1 shows the effect of the aforementioned probiotic composition on the growth of butyric acid bacteria, and the number of bacteria in the blank group has an OD600 of 1.1. According to Fig. 1, compared with the blank control group, the addition of the probiotic composition significantly increased the relative number of butyric acid bacteria to approximately 3.4 respectively. Therefore, it can be known that the combination of okra fermentation product, xylo-oligosaccharide, and lactitol can most effectively promote the growth of butyric acid bacteria when the weight ratio is 3~5:2~4:2~4. Lactitol has the lowest growth-promoting effect shown in Figure 1.

Example 3 Physiological effects of probiotic composition

This example explores the physiological effects of continuous consumption of a probiotic composition capsule containing 200 mg of okra fermented product, 150 mg of xylo-oligosaccharides, and 150 mg of lactitol on subjects, where the subject has a BMI greater than 24 or a body fat greater than 25 Healthy men or women. Nine subjects aged 20 to 55 years old were given the probiotic composition orally after lunch every day for four consecutive weeks, at a dose of 400 mg/person/ The subjects’ bowel difficulties were assessed before and four weeks after taking, including the average number of days of defecation, clean defecation frequency, stool status and smoothness of defecation, and waist circumference measured by body composition meter (TANITA BC-545F) , Weight and body fat.

In order to verify that the fermented okra or its composition of the present invention has the effect of improving the degree of difficulty in defecation, the subjects filled in the gastrointestinal questionnaire before taking the probiotic composition and four weeks after taking the probiotic composition (Table 1) And the questionnaire about the difficulty of defecation (Table 2). Figures 2A to 5B show the statistical results of the experiment when the subjects have completed Table 1 and Table 2.

Please refer to Figure 2A and Figure 2B, respectively, according to the first question in Table 1, the results of the subjects before taking the capsule and four weeks after taking the capsule. Please refer to Figure 2A. Before taking the capsule, 22% of the subjects had a bowel movement once every three days. Please refer to Figure 2B. After the subjects took the capsule for four weeks, the number of subjects who had bowel movements once every three days was reduced to 0%, the number of bowel movements increased to 45%, and the number of bowel movements was twice a day. Increased to 22%, it can be seen that the probiotic composition capsule of the present invention has a significant improvement in the number of days of defecation.

Please refer to Figure 3A and Figure 3B, respectively, according to the fourth question in Table 1, the results of the subjects before and four weeks after taking the capsule. Please refer to Figure 3A. Before the subjects took the capsules, 11% of the subjects had the trouble of always having unclean defecation, and 78% of the subjects sometimes had the trouble of having unclean defecation. Please refer to Figure 3B. After the subjects took the capsule for four weeks, the number of subjects who always felt unclean and often unclean defecation was reduced to 0%. All subjects rarely felt unclean defecation. Troubled. Therefore, it can be known that the probiotic composition capsule of the present invention can effectively improve the frequency of unclean defecation.

Please refer to Figures 4A and 4B, respectively, according to the sixth question in Table 1, the results of the subjects before taking the capsule and four weeks after taking the capsule. Please refer to Figure 4A. Before the subjects took the capsules, 56% of the subjects had moderate stools, and 33% of the subjects had mild stools. Please refer to Figure 4B. After the subjects took the capsules for four weeks, the number of subjects with moderate stool conditions decreased to 11%, and the number of subjects with mild and no stool conditions increased to 56% and 33%, respectively. Therefore, it can be known that the probiotic composition capsule of the present invention can improve the stool condition.

Please refer to Figures 5A and 5B, respectively, according to the rectal symptoms in Table 2 before and four weeks after taking the capsule. Please refer to Figure 5A. Before taking the capsules, 56% of the subjects felt a moderate level of difficulty when they intended to defecate, and 11% of the subjects had Feeling a mild level of difficulty when you want to defecate. Please refer to Figure 5B. After the subjects took the capsule for four weeks, only 11% of the subjects felt a moderate level of difficulty when they intended to defecate, and the remaining 89% of the subjects had already No difficulty. Therefore, it can be seen that the probiotic composition capsule of the present invention can effectively improve the smoothness of defecation.

According to Figures 6 to 8, compared with before taking the probiotic composition, taking the probiotic composition for four weeks reduces the average waist circumference by about 1 cm, reduces the average weight by about 0.3 kg, and reduces the average body fat percentage by about 0.2%. This result confirms that long-term use of the probiotic composition of the present invention can improve the body condition of obese people.

In summary, the present invention discloses multiple probiotics and their combinations that are beneficial to the growth of butyric acid bacteria. The probiotic combination can be used to prepare a probiotic composition that promotes the growth of butyric acid bacteria, such as a medium supplement for in vitro bacterial culture, or a probiotic product for promoting the proliferation of butyric acid bacteria in the human intestinal tract. The probiotic composition can have a dosage form of powder, granule, solution, or capsule, and can be made into food, drink, medicine, or nutritional supplement, and administered to a body by oral administration.

Claims (9)

A use of a composition containing a fermented okra obtained by fermentation with a yeast and a Lactobacillus embryo for preparing a probiotic composition for promoting the growth of butyric acid bacteria, wherein the yeast and the Lactobacillus embryo are inoculated The amounts are respectively 0.1% to 0.4% (w/v) and 0.2% to 0.5% (w/v). The use according to claim 1, wherein the okra fermentation product is an okra pulp and an air culture solution obtained by simultaneously fermenting the yeast (Saccharomyces cerevisiae) and the embryonic Lactobacillus plantarum. The use according to claim 2, wherein the air culture solution is obtained by mixing okra pulp and water in a weight ratio of 1:1 to 1:20. The use according to claim 1, wherein the composition comprising the fermented okra further comprises an oligosaccharide selected from the group consisting of xylo-oligosaccharides, fructo-oligosaccharides, and combinations thereof. The use according to claim 4, wherein the composition containing the fermented okra further contains a sugar alcohol. The use according to claim 5, wherein the oligosaccharide is xylo-oligosaccharide, and the sugar alcohol is lactitol. The use according to claim 6, wherein the weight ratio of the fermented okra, the xylo-oligosaccharide, and the lactitol ranges from 3 to 5: 2 to 4: 2 to 4. A probiotic composition for promoting the growth of butyric acid bacteria, comprising a fermented okra, a xylo-oligosaccharide, and a lactitol, wherein the fermented okra includes a yeast and a lactobacillus embryo, and the okra is fermented The weight ratio of the substance, the xylo-oligosaccharide, and the lactitol ranges from 3 to 5: 2 to 4: 2 to 4. A use of the probiotic composition according to claim 8 for preparing a composition for reducing waist circumference.

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