CN112626001B - Probiotic composition for promoting growth of butyric acid bacteria and application thereof - Google Patents

Abstract

The invention relates to the field of microbial fermentation, in particular to the use of a composition containing fermented okra and a prebiotic composition. The present invention provides an application of a composition containing okra fermented product for preparing a prebiotic composition for promoting the growth of butyric acid bacteria, and a prebiotic composition containing okra fermented product. The prebiotic composition promotes the growth of butyric acid bacteria in the intestinal tract, reduces the waistline of the user, and improves defecation.

Description

Prebiotic composition for promoting growth of butyric acid bacteria and use thereof

technical field

The invention relates to the field of microbial fermentation, in particular to a prebiotic composition for promoting the growth of butyric acid bacteria.

Background technique

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 is densely covered with nerve branches to collect physiological information and feed back to the central nervous system. Therefore, intestinal health has a great impact on human health. In the past ten years, studies have found that there are more than a thousand kinds of intestinal bacteria living in the intestinal mucosa, they communicate with the body organs and give appropriate responses, such as supplying the nutrients needed by the body, regulating the development of intestinal cells, and inducing the immune system Development, so intestinal bacteria can be described as an essential organ of the body.

The bacteria that make up the intestinal flora have specific functions. When their composition is out of balance due to unhealthy living habits, diseases, and the use of antibiotics, the body may lose its health. For example, if a person consumes a high-fat diet on a daily basis, the fat-eating Gram-negative bacteria in the large intestine will proliferate and produce lipopolysaccharides (lipopolysaccharides, LPS), which can cause chronic inflammation throughout the body through blood circulation, and eventually It can lead to chronic diseases such as type 2 diabetes, cardiovascular disease, and even neurodegenerative diseases and cancer. Studies have also shown that mice with disrupted gut microbiota have memory loss in stressful situations. Therefore, maintaining a constant composition of gut bacteria can help maintain physical and mental health.

In addition to Bifidobacterium and Lactobacillus bacteria, butyric acid bacteria (Clostridium butyricum) are also known to be beneficial to human health. The bacteria account for 1% to 4% of the intestinal bacteria in adults, and are anaerobic Gram-positive bacteria that grow in the large intestine. Butyric acid bacteria can produce butyric acid, which can help defecation and promote the growth of lactic acid bacteria. In the mouse animal experiment, when the mice ingested butyric acid bacteria under a high-fat diet, the range of weight gain of the mice was reduced.

In view of the importance of butyric acid bacteria to maintain the integrity of the intestinal tract, it is necessary to develop a prebiotic composition that can promote the growth of butyric acid bacteria in the intestinal tract.

Contents of the invention

Therefore, an object of the present invention is to provide a composition comprising fermented okra for the preparation of a prebiotic composition for promoting the growth of butyric acid bacteria.

In one embodiment of the present invention, the okra fermented product is obtained by simultaneously fermenting an empty culture solution of okra pulp and water with a yeast (Saccharomyces cerevisiae) and a lactic acid bacterium (Lactobacillus plantarum).

In one embodiment of the present invention, the aerial culture solution is obtained by mixing okra pulp and water at a weight ratio of 1:1 to 1:20, and the inoculum amounts of the yeast and the lactic acid bacteria are respectively 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 comprising fermented okra further comprises an oligosaccharide selected from xylooligosaccharides, fructooligosaccharides, and combinations thereof.

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

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

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

Another object of the present invention is to provide a prebiotic composition that promotes the growth of butyric acid bacteria, comprising a okra fermented product, an oligosaccharide, and a sugar alcohol, wherein the oligosaccharide is selected from xylooligosaccharide, Fructooligosaccharides, and combinations thereof.

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

In one embodiment of the present invention, the prebiotic composition increases the number of butyric acid bacteria in the intestinal tract of obese subjects, and reduces the body weight, body fat percentage and waist circumference of obese subjects.

The invention discloses a variety of prebiotics and combinations thereof that are beneficial to the growth of butyric acid bacteria. The prebiotic combination can be used to prepare a prebiotic composition that effectively promotes the growth of butyric acid bacteria, for example, as a medium supplement for in vitro bacterial culture, or as a prebiotic that promotes the proliferation of butyric acid bacteria in the intestinal tract of a living body (such as a human body) product. The prebiotic composition can be in the dosage form of powder, granule, solution, or capsule, and can be made into food, drink, medicine, or nutritional supplement, and administered to an individual orally.

The implementation of the present invention will be further described in conjunction with the drawings below. 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 who is skilled in the art will not depart from Within the spirit and scope of the present invention, some changes and modifications can be made, so the scope of protection of the present invention should be defined by the scope of the appended patent application.

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments, but not as a limitation of the present invention.

Description of drawings

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

Fig. 2A shows the statistical results of the average defecation days before subjects take the prebiotic composition of an embodiment of the present invention.

Fig. 2B shows the statistical results of the average defecation days after the subject took the prebiotic composition of an embodiment of the present invention for four weeks.

Fig. 3A shows the statistical results of the frequency of unclean defecation experienced by the subjects before taking the prebiotic composition according to an embodiment of the present invention.

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

Fig. 4A shows the statistical results of the stool status of the subject before taking the prebiotic composition of an embodiment of the present invention.

Fig. 4B shows the statistical results of the stool status of the subject after taking the prebiotic composition of an embodiment of the present invention for four weeks.

FIG. 5A shows the statistical results of defecation and excretion of subjects before taking the prebiotic composition of an embodiment of the present invention.

FIG. 5B shows the statistical results of defecation and excretion of subjects after taking the prebiotic composition of an embodiment of the present invention for four weeks.

Fig. 6 shows the change of the average waist circumference of the subjects after taking the prebiotic composition of an embodiment of the present invention for four weeks.

Fig. 7 shows the average body weight changes of the subjects after taking the prebiotic composition of an embodiment of the present invention for four weeks.

Fig. 8 shows the change of body fat percentage of the subject after taking the prebiotic composition of an embodiment of the present invention for four weeks.

detailed description

The present invention provides an application of a composition comprising fermented okra for preparing a prebiotic composition for promoting the growth of butyric acid bacteria. In addition to the fermented okra, the prebiotic composition may further comprise an oligosaccharide selected from xylooligosaccharides, fructooligosaccharides, and combinations thereof, and a sugar alcohol, such as sorbitol, mannitol, erythritol Sugar alcohols, maltitol, lactitol, and xylitol. The prebiotic composition preferably comprises okra fermented product, xylooligosaccharide, and lactitol, and the weight ratio range thereof is preferably 3-5:2-4:2-4. The following examples show the growth-promoting effect of each component in the prebiotic composition and their combinations on butyric acid bacteria, and also show that the prebiotic composition can reduce the body weight, body fat percentage, and waist circumference of obese subjects .

definition

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

The so-called "okra" in this article refers to the annual or perennial herbaceous plant of the genus Abelmoschus in the Malvaceae family, also known as hollyhock, carob, hair eggplant, apingtian, okra, okra, Vietnamese sesame, lady's finger and other aliases.

The so-called "xylooligosaccharide" herein refers to oligosaccharides containing 3 to 10 xylose units, which can be obtained from plants, or obtained by microbial fermentation, enzyme conversion, or chemical synthesis.

According to the present invention, the food product can be regarded as a food additive (food additive), which is added during the preparation of raw materials by known methods, or added during the production process of food, and formulated with any edible material for Food products for ingestion by humans and non-human animals.

According to the present invention, the types of food products include but 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, respectively, Saccharomyces cerevisiae and Lactobacillus plantarum which are readily available to those skilled in the art (for example, commercially available from Deposit institutions at home or abroad), or Saccharomyces cerevisiae and lactic acid bacteria strains isolated and purified from natural sources by the usual microbial isolation methods in this art.

Materials and Methods

Preparation of fermented okra

Okra (Abelmoschus esculentus) is an annual or perennial herbaceous plant of the Malvaceae genus Abelmoschus, also known as hollyhock, carob, hair eggplant, apin field, okra, okra, Vietnamese sesame , croissants and other aliases. It is native to Abyssinia, Egypt and the Caribbean in Northeast Africa or tropical Asia, and was introduced to Taiwan, China in 1901. The production areas are distributed in Changhua, Yunlin, Chiayi and other counties, and it is abundant from April to September every year. The okra plant is upright, about one meter high, the whole plant is covered with fluff, the stem is lignified, the flowers are complete and light yellow, and the tender fruits can be harvested about three to four days after the flowers fade. The fruit of sunflower is a capsule, about ten centimeters long, slender like a horn or pepper, with thin and leathery skin, tapered apex, fruit color from light green to dark green, and some are purple-red. The okra fruit contains a special sticky substance and has a special aroma or flavor. Fermenting the okra in the present invention will obtain a fermented product with richer constituent molecules.

The okra fermented product used in Examples 1-3 herein, its preparation method 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 be used as an air culture solution. After the air culture solution is treated by pasteurization, it is cooled to room temperature for subsequent fermentation. Then, 0.01-0.5% (w/w) yeast (Saccharomyces cerevisiae, purchased from Biological Resource Conservation and Research Center, Taiwan, China, No. /w) lactic acid bacteria (Lactobacillus plantarum, purchased from Biological Resource Conservation and Research Center, Taiwan, China, No. 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, it is judged that the fermentation is complete and obtained Described fermented product is also the experimental sample of the okra fermented product used in embodiment 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% isomaltooligosaccharide was added to adjust the specification, and then heated at 95-105°C for 70 -90 minutes for sterilization. The okra fermented liquid can be made into a powder product by spray drying and other drying methods.

Oligosaccharides, polysaccharides and sugar alcohols

The oligosaccharides used in the examples herein include xylooligosaccharides (purchased from Shandong Longli Biotechnology Co., Ltd.), fructooligosaccharides (purchased from MEIJI CO., Ltd.). Polysaccharides used included inulin (available from Cosucra Groupe Warcoing SA). The sugar alcohols used include lactitol (purchased from Hongwei Biotechnology Co., Ltd.), sorbitol, mannitol, erythritol, maltitol, and xylitol.

Source and sample preparation of butyric acid bacteria

The following examples use butyric acid bacteria (Clostridium butyricum, BCRC 81048) purchased from the Bioresource Collection and Research Center (BCRC), Food Industry Development Institute.

Carry out an activation procedure to activate the butyric acid bacteria. First, butyric acid 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)) with a bacterial volume of 10% by volume, and in Cultivate at 37 for 16 hours to activate butyric acid bacteria. After the bacteria are thawed and activated, they can be used for prebiotic tests.

Example 1

Prebiotic 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 according to the inoculum of 3%, at 37 ° C under anaerobic conditions After culturing for 24 hours, the resulting bacterial solution was spread on the MRS medium and cultured for 72 hours to estimate the number of bacteria. The candidate prebiotics are xylooligosaccharides, lactitol, inulin, fructooligosaccharides, or fermented okra. As a control, butyric acid bacteria were additionally cultured in the MRS medium (blank group) without 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 viable concentration of butyric acid bacteria. According to Figure 1, the OD600 of the blank group was 1.1. Compared with the blank group, the candidate probiotics that significantly increased the number of bacteria included autumnal oligosaccharides, inulin, fructooligosaccharides, and okra fermented products, and their OD600 increased to about 2.2, 1.8, 1.7, and 2.9, respectively. The results indicated that any of okra fermented product, xylooligosaccharides, fructooligosaccharides, and lactitol could be used as prebiotics for butyric acid bacteria.

Example 2

Prebiotic composition for promoting growth of butyric acid bacteria

In order to evaluate the promotion effect of different combinations of the aforementioned butyric acid bacteria prebiotics on the growth of butyric acid bacteria, the pre-cultured butyric acid bacteria were added to the MRS medium containing 1% (w/w) prebiotic composition according to the inoculum size of 3%. , cultivated at 37°C and under anaerobic conditions for 24 hours, and then spread the obtained bacterial liquid on MRS agar medium and cultivated for 72 hours to estimate the number of bacteria. The prebiotic composition in the MRS medium had the following composition: 0.4% okra ferment, 0.3% xylooligosaccharide, and 0.3% lactitol. As a control, butyric acid bacteria were additionally cultured in MRS medium without prebiotics (blank group).

Figure 1 shows the effect of the aforementioned prebiotic composition on the growth of butyric acid bacteria, and the OD600 of the bacterial count in the blank group was 1.1. According to Figure 1, compared with the blank control group, the addition of the prebiotic composition significantly increased the relative bacterial counts of butyric acid bacteria to about 3.4 respectively. Therefore, it can be seen that the combination of fermented okra, xylooligosaccharide, and lactitol can most effectively promote the growth of butyric acid bacteria when the weight ratio is 3-5:2-4:2-4, even Lactitol had the lowest growth-promoting effect shown in Figure 1.

Example 3

Physiological Effects of Prebiotic Compositions

This example investigates the physiological effects of continuously taking a capsule of a prebiotic composition containing 200 mg okra fermented product, 150 mg xylooligosaccharide, and 150 mg lactitol on a subject, wherein 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 were orally administered the prebiotic composition after lunch every day for four consecutive weeks at a dose of 400 mg/person/day, and the defecation of these subjects was evaluated before and after four weeks of taking Difficulty, including the average number of days of defecation, frequency of clean defecation, stool status and smoothness of defecation, as well as waist circumference, weight and body fat measured with a body composition meter (TANITA BC-545F).

In order to confirm that the okra fermented product of the present invention or its composition has the effect of improving the degree of difficulty in defecation, before taking the prebiotic composition and after taking the prebiotic composition for four weeks, the subjects filled out the gastrointestinal questionnaire (Table 1) and the questionnaire on the degree of defecation difficulty (Table 2). Fig. 2A to Fig. 5B are the experimental results obtained by the subjects filling in Table 1 and Table 2.

Table 1_ Gastrointestinal tract questionnaire

Form 2_Survey of difficulty in defecation (please tick the box) (single choice)

Please refer to Fig. 2A and Fig. 2B, respectively, according to the first question in Table 1, the statistical results of the subject before taking the capsule and after taking the capsule for four weeks. Please refer to FIG. 2A , before taking the capsule, 22% of the subjects defecated once every three days. Please refer to Figure 2B. After the subjects took the capsule for four weeks, the subjects who originally defecated once every three days decreased to 0%, the subjects who defecated once a day increased to 45%, and the subjects who defecated twice a day Increased to 22%, it can be seen that the prebiotic composition capsule of the present invention has a significant improvement in the number of days of defecation.

Please refer to Fig. 3A and Fig. 3B, respectively, according to the fourth question in Table 1, the statistical results of the subject before taking the capsule and after taking the capsule for four weeks. Please refer to FIG. 3A , before taking the capsules, 11% of the subjects always had trouble with unclean defecation, and 78% of the subjects sometimes had trouble with unclean defecation. Please refer to Figure 3B. After the subjects took the capsule for four weeks, the number of subjects who always felt unclean defecation and those who often felt unclean defecation decreased to 0%, and all the subjects rarely felt that they had unclean defecation. troubled. Therefore, it can be known that the prebiotic composition capsule of the present invention can effectively improve the frequency of unclean defecation.

Please refer to Fig. 4A and Fig. 4B, respectively, according to the sixth question in Table 1, the statistical results of the subject before taking the capsule and after taking the capsule for four weeks. Please refer to FIG. 4A , before the subjects took the capsule, 56% of the subjects had a moderate stool condition, and 33% of the subjects had a mild stool condition. Please refer to FIG. 4B , after taking the capsule for four weeks, the subjects with moderate stool condition decreased to 11%, and the subjects with mild and no stool condition increased to 56% and 33% respectively. Therefore, it can be known that the capsule of the prebiotic composition of the present invention can improve the stool condition.

Please refer to Fig. 5A and Fig. 5B, respectively, according to the rectal symptoms in Table 2, the statistical results of the subject before taking the capsule and after taking the capsule for four weeks. Please refer to FIG. 5A , before taking the capsule, 56% of the subjects felt moderate difficulty in defecation, and 11% of the subjects felt mild difficulty in defecation. Please refer to Figure 5B. After taking the capsule for four weeks, only 11% of the subjects felt moderate difficulty when defecating, and the remaining 89% of the subjects felt difficulty defecating when defecating. No difficulty felt. Therefore, it can be known that the capsule of the prebiotic composition of the present invention can effectively improve the smoothness of defecation.

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

In summary, the present invention discloses a variety of prebiotics and combinations thereof that are beneficial to the growth of butyric acid bacteria. The prebiotic combination can be used to prepare a prebiotic composition that promotes the growth of butyric acid bacteria, such as a medium supplement for in vitro bacterial culture, or a prebiotic product for promoting the proliferation of butyric acid bacteria in the human intestinal tract. The prebiotic composition can be in the dosage form of powder, granule, solution, or capsule, and can be made into food, drink, medicine, or nutritional supplement, and administered to an individual orally.

Certainly, the present invention also can have other various embodiments, without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and deformations according to the present invention, but these corresponding changes All changes and modifications should belong to the protection scope of the claims of the present invention.

Claims (8)

1. A composition comprising okra fermented product is used for preparing the purposes of the prebiotic composition that promotes the growth of butyric acid bacteria, wherein said okra fermented product is a okra pulp and water with 1:1 to 1:20 The blank culture solution obtained by mixing the weight ratio is obtained by simultaneous fermentation of a yeast with a preservation number of BCRC20271 and a lactic acid bacterium with a preservation number of BCRC910760. 2. The use according to claim 1, characterized in that, the inoculum amounts of the yeast and the lactic acid bacteria are respectively 0.1% to 0.4% (w/v) and 0.2% to 0.5% (w/v). 3. A composition comprising okra fermented product and oligosaccharide is used to prepare the purposes of the prebiotic composition that promotes the growth of butyric acid bacteria, it is characterized in that, described okra fermented product is a okra pulp and water with 1 : 1 to 1: 20 in a weight ratio to obtain an empty culture solution obtained by simultaneous fermentation of a yeast with a preservation number of BCRC20271 and a lactic acid bacterium with a preservation number of BCRC910760, and the oligosaccharides are selected from xylooligosaccharides , fructooligosaccharides, and combinations thereof. 4. A composition comprising okra fermented product, oligosaccharide and sugar alcohol is used to prepare the purposes of the prebiotic composition that promotes the growth of butyric acid bacteria, it is characterized in that, described okra fermented product is a okra pulp and An empty culture solution obtained by mixing water at a weight ratio of 1:1 to 1:20 is obtained through simultaneous fermentation of a yeast with a preservation number of BCRC20271 and a lactic acid bacterium with a preservation number of BCRC910760, and the oligosaccharide is selected from Xylooligosaccharides, fructooligosaccharides, and combinations thereof. 5. The use according to claim 4, characterized in that the oligosaccharide is xylooligosaccharide, and the sugar alcohol is lactitol. 6 . The use according to claim 5 , characterized in that the weight ratio of the okra fermented product, the xylooligosaccharide, and the lactitol ranges from 3 to 5:2 to 4:2 to 4. 7. A prebiotic composition that promotes the growth of butyric acid bacteria, comprising a okra fermented product, an oligosaccharide, and a sugar alcohol, wherein the okra fermented product is an okra pulp and water at a ratio of 1:1 to An empty culture solution mixed at a weight ratio of 1:20 is obtained through simultaneous fermentation of a yeast with a preservation number of BCRC20271 and a lactic acid bacterium with a preservation number of BCRC910760. The oligosaccharides are selected from xylooligosaccharides, fructooligosaccharides sugars, and combinations thereof. 8. A use of the prebiotic composition according to claim 7 for preparing a pharmaceutical composition for reducing waist circumference.

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