Use of a prebiotic composition to assist a recipient in weight loss

Abstract

The application discloses a probiotic composition and application thereof, and the probiotic composition comprises kiwi fruit ferment, inulin and fructo-oligosaccharide, and the weight ratio of the kiwi fruit ferment, the inulin and the fructo-oligosaccharide is 3-4:2.5-4:2.5-4. Therefore, the probiotic composition prepared from the kiwi fruit ferment and the saccharide can be used for weight loss.

Description

[0001] This application is a divisional application of Chinese national patent application No. 202111303863.2, filed on November 5, 2021, entitled "Prebiotic Composition and Its Use". Technical Field

[0002] This invention relates to the use of a prebiotic composition, which involves using kiwifruit ferment and sugars to prepare the prebiotic composition, and to using the prebiotic composition for weight loss. Background Technology

[0003] Prebiotics, also known as prebiotics or probiotics, are polysaccharide components in natural foods that are not easily digested by human enzymes. However, they can be utilized by probiotics in the digestive system (mainly the large intestine) for the growth, expansion, and metabolism of the gut microbiota to produce short-chain fatty acids (SFCAs).

[0004] According to the joint statement on prebiotics published in Nature Report in 2017 by the International Scientific Association for Probiotics and Prebiotics (ISAPP), prebiotics are defined as "substances that can be selectively utilized by microorganisms that live in symbiosis with the host, thereby promoting the health of the host."

[0005] Specifically, prebiotics help probiotics grow and help suppress harmful bacteria in the gut. Probiotics in the gut also metabolize prebiotics into short-chain fatty acids, which are then provided as an energy source for both the probiotics and the host. Summary of the Invention

[0006] In view of this, the present invention provides a prebiotic composition comprising kiwifruit ferment and sugars, which can be used to assist in receptor weight loss.

[0007] In some embodiments, a prebiotic composition includes kiwifruit ferment, inulin, and fructooligosaccharides, wherein the weight ratio of kiwifruit ferment, inulin, and fructooligosaccharides is 3-4:2.5-4:2.5-4.

[0008] In some embodiments, the use of a kiwifruit ferment and sugars for preparing a prebiotic composition for improving the gut microbiota of receptors, wherein the prebiotic composition comprises kiwifruit ferment, inulin and fructooligosaccharides, and the weight ratio of kiwifruit ferment, inulin and fructooligosaccharides is 3-4:2.5-4:2.5-4.

[0009] In some embodiments, the use of a kiwifruit ferment and sugars in preparing a prebiotic composition for assisting receptor weight loss, wherein the prebiotic composition comprises kiwifruit ferment, inulin and fructooligosaccharides, and the weight ratio of kiwifruit ferment, inulin and fructooligosaccharides is 3-4:2.5-4:2.5-4.

[0010] In some embodiments, the prebiotic composition comprises kiwifruit ferment, inulin, and fructooligosaccharides in a weight ratio of 4:3:3.

[0011] In some embodiments, the preparation process of kiwifruit fermentation product includes: mixing whole kiwifruit with water and extracting at 80°C to 100°C for 0.5 to 1.5 hours to obtain kiwifruit extract; and fermenting sequentially with 0.1% brewer's yeast and 0.05% thermophilic streptococcus for 0.5 to 2 days to form the kiwifruit fermentation product.

[0012] In some embodiments, improving the recipient gut includes improving the gut microbiota, improving the recipient's defecation status, enhancing the recipient's intestinal barrier, or a combination thereof.

[0013] In some embodiments, the bacterial community includes Akkermansia muciniphila, Parabacteroides goldsteinii, and Bifidobacterium.

[0014] In some embodiments, the prebiotic composition is used to promote the growth of *Pseudomonas gondii*.

[0015] In some embodiments, improving the recipient's defecation status means improving the recipient's defecation difficulty, promoting the recipient's intestinal peristalsis frequency, reducing the recipient's incomplete defecation, reducing the recipient's defecation time, or a combination thereof.

[0016] In some embodiments, the prebiotic composition is used to increase the concentration of tight structural proteins in the recipient's blood.

[0017] In some embodiments, the tight structural protein is Claudin 3 (CLDN3).

[0018] In some embodiments, the prebiotic composition is used to reduce a recipient’s weight, waist circumference, body fat percentage, or a combination thereof.

[0019] In some embodiments, the prebiotic composition is used to reduce a receptor’s weight, waist circumference, total body fat percentage, trunk body fat percentage, abdominal fat, or a combination thereof.

[0020] In some embodiments, the prebiotic composition is used to suppress the appetite of receptors.

[0021] In some embodiments, the prebiotic composition is used to reduce fat storage in receptors.

[0022] In some embodiments, the prebiotic composition is used to enhance lipid metabolism in receptors.

[0023] In some embodiments, the prebiotic composition is used to activate brown cells of the receptor.

[0024] In some embodiments, the prebiotic composition is used to increase the levels of Kermania myxotroph and Parabacterium goeringii in the recipient's gut.

[0025] In summary, the prebiotic composition of any embodiment includes kiwifruit ferment, inulin, and fructooligosaccharides. That is, kiwifruit ferment and polysaccharides can be used to prepare a prebiotic composition that improves the recipient's gut microbiota. In some embodiments, the prebiotic composition can be used to improve the gut microbiota, improve the recipient's bowel movements, enhance the recipient's intestinal barrier, or a combination thereof. For example, the prebiotic composition can enhance the growth of beneficial bacteria such as *Kermania*, *Parabacterium goeringii*, and *Bifidobacterium*, and promote their abundance in the recipient's gut. In some embodiments, the prebiotic composition can improve the recipient's bowel movement difficulties, promote the recipient's intestinal peristalsis frequency, reduce the recipient's incomplete bowel movements, reduce the recipient's bowel movement time, or a combination thereof. In some embodiments, the prebiotic composition can increase the concentration of tight structural proteins (e.g., claudin 3, CLDN3) in the recipient's blood. In some embodiments, the prebiotic composition is used to reduce the recipient's weight, waist circumference, body fat percentage, or a combination thereof, thereby achieving a weight loss effect.

[0026] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the present invention. Attached Figure Description

[0027] Figure 1 This is a graph showing the experimental results of analyzing the effect of a single component on the growth of probiotics;

[0028] Figure 2 This is a graph showing the results of an analysis of the effects of multiple different components on the growth of probiotics.

[0029] Figure 3 This is a graph showing the experimental results analyzing the effects of multiple combinations of prebiotics in different proportions on the growth of probiotics;

[0030] Figure 4 This is a graph showing the experimental results of the prebiotic composition on the expression of tight structural proteins in the recipient's blood.

[0031] Figure 5 This is a graph showing the experimental results of the probiotic composition's ability to inhibit the growth of Kermania pseudomallei.

[0032] Figure 6 This is a graph showing the experimental results of the probiotic composition's ability to inhibit the growth of *Pseudomonas gondii*.

[0033] Figure 7 This is a graph showing the experimental results of the probiotic composition's ability to inhibit the growth of Bifidobacterium.

[0034] Figure 8 This is a graph showing the analysis results of the prebiotic composition on the average number of bowel movements in the subjects;

[0035] Figure 9 This is a graph showing the analysis results of the prebiotic composition on the average defecation time of the subjects;

[0036] Figure 10 This is a graph showing the analysis results of the frequency of incomplete bowel movements in subjects affected by the prebiotic composition;

[0037] Figure 11 This is a graph showing the results of the analysis of the frequency of intestinal peristalsis in the subjects affected by the prebiotic composition;

[0038] Figure 12 This is an analysis chart of the average weight of the subjects in weeks 0, 2, and 4;

[0039] Figure 13 This is an analysis chart of the subjects' average total body fat percentage in weeks 0, 2, and 4;

[0040] Figure 14 This is an analysis chart of the average trunk body fat percentage of the subjects in weeks 0, 2, and 4;

[0041] Figure 15 This is an analysis chart of the average waist circumference of the subjects in weeks 0, 2, and 4; and

[0042] Figure 16 These are photographs analyzing the abdominal fat mass of the subjects at weeks 0 and 4.

[0043] In the attached figures, the following labels are used:

[0044] FA0: Abdominal fat in week 0

[0045] FA4: Abdominal fat in week 4 Detailed Implementation

[0046] In the following description of the embodiments, unless otherwise stated, the "%" symbol refers to weight percentage and the symbol "volume %" usually refers to volume percentage concentration.

[0047] The prebiotic composition includes kiwifruit ferment and sugars. The sugars are oligosaccharides, dietary fiber, and other polysaccharides, such as fructose oligosaccharides and inulin. The kiwifruit ferment is prepared by fermenting the juice of kiwifruit (Actinidia deliciosa) using multiple bacterial strains.

[0048] In some embodiments, the prebiotic composition includes kiwifruit ferment, inulin, and fructooligosaccharides, and the weight ratio of kiwifruit ferment, inulin, and fructooligosaccharides is 3-4:2.5-4:2.5-4. For example, the prebiotic composition includes kiwifruit ferment, inulin, and fructooligosaccharides in a weight ratio of 4:3:3.

[0049] In some embodiments, the kiwifruit fermentation preparation process is as follows: glucose and kiwifruit fruit are mixed with water to obtain a culture medium, wherein the weight of water is 3-6 times the total weight of the kiwifruit fruit. Next, the culture medium and a plurality of microbial strains are fermented for 1-4 days to obtain kiwifruit fermented juice. Here, the plurality of microbial strains include yeast and lactic acid bacteria. Here, "kiwifruit fruit" can include its pulp / fruit, pulp / fruit containing the peel, or pulp / fruit containing seeds.

[0050] The yeast used can be commercially available brewer's yeast (Saccharomyces cerevisiae). For example, it can be brewer's yeast strain BCRC20271 (international deposit number ATCC26602) purchased from the Food Research and Development Institute in Taiwan.

[0051] The lactic acid bacteria used can be commercially available Lactobacillus helveticus, Streptococcus thermophiles, or Lactobacillus plantarum. For example, Lactobacillus helveticus TCI357 (Taiwan Food Research Institute Depository No. BCRC910846, International Depository No. DSM33107), Streptococcus thermophiles TCI028 (depository No. BCRC910805, International Depository No. DSM33108), Streptococcus thermophiles TCI378 (depository No. BCRT910760, International Depository No. DSM32451), or Streptococcus thermophiles TCI633 (depository No. BCRC910636, International Depository No. DSM28121) can also be used.

[0052] For example, kiwifruit is first mixed with water and extracted at 80-100°C for 0.5-1.5 hours to obtain a kiwifruit extract. Glucose is then added to the kiwifruit extract to obtain a culture medium for subsequent fermentation. The amount of glucose added is 8-10% of the total weight of the kiwifruit and water. Adding glucose ensures sufficient sugar content in the culture medium to provide adequate nutrients for the microorganisms during subsequent fermentation and to ensure successful fermentation. Next, yeast is added to the culture medium and fermented for 0.5-2 days to form a primary fermentation broth. Lactic acid bacteria are then added to the primary fermentation broth and fermented for another 0.5-2 days to form a kiwifruit fermentation broth. The kiwifruit fermentation broth is then filtered, concentrated, freeze-dried, and pulverized to obtain the kiwifruit fermented product. In some embodiments, the total polyphenol content of the kiwifruit fermentation broth is 200-220 µg / ml.

[0053] Therefore, a prebiotic composition can be obtained by mixing kiwifruit fermented product obtained through a specially customized process with inulin and fructooligosaccharides in a specific ratio. The weight ratio of kiwifruit fermented product, inulin, and fructooligosaccharides is 3-4:2.5-4:2.5-4. Furthermore, the prebiotic composition can increase the number of probiotics in the recipient's gut by at least three times.

[0054] In some embodiments, the prebiotic composition can improve the gut microbiota. For example, the prebiotic composition can increase the abundance of Akkermansia muciniphila in the recipient's gut by at least 3 times, the abundance of Parabacteroides goldsteinii in the recipient's gut by at least 1.5 times, and the abundance of Bifidobacterium in the recipient's gut by at least 3 times.

[0055] In some embodiments, the prebiotic composition can improve the recipient's bowel movements. For example, by taking the prebiotic composition, the recipient can reduce the frequency and duration of bowel movements to improve bowel movement difficulty, reduce the frequency of incomplete bowel movements, and promote the frequency of intestinal peristalsis, thereby improving their bowel movements.

[0056] In some embodiments, the prebiotic composition can increase the concentration of tight structural proteins in the recipient's blood, thereby enhancing the recipient's intestinal barrier. For example, the tight structural protein can be claudin 3 (CLDN3).

[0057] Therefore, it can be concluded that the prebiotic composition can improve the receptor's gut. Here, the receptor is a human being.

[0058] In some embodiments, the prebiotic composition can improve the recipient's gut health, thereby achieving a weight loss effect. For example, by taking the prebiotic composition, the recipient can reduce weight, waist circumference, body fat percentage, or a combination thereof. Body fat percentage refers to total body fat percentage and trunk body fat percentage.

[0059] In some embodiments, the prebiotic composition may be solid, such as powder, tablets, capsules, etc.

[0060] In some embodiments, the dosage of the prebiotic composition is 800 mg / day. For example, the prebiotic composition is mainly composed of kiwifruit ferment, inulin, and fructooligosaccharides, and the daily dosage of 800 mg of the prebiotic composition refers to a total amount of 800 mg of kiwifruit ferment, inulin, and fructooligosaccharides.

[0061] Any of the aforementioned prebiotic compositions may be a pharmaceutical product. In other words, this pharmaceutical product contains effective amounts of kiwifruit ferment, inulin, and fructooligosaccharides in specific proportions.

[0062] In some embodiments, the aforementioned pharmaceutical products may be manufactured using techniques known to those skilled in the art into dosage forms suitable for oral, parenterally, oral, or topically administration.

[0063] In some embodiments, enteral or oral dosage forms may be, but are not limited to, tablets, troche, lozenges, pills, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries, or the like. In some embodiments, non-enteral or localized dosage forms may be, but are not limited to, injections, sterile powders, external preparations, or the like. In some embodiments, injections may be administered via subcutaneous injection, intraepidermal injection, intradermal injection, or intralesional injection.

[0064] In some embodiments, the aforementioned pharmaceutical product may comprise a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing techniques. In some embodiments, a pharmaceutically acceptable carrier may be one or more of the following: solvent, buffer, emulsifier, suspending agent, decomposer, disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome, and the like. The type and quantity of carrier selected fall within the scope of professional competence and routine technique of a person skilled in this art. In some embodiments, the solvent that serves as a pharmaceutically acceptable carrier may be water, normal saline, phosphate-buffered saline (PBS), or an aqueous solution containing alcohol.

[0065] In some embodiments, any of the aforementioned prebiotic compositions may be an edible product. In other words, the edible product contains specific amounts of fermented kiwifruit, inulin, and fructooligosaccharides in specific proportions. In some embodiments, the edible product may be a general food, health food, or dietary supplement.

[0066] In some embodiments, the aforementioned edible products may be manufactured into dosage forms suitable for oral administration using techniques known to those skilled in the art. In some embodiments, the aforementioned general food may be the edible product itself. In some embodiments, general food may be, but is not limited to, beverages, fermented foods, bakery products, or seasonings.

[0067] In some embodiments, the resulting prebiotic composition can be further used as a food additive to prepare a food composition containing a prebiotic composition prepared from kiwifruit ferment, inulin, and fructooligosaccharides in specific proportions. Thus, the prebiotic composition of any embodiment can be added during the preparation of raw materials or during the food manufacturing process using conventional methods, and can be formulated with any edible material to produce an edible product (i.e., a food composition) for human and non-human animal consumption.

[0068] Example 1: Preparation of Kiwi Fermented Products

[0069] First, thoroughly wash the kiwifruit (Actinidia deliciosa) and crush the whole fruit into kiwifruit particles with a diameter of less than 12mm. Mix the kiwifruit particles with water at a ratio of 1:5 to obtain a raw material mixture. Then, based on the total weight of the raw material mixture, add 7.5% glucose to the raw material mixture and extract the mixture at 95°C for 0.5 hours to obtain the kiwifruit culture medium. At this point, the sugar content of the culture medium is 8.0°Bx.

[0070] After the culture medium cooled to room temperature, the fermentation process was initiated. First, 0.1% of *Saccharomyces cerevisiae* (purchased from the Bioresource Conservation and Research Center (BCRC), Food Industry Development Research Institute, registration number BCRC20271) was added to the kiwifruit culture medium, and fermentation was carried out at 30°C for one day to form the initial fermentation broth. Then, 0.05% of *Streptococcus thermophilus* (purchased from BCRC, registration number BCRC910636) was added to the initial fermentation broth, and fermentation was carried out at 30°C for one day to obtain the kiwifruit fermentation broth. The specifications for this kiwifruit fermentation broth were set as follows: pH value 3.5 ± 0.3, sugar content less than 4.0°Bx. Fermentation was considered complete when these specifications were met. At this point, most of the sugar in the kiwifruit fermentation broth had been depleted.

[0071] Next, the kiwifruit fermentation liquid was filtered through a 200-mesh sieve to remove fruit residue from the fermentation juice, and then freeze-dried and pulverized to obtain the kiwifruit fermentation product.

[0072] Example 2: The effect of a single ingredient on the growth of probiotics

[0073] Here, the single component refers to the kiwifruit ferment, fructooligosaccharide (purchased from Meiji), and inulin (purchased from Cosucra) prepared in Example 1. The probiotic refers to *Parabacteroides goldsteinii* (purchased from ATCC). The liquid culture medium used was Tryptone Soy Broth (TSB; hereinafter referred to as TSB medium) with 5% sheep blood (purchased from BD).

[0074] The groups were divided into a control group and three experimental groups. The control group used a simple liquid culture medium (95% TSB medium and 5% sheep blood), while the three experimental groups were a kiwifruit fermentation group, an inulin group, and a fructooligosaccharide group, each containing a single component to be tested. Specifically, the kiwifruit fermentation group used a medium consisting of 94% TSB medium, 5% sheep blood, and 1% of the kiwifruit fermentation prepared in Example 1. The inulin group used a medium consisting of 94% TSB medium, 5% sheep blood, and 1% inulin. The fructooligosaccharide group used a medium consisting of 94% TSB medium, 5% sheep blood, and 1% fructooligosaccharides.

[0075] 1% of the activated *Pseudomonas gondii* was added to 15 mL test tubes containing 5 mL of culture medium for each group, and the tubes were anaerobically cultured at 37°C for 48 hours. After 48 hours of culture, 100 μL of each bacterial culture was transferred to solid TSB medium containing 5% sheep blood, and the colony-forming units (CFU) for each group were calculated. The CFU of the control group was considered as 100% of the growth rate, and the corresponding growth rates (%) for the other groups were calculated.

[0076] Please see Figure 1 The growth rate of the control group without the single ingredient to be tested was 100%, while in the other three experimental groups, the growth rate of the kiwifruit fermentation group was 106%, the growth rate of the inulin group was 107%, and the growth rate of the fructooligosaccharide group was 100%. This indicates that 1% fructooligosaccharide does not promote the growth of *Parabacterium goeringii*, but 1% inulin and kiwifruit fermentation can both increase its bacterial count and enhance its growth capacity.

[0077] Example 3: The effect of different components on the growth of probiotics

[0078] The prebiotic ingredients used included inulin (purchased from Cosucra), xylooligosaccharide (purchased from Shandong Longli Biotechnology Co., Ltd.), fructooligosaccharide (purchased from Meiji), pomegranate enzyme, and the kiwifruit ferment prepared in Example 1. The liquid culture medium used was tryptone soybean broth (TSB; hereinafter referred to as TSB medium) with 5% sheep blood (purchased from BD).

[0079] The process for producing pomegranate enzyme is as follows: Pomegranates (Punica granatum) are thoroughly washed and the entire fruit is crushed into particles smaller than 12mm. The pomegranate particles are then mixed with water at a ratio of 1:5 to obtain a raw material mixture. Next, based on the total weight of the raw material mixture, 7.5% glucose is added to the mixture, and the mixture is extracted at 95℃ for 0.5 hours to obtain a pomegranate culture medium. At this point, the sugar content of the culture medium is 8.0°Bx. After the culture medium cooled to room temperature, the fermentation process was initiated. First, 0.1% of *Saccharomyces cerevisiae* (purchased from the Bioresource Conservation and Research Center (BCRC), Food Industry Development Research Institute, registration number BCRC20271) was added to the pomegranate culture medium, and fermentation was carried out at 30°C for one day to form the initial fermentation broth. Then, 0.05% of *Streptococcus thermophilus* (purchased from BCRC, registration number BCRC910636) was added to the initial fermentation broth, and fermentation was carried out at 30°C for one day to obtain the pomegranate fermentation broth. The specifications for this pomegranate fermentation broth were set as follows: pH 3.5 ± 0.3, sugar content less than 4.0°Bx. Fermentation was considered complete when these specifications were met. At this point, most of the sugar in the pomegranate fermentation broth had been depleted. Next, the pomegranate fermentation liquid was filtered through a 200-mesh sieve to remove fruit residue from the fermentation juice, and then freeze-dried and pulverized to obtain pomegranate enzyme.

[0080] The groups were divided into a control group, an experimental group, and seven control groups (control groups A to G). The composition of the culture medium used in each group is shown in Table 1.

[0081] Table 1

[0082]

[0083] As shown in the table above, except for the control group which used only liquid culture medium (i.e., 95% TSB medium + 5% sheep blood), the culture medium for the other experimental groups and control groups (control groups A~G) was 94% TSB medium + 5% sheep blood + 1% prebiotic combination (experimental group prebiotic combination (i.e., prebiotic composition), prebiotic combinations A~G). Taking the experimental group's experimental culture medium as an example, 100mL of experimental culture medium contains 99mL of liquid culture medium (94mL of TSB medium and 5mL of sheep blood) and 1mL of experimental group prebiotic combination. The 1mL of experimental group prebiotic combination consists of 400μL of 10% kiwifruit fermentation product, 300μL of 10% fructooligosaccharide solution, and 300μL of 10% inulin solution. The culture media for the other groups follow the same principle.

[0084] 1% of the activated *Pseudomonas gondii* was added to 15 mL test tubes containing 5 mL of culture medium for each group, and the tubes were anaerobically cultured at 37°C for 48 hours. After 48 hours of culture, 100 μL of each bacterial culture was transferred to solid TSB medium containing 5% sheep blood, and the colony-forming units (CFU) for each group were calculated. The CFU of the control group was considered as 100% of the growth rate, and the corresponding growth rates (%) for the other groups were calculated.

[0085] Please see Figure 2 The growth rate of the control group without the added prebiotic combination was 100%, while the growth rates of the other groups were as follows: experimental group 409.8% (nearly 4.1 times), control group A 207.7%, control group B 72.7%, control group C 80.8%, control group D 35.3%, control group E 124.1%, control group F 209.4%, and control group G 227.3%. This indicates that the prebiotic combination consisting of 40% kiwifruit ferment, 30% inulin, and 30% fructooligosaccharides can significantly improve the growth ability of *Parabacterium goeringii*, increasing its bacterial count.

[0086] Therefore, specific combinations of prebiotics can effectively and significantly enhance the growth capacity of probiotics (e.g., Parabacterium griseus), and not all common combinations of prebiotics (e.g., inulin, fructooligosaccharides, xylooligosaccharides, etc.) can effectively enhance the growth capacity of any probiotic (e.g., Parabacterium griseus).

[0087] Example 4: The effect of different proportions of prebiotic compositions on probiotic growth

[0088] The prebiotic combination used here consisted of inulin (purchased from Cosucra), fructooligosaccharides (purchased from Meiji), and the kiwifruit fermentation product prepared in Example 1. The probiotic was Parabacteroides goldsteinii (purchased from ATCC). The liquid culture medium used was Tryptone Soy Broth (TSB; hereinafter referred to as TSB medium) with 5% sheep blood (purchased from BD).

[0089] The groups were divided into a control group and four experimental groups (experimental groups a~d). The composition of the culture medium used in each group is shown in Table 2.

[0090] Table 2

[0091]

[0092] As shown in the table above, the weight ratio of kiwifruit ferment, fructooligosaccharides, and inulin in each group is 3-4:2.5-4:2.5-4.

[0093] 1% of the activated *Pseudomonas gondii* was added to 15 mL test tubes containing 5 mL of culture medium for each group, and the tubes were anaerobically cultured at 37°C for 48 hours. After 48 hours of culture, 100 μL of each bacterial culture was transferred to solid TSB medium containing 5% sheep blood, and the colony-forming units (CFU) for each group were calculated. The CFU of the control group was considered as 100% of the growth rate, and the corresponding growth rates (%) for the other groups were calculated.

[0094] Please refer to Figure 3 The growth rate of the control group without the added prebiotic combination was 100%, while the growth rates of the other groups were: experimental group a 312.0% (nearly 3.12 times), experimental group b 354.0% (nearly 3.54 times), experimental group c 409.8% (nearly 4.1 times), and experimental group d 388.0% (nearly 3.88 times). In other words, the prebiotic combination composed of kiwifruit ferment, fructooligosaccharides, and inulin can effectively increase the growth capacity of *Parabacterium goeringii* by at least 3 times. Furthermore, when the proportion of the prebiotic combination is 40% kiwifruit ferment, 30% inulin, and 30% fructooligosaccharides, it can significantly increase the growth capacity of *Parabacterium goeringii* by at least 4 times.

[0095] Therefore, a certain proportion and specific types of prebiotic combinations can effectively and significantly improve the growth capacity of probiotics (such as Parabacterium goeringii).

[0096] Example 5: Human Experimentation

[0097] To further confirm the effects of a specific type of prebiotic composition on the human body, eight subjects were given capsules containing 400 mg of a prebiotic composition, consisting of kiwifruit ferment, inulin (purchased from Cosucra), and fructooligosaccharides (purchased from Meiji) prepared in Example 1 in a weight ratio of 4:3:3. Each subject took two capsules daily for four weeks. In other words, the daily dose per subject was 800 mg of the prebiotic composition. Blood and stool samples were collected, questionnaires were administered, and body composition was measured at week 0 (before administration), week 2 (after two weeks of administration), and week 4 (after four weeks of administration).

[0098] The blood test was to measure the content of intestinal barrier proteins. The basic structure of the intestinal barrier is composed of intestinal epithelial cells made up of tight structural proteins, also known as tight junctions (TJs). Therefore, tight structural proteins (such as CLDN3 protein and OCLN protein) are also called intestinal barrier proteins. The indicator protein in this blood test was CLDN3 protein. It contains four transmembrane structures and is a major component of the tight junction structure.

[0099] Fecal sampling was used to determine the growth capacity of intestinal flora. The bacterial species tested in this study were *Kermania pseudomallei*, *Parabacteroides gossypii*, and *Bifidobacterium*.

[0100] The questionnaire feedback included feedback on bowel movement bacteria.

[0101] Body composition measurements include weight, body fat percentage, and waist circumference.

[0102] Example 5-1. Analysis of CLDN3 protein content in the blood of subjects

[0103] Eight subjects had 6 mL of venous blood collected from each subject before taking the prebiotic composition (week 0) and after taking the prebiotic composition (weeks 2 and 4) using purple-tipped blood collection tubes containing EDTA anticoagulant. The results were then sent to TCI GENE for performance analysis of the intestinal shielding protein content in the blood.

[0104] Therefore, the intestinal shielding protein detected was CLDN3 protein.

[0105] Please see Figure 4At week 0, the average concentration of CLDN3 protein in the blood of the 8 subjects was 8.9 ng / mL. After 2 weeks of taking the prebiotic combination, the average concentration of CLDN3 protein in the blood increased to 9.96 ng / mL, and after 4 weeks of taking the prebiotic combination, the average concentration of CLDN3 protein in the blood increased to 12.70 ng / mL. That is, after 2 weeks of taking the prebiotic combination, the average concentration of CLDN3 protein in the blood increased by 1.06 ng / mL, and after 4 weeks of taking the prebiotic combination, the average concentration of CLDN3 protein in the blood increased by 3.8 ng / mL. Therefore, taking the prebiotic combination can effectively increase the concentration of CLDN3 protein, thereby improving intestinal barrier function.

[0106] Example 5-2. Analysis of the gut microbiota of the subjects

[0107] Stool samples were collected from eight subjects before taking the prebiotic combination (week 0) and after taking the prebiotic combination (weeks 2 and 4). The fecal microbiota was analyzed by BIOTOOLS (NGS sequencing of 16S rRNA V3-V4). The analysis results from the eight subjects are summarized as follows: Figures 5 to 7 As shown.

[0108] The bacterial species analyzed were Akkermansia muciniphila (AKK), Parabacteroides goldsteinii (PG), and Bifidobacterium (BF).

[0109] Please see Figure 5The average AKK bacteria count of the eight subjects at week 0 was considered as 1, and the average AKK bacteria count ratio of the eight subjects at weeks 2 and 4 was calculated based on this. Thus, the average AKK bacteria count ratio of the eight subjects at week 2 was 3.44, and the average AKK bacteria count ratio at week 4 was 4.87. This means that after four weeks of continuous use of the prebiotic composition, the AKK bacteria count in the intestines of the eight subjects increased by 4.87 times. AKK bacteria metabolites can suppress the host's appetite and induce the expression of the host's FIAF (Fasting-Induced Adipose Factor) gene, thereby reducing the host's ability to store fat. Furthermore, the increased AKK bacteria count in the intestines can reverse obesity caused by a high-fat diet, reduce the concentration of lipopolysaccharides from harmful bacteria in the blood, thereby reducing chronic inflammation, and alleviate insulin resistance. Therefore, by taking a prebiotic combination composed of kiwifruit ferment, inulin and fructooligosaccharides, the amount of AKK bacteria in the user's intestines can be effectively increased, thereby achieving multiple effects such as weight loss, reducing chronic inflammation, and alleviating insulin resistance.

[0110] Please see Figure 6 The average PG bacteria count of the 8 subjects at week 0 was considered as 1, and the average PG bacteria count ratio of the 8 subjects at weeks 2 and 4 was calculated based on this. Thus, the average PG bacteria count ratio of the 8 subjects at week 2 was 3.76, and the average PG bacteria count ratio at week 4 was 1.93. This means that after 4 weeks of continuous use of the prebiotic composition, the PG bacteria count in the gut of the 8 subjects increased by 1.93 times. Furthermore, the increase in PG bacteria helps improve metabolic syndrome, reduces leaky gut, and improves intestinal inflammation. Specifically, when the PG bacteria count in the gut increases, it can reduce host weight, alleviate host insulin resistance, improve host fat metabolism, and activate brown cells in the host, thereby achieving a weight loss effect. Therefore, by taking the prebiotic composition composed of kiwifruit ferment, inulin, and fructooligosaccharides, the PG bacteria count in the user's gut can be effectively increased, thereby achieving multiple effects such as weight loss, reducing leaky gut, and improving intestinal inflammation.

[0111] Please see Figure 7The average BF bacteria count of the 8 subjects at week 0 was considered as 1, and the average BF bacteria count ratio of the 8 subjects at weeks 2 and 4 was calculated based on this. Thus, the average BF bacteria count ratio of the 8 subjects at week 2 was 3.33, and the average BF bacteria count ratio at week 4 was 3.27. That is to say, after 4 weeks of continuous use of the prebiotic composition, the BF bacteria count in the intestines of the 8 subjects increased by 3.27 times. Furthermore, BF bacteria, as a physiologically beneficial bacterium, have many important physiological functions for host health, including improving the biological barrier, enhancing nutritional function, anti-tumor effects, immune enhancement, improving gastrointestinal function, and anti-aging. Therefore, by taking the prebiotic composition composed of kiwifruit ferment, inulin, and fructooligosaccharides, the BF bacteria count in the user's intestines can be effectively increased, thereby achieving multiple effects such as improving biological barrier capacity, enhancing nutrient absorption, anti-tumor effects, enhancing immunity, and improving gastrointestinal function.

[0112] Example 5-3. Analysis of the subject's bowel movement questionnaire

[0113] Eight participants underwent a questionnaire survey to assess their bowel movements before taking the prebiotic composition (week 0) and after taking the composition (weeks 2 and 4). The questionnaire feedback results from the eight participants were compiled as follows: Figures 8 to 11 As shown.

[0114] The questionnaire items analyzed included: degree of defecation difficulty (e.g., frequency of defecation, time required for defecation, and whether defecation was complete) and frequency of intestinal peristalsis, as shown in Table 3.

[0115] Table 3

[0116]

[0117] Please see Figure 8 Before taking the prebiotic composition at week 0, 4 out of 8 subjects (50%) had bowel movements more than once every two days. However, after 2 weeks of taking the prebiotic composition, the number of subjects with bowel movements more than once every two days dropped to 1, with 4 subjects having bowel movements once a day and 3 subjects having bowel movements more than twice a day. After 4 weeks of taking the prebiotic composition, all subjects (100%) had bowel movements daily, with 5 subjects having bowel movements once a day and 3 subjects having bowel movements more than twice a day. This indicates that taking the prebiotic composition can improve the frequency of bowel movements (to at least once a day), thus making it easier for the subjects to clear accumulated stool.

[0118] Please see Figure 9Before taking the prebiotic composition in week 0, 1 out of 8 subjects required 10-20 minutes to defecate, 4 subjects required 5-10 minutes, and 3 subjects required less than 5 minutes. However, after 2 weeks of taking the prebiotic composition, no subject required more than 10 minutes to defecate, and 5 subjects required 5-10 minutes, while 3 subjects required less than 5 minutes. Furthermore, after 4 weeks of taking the prebiotic composition, the results were the same as in week 2: no subject required more than 10 minutes to defecate, and 4 out of 8 subjects required 5-10 minutes and 4 required less than 5 minutes. In other words, 50% of the subjects required less than 5 minutes to defecate, and 100% of the subjects required less than 10 minutes. Therefore, it can be seen that taking a prebiotic combination can improve the defecation time of the receptor, thereby avoiding problems caused by prolonged sitting on the toilet (such as numbness in the legs and nerve compression), and can reduce the time spent using the toilet.

[0119] Please see Figure 10 Before taking the prebiotic composition at week 0, 50% of the 8 participants felt "sometimes" incomplete bowel movements, 12.5% ​​felt "frequently" incomplete bowel movements, only 25% felt "rarely" incomplete bowel movements, and 12.5% ​​felt "no" incomplete bowel movements. After 2 weeks of taking the prebiotic composition, 50% of the participants felt "rarely" incomplete bowel movements, 12.5% ​​felt "no" incomplete bowel movements, and no participants felt "frequently" incomplete bowel movements. Furthermore, only 37.5% of the participants felt "sometimes" incomplete bowel movements. After 4 weeks of taking the prebiotic composition, 62.5% of the participants felt "rarely" incomplete bowel movements, 25% felt "no" incomplete bowel movements, and no participants felt "frequently" incomplete bowel movements. Furthermore, only 12.5% ​​of the participants felt "sometimes" incomplete bowel movements. In other words, taking a prebiotic combination can reduce the feeling of incomplete bowel movements, thereby making the recipient feel that bowel movements are smooth.

[0120] Please see Figure 11Before taking the prebiotic composition at week 0, none of the 8 subjects experienced frequent bowel movements, while 1 subject experienced no bowel movements. Two subjects rarely experienced bowel movements, and 5 subjects sometimes experienced them. After 2 weeks of taking the prebiotic composition, 2 subjects frequently experienced bowel movements, no subjects did not experience any bowel movements, and a total of 6 subjects rarely or sometimes experienced them. After 4 weeks of taking the prebiotic composition, 3 subjects frequently experienced bowel movements, no subjects did not experience any bowel movements, 3 subjects sometimes experienced them, and 2 subjects rarely experienced them. Therefore, all 8 subjects experienced an increased frequency of bowel movements after 4 weeks of taking the prebiotic composition. Therefore, it can be seen that taking the prebiotic combination can promote the frequency of intestinal peristalsis of the receptor, thereby improving the intestines and defecation, and making the receptor defecation smooth.

[0121] Example 5-4. Analysis of Subject Body Composition

[0122] Eight subjects had their body composition measured using a body fat analyzer (brand: TANITA BC-601FS) and measuring tape before taking the prebiotic composition (week 0) and after taking the composition (weeks 2 and 4). The body composition results of the eight subjects were then compiled as follows: Figures 12 to 15 As shown.

[0123] The body composition items analyzed here include: weight, total body fat percentage, trunk body fat percentage, and waist circumference.

[0124] Please see Figure 12 Before taking the prebiotic composition at week 0, the average weight of the 8 subjects was 73.6 kg. After 2 weeks of taking the prebiotic composition, the average weight of the 8 subjects decreased to 72.7 kg. Furthermore, after 4 weeks of taking the prebiotic composition, the average weight of the 8 subjects further decreased to 72.5 kg. In other words, the subjects' average weight decreased by 0.9 kg after 2 weeks of taking the prebiotic composition, and by 1.1 kg after 4 weeks. This demonstrates that taking the prebiotic composition composed of kiwifruit ferment, inulin, and fructooligosaccharides can effectively reduce the subjects' weight, achieving a weight loss effect.

[0125] Please see Figure 13Before taking the prebiotic composition at week 0, the average total body fat percentage of the 8 subjects was 32%. After 2 weeks of taking the prebiotic composition, the average total body fat percentage of the 8 subjects decreased to 31.5%. Furthermore, after 4 weeks of taking the prebiotic composition, the average total body fat percentage of the 8 subjects further decreased to 31.2%. In other words, the subjects' average total body fat percentage decreased by 0.5% after 2 weeks of taking the prebiotic composition, and decreased by 0.8% after 4 weeks. This demonstrates that taking the prebiotic composition composed of kiwifruit ferment, inulin, and fructooligosaccharides can effectively reduce the subjects' total body fat percentage, achieving a weight loss effect.

[0126] Please see Figure 14 Before taking the prebiotic composition at week 0, the average trunk body fat percentage of the 8 subjects was 33.6%. After 2 weeks of taking the prebiotic composition, the average trunk body fat percentage of the 8 subjects decreased to 32.9%. Furthermore, after 4 weeks of taking the prebiotic composition, the average trunk body fat percentage of the 8 subjects further decreased to 32.7%. In other words, the subjects' average trunk body fat percentage decreased by 0.7% after 2 weeks of taking the prebiotic composition, and decreased by 0.9% after 4 weeks. This indicates that taking the prebiotic composition composed of kiwifruit ferment, inulin, and fructooligosaccharides can effectively reduce the subjects' trunk body fat percentage, achieving a weight loss effect.

[0127] Please see Figure 15 Before taking the prebiotic composition at week 0, the average waist circumference of the 8 subjects was 91.9 cm. After 2 weeks of taking the prebiotic composition, the average waist circumference of the 8 subjects decreased to 89.4 cm. Furthermore, after 4 weeks of taking the prebiotic composition, the average waist circumference of the 8 subjects further decreased to 87.9 cm. In other words, the subjects' average waist circumference decreased by 1.7 cm after 2 weeks of taking the prebiotic composition, and by 3.2 cm after 4 weeks. This demonstrates that taking the prebiotic composition composed of kiwifruit ferment, inulin, and fructooligosaccharides can effectively reduce the subjects' waist circumference, thereby achieving a weight loss effect.

[0128] Furthermore, one of the subjects underwent changes in abdominal fat measurement using a bone mineral density and body composition analyzer (DXA) at weeks 0 and 4. Figure 16 As shown. FA0 represents the subject's abdominal fat at week 0. Figure 16 The white area above the spine on both sides in the left image represents the subject's abdominal fat at week 4. Figure 16The white area above the spine on both sides (right image) shows a significant reduction in abdominal fat in this subject when comparing the sizes of FA0 and FA4. In other words, consuming a prebiotic combination composed of kiwifruit ferment, inulin, and fructooligosaccharides can effectively reduce the recipient's abdominal fat, thereby achieving weight loss.

[0129] In summary, the prebiotic composition comprising kiwifruit ferment, inulin, and fructooligosaccharides according to any embodiment of the present invention can be used to improve the intestinal tract of a recipient. The weight ratio of kiwifruit ferment, inulin, and fructooligosaccharides in the prebiotic composition is 3-4:2.5-4:2.5-4. In some embodiments, the prebiotic composition comprising kiwifruit ferment, inulin, and fructooligosaccharides can be used to improve the intestinal flora (e.g., Kermansia myxobolus, Parabacterium goeringii, and Bifidobacterium), improve the recipient's bowel movements (e.g., improve the recipient's difficulty in defecation, promote the recipient's intestinal peristalsis frequency, reduce the recipient's incomplete defecation, reduce the recipient's defecation time, or combinations thereof), enhance the recipient's intestinal barrier (e.g., increase the concentration of tight structural proteins in the recipient's body), or combinations thereof. Furthermore, in some embodiments, the prebiotic composition comprising kiwifruit ferment, inulin, and fructooligosaccharides can be used to reduce the recipient's weight, waist circumference, body fat percentage, or combinations thereof, thereby achieving weight loss.

[0130] Although the technical content of the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any modifications and refinements made by those skilled in the art without departing from the spirit of the present invention should be included within the scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

[0131] Of course, the present invention may 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 modifications according to the present invention, but these corresponding changes and modifications should all fall within the protection scope of the claims of the present invention.

Claims

1. The use of a fermented kiwi fruit product and sugars in the preparation of a prebiotic composition for assisting receptor weight loss, characterized in that, The prebiotic composition includes kiwifruit ferment, inulin, and fructooligosaccharides, and the weight ratio of the kiwifruit ferment, inulin, and fructooligosaccharides is 3-4:2.5-4:2.5-4.

2. The use according to claim 1, characterized in that, The preparation process of the kiwifruit ferment includes: mixing whole kiwifruit with water and extracting at 80°C to 100°C for 0.5 to 1.5 hours to obtain kiwifruit extract; and fermenting with 0.1% brewer's yeast and 0.05% thermophilic streptococci in sequence for 0.5 to 2 days to form the kiwifruit ferment.

3. The use according to claim 1, characterized in that, The weight ratio of the kiwifruit ferment, the inulin, and the fructooligosaccharide is 4:3:

3.

4. The use according to claim 1, characterized in that, The prebiotic composition is used to reduce the recipient's weight, waist circumference, total body fat percentage, trunk body fat percentage, abdominal fat, or a combination thereof.

5. The use according to claim 1, characterized in that, The prebiotic composition is used to suppress the appetite of the receptor.

6. The use according to claim 1, characterized in that, The prebiotic composition is used to reduce fat storage in the receptor.

7. The use according to claim 1, characterized in that, The prebiotic composition is used to enhance the lipid metabolism of the receptor.

8. The use according to claim 1, characterized in that, The prebiotic composition is used to activate the brown cells of the receptor.

9. The use according to claim 1, characterized in that, The probiotic composition is used to increase the content of Kermania gravidarum and Parabacterium goeringii in the recipient's intestine.

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