KR102401050B1 - Functional food evaluation method and kit - Google Patents

Abstract

After fermenting functional food using simulated digestion and fecal microorganisms, it provides a method for evaluating food by analyzing changes in microbial ecology or changes in short-chain fatty acids.

Description

Functional food evaluation method and kit {Functional food evaluation method and kit}

The present invention relates to a functional food evaluation method and a kit using the same. Specifically, it relates to a method of evaluating the functionality of food by artificially digesting food and fermenting it using fecal microorganisms to analyze the effect of food on changes in the intestinal environment by analyzing microorganisms or short chain fatty acids (SCFA) .

Functional health food is a food manufactured using raw materials or ingredients (hereinafter referred to as functional raw materials) that are easily deficient in daily meals or have useful functions to the human body and are foods that help maintain health. The Ministry of Food and Drug Safety evaluates scientific evidence such as animal testing and human application testing to recognize functional ingredients. However, the same effect cannot be obtained even if the same food is consumed depending on the individual intestinal microflora.

The aggregate of all microorganisms naturally present in the human body is called the human microbiome. Human microbiota exist in various parts of the body, such as the skin, oral cavity, teeth, genitals, respiratory tract, and gastrointestinal tract, but the gastrointestinal tract contains the largest and most diverse types of microorganisms.

In general, intestinal bacteria are in a certain balanced state. However, if various factors that disturb the balance, such as a large amount of meat intake, abuse of antibiotics, stress, etc., are applied over a long period of time or with aging, the activity of restoring the flora to a normal state becomes weak. When the balance of the intestinal flora is disrupted, bifidobacterium , a representative beneficial bacteria in the intestinal flora, decreases, and representative harmful bacteria such as Clostridium and pathogenic E. coli increase.

In the short term, when pathogens invade or abnormal growth of harmful bacteria occurs, diarrhea may occur as a phenomenon of excretion. Various decomposing substances or carcinogens produced combine to decrease immunity, and various adult diseases such as hyperlipidemia, arteriosclerosis, and cancer and aging are easy to occur.

The gut microbes break down foods that are difficult for humans to digest into digestible forms. By decomposing plant polysaccharides that cannot be broken down by human enzymes, short chain fatty acids (SCFAs) such as acetate, propionate, and butyrate are produced. These products have a beneficial effect on health by performing various functions related to the maturation and development of the mucosal immune system, interaction with the nervous system, and maintenance of homeostasis in the body, in addition to regulating metabolic functions such as securing energy and helping digestion.

Research that reveals the relationship between the intestinal microbiota and other diseases such as atopic dermatitis, obesity, cancer, and diabetes is progressing rapidly, and the market is predicting that personalized medicine using the microbiome will become a reality within a few years.

However, the market for personalized functional food based on intestinal microbes is progressing insignificantly. Accordingly, there is a need to develop a system for developing personalized functional food based on intestinal microbes.

Republic of Korea Patent No. 10-1875311

An object of the present invention is to prepare a digestion product by adding artificial digestive juice to a target food, to prepare a fermentation product by adding fecal microorganisms to the digestion product, and the base sequence of the V4 variable region of the 16S rDNA gene in the fermentation product It is to provide a functional food evaluation method comprising the steps of analyzing the microbial ecology and comparing the microbial ecology of the target food fermented product and the indicator beneficial bacteria or indicator harmful bacteria of the standard food fermented product.

Another object of the present invention is to provide a kit for functional food evaluation comprising an artificial digestive juice, an intestinal composition medium and fecal microorganisms.

As one aspect for achieving the above object, the present invention provides a method for evaluating functional food.

The evaluation method of the functional food includes the steps of adding an artificial digestive juice to the target food to prepare a digestive product, adding a fecal microorganism to the digestive product to prepare a fermentation product, and in the fermentation product, the V4 variable region of the 16S rDNA gene. A step of confirming the microbial ecology by analyzing the nucleotide sequence and comparing the microbial ecology of the target food fermented product with the indicator beneficial bacteria or indicator harmful bacteria of the standard food fermented product.

The step of preparing the digestion product is a process of artificially digesting the target food by adding an artificial digestive juice to the target food. Specifically, in a test tube, simulated saliva is added to the target food to perform a digestion process in the oral cavity, and after gastric digestion is performed by adding simulated gastric juice to the oral digestion product, simulated small intestine fluid is added to the gastric digestion product to add simulated small intestine. It is characterized in that it performs the digestion process in

In the present invention, "target food" means a food that is estimated to have functionality or randomly selected according to a conventional selection method.

In the present invention, "functional" means to maintain, promote, prevent or prevent disease by effectively acting on human health, and specifically increase the specific gravity of beneficial bacteria in the intestine and reduce the specific gravity of harmful bacteria, or By increasing the intestinal production of short-chain fatty acids, it may be to maintain, promote health, prevent or prevent disease.

In the present invention, "artificial digestive fluid" includes simulated saliva, simulated gastric juice, and simulated small intestine fluid artificially prepared to reproduce the digestive process in a test tube. The artificial digestive fluid includes digestive enzymes secreted from each oral cavity, stomach or small intestine.

In an embodiment of the present invention, simulated saliva was put in a test tube blocked from light and oral digestion was carried out for 2 minutes. . Next, simulated small intestine fluid was added to the above digestion product, and the digestion process was performed for 2 hours.

Therefore, the digestion product of the present invention is the result of sequentially digesting the target food with the digestive juices of the oral cavity, stomach, and small intestine.

The step of preparing the fermentation product is a step of fermenting by adding a fecal-derived microorganism to the digestion product, and it is characterized in that the digestion product and the fecal microorganism are anaerobically cultured using an intestinal composition medium.

In one embodiment of the present invention, human feces collected on the day of fecal fermentation were suspended in PBS, suspended matter was removed, and only fecal microorganisms were collected and used. The PBS was used by removing all oxygen in the anaerobic chamber.

Therefore, the "fecal microorganism" of the present invention may be a microorganism collected from feces, which may be obtained by suspending feces in PBS from which oxygen has been removed to remove suspended matter, but is not limited thereto.

Preferably, it is possible to use feces separated from subjects who do not consume functional food. When a functional food is evaluated using fecal microorganisms isolated from a subject, the functional effect of the subject food can be evaluated on an individual subject, which can be usefully used for personal customized food prescription and development.

In an embodiment of the present invention, the fecal microorganism and the intestinal composition medium were added to the digestion product and cultured in an anaerobic chamber. At this time, the intestinal composition medium was used by removing nutrients such as peptone and yeast extract to evaluate only the fermentation caused by food.

Therefore, the fermentation product of the present invention can be prepared by adding fecal microorganisms and intestinal composition medium to the digestion product digested with artificial digestive juice and fermenting it under anaerobic conditions.

The step of identifying the microorganism is a step of isolating DNA from the fermentation product and analyzing the nucleotide sequence of the 16S rDNA gene to analyze the microbial ecology.

The nucleotide sequence of the V4 variable region of the 16S rDNA gene of a known microorganism may be used for the analysis of the microorganism.

In the present invention, "microbial ecology" refers to various microbial populations, communities or groups inhabiting. More specifically, the microbial ecology refers to various microbial populations, communities or groups living in an anaerobic state for producing fermentation products.

In the step of confirming the microbial ecology, based on the base sequence of the 16S rDNA gene obtained in the step of identifying the microorganism, the microorganisms present in the environment are classified according to Taxonomy (Genus) level or species (Species) level It is a step to classify and confirm the distribution ratio of each microorganism.

In the step of comparing the indicator beneficial bacteria or indicator harmful bacteria, the beneficial bacteria or harmful bacteria of the fermented product of the target food and the indicator beneficial bacteria or indicator harmful bacteria of the fermented product of the standard food using the microbial distribution analysis result analyzed in the step of confirming the microbial ecology is a step to compare Specifically, it may be to compare the abundance of beneficial bacteria in the fermented product of the target food and the abundance of beneficial bacteria in the index of the fermented standard food, and compare the abundance of harmful bacteria in the fermented product of the target food and the abundance of harmful bacteria in the index of the fermented standard food. it could be

In the present invention, "beneficial bacteria" are bacteria that have a beneficial effect on human health when ingested, and may promote the production of short-chain fatty acids in the intestine or inhibit the intestinal proliferation of harmful bacteria by producing lactic acid.

In the present invention, "harmful bacteria" refers to bacteria known to cause diseases by accumulating toxins and waste products including ammonia, hydrogen emulsion, and lipid peroxide in the intestine.

In the present invention, "indicator beneficial bacteria" means beneficial bacteria contained in the fermented product of standard food.

In the present invention, "indicator harmful bacteria" means harmful bacteria included in the fermented product of standard food.

In one embodiment of the present invention, after fermenting only fecal microorganisms, it was confirmed that Chao 1 decreased after 6 hours to decrease species richness, and Shannon also decreased after 6 hours, resulting in species uniformity (species richness). evenness) was confirmed to decrease.

In another embodiment of the present invention, as a result of analyzing the change in microbial ecology according to the fermentation time of food in fermented products prepared using commercially available cheonggukjang in a test tube, it was confirmed that food affects the microbial ecology from 2 hours after fermentation. As a result of non-metric multidimensional scaling (NMDS), it was confirmed that the distribution shifted to the right as the fermentation time increased for both the commercially available cheonggukjang fermented product and the fermented product fermented only with fecal microorganisms. This is judged to be a change in the microbial ecology due to in vitro digestion and fermentation. As a result of comparing the microorganisms of commercially available cheonggukjang and fermented products of fecal microorganisms, the red arrow shows a tendency to move in the upward direction (upward direction), so food affects the microbial ecology. was confirmed to be crazy.

That is, it was confirmed that the effect of the target food on the microbial ecology of the intestine can be simulated by fermenting the digested product of the target food with fecal microorganisms.

As a result of analyzing the microbial ecology before and after fermentation for 2 hours in the fermentation product of fecal microorganisms in an embodiment of the present invention, when only feces were fermented for 2 hours, Parabacteroides spp ., Bilophila spp ., Sutterella spp ., Oscillospira spp. was significantly increased, and microorganisms of the Lachnospiraceae and Ruminococcaceae families were significantly decreased.

Next, the abundance of microorganisms in the fermented product of fecal microorganisms fermented for 2 hours and the fermented product of commercial cheonggukjang was analyzed. Collinsella spp ., Bacteroides spp ., Lachnospiraceae , Bifidobacterium spp., Ruminococcus , and Phascolarctobacterium spp . microorganisms were significantly increased in commercial fermented soybean paste, and relatively spp. Anaerostipes spp ., Peptospira spp . , Rikenellaceae , Sutterella spp ., Faecalibacterium spp. , Prevotella spp. It was confirmed that significantly decreased.

In the microbial strain, Bilophila spp . and Sutterella spp . are harmful bacteria, and the Oscillospira spp ., Collinsella spp ., Bacteroides spp ., Bifidobacterium spp. , Phascolarctobacterium spp ., Anaerostipes spp. , Ruminococcus bromii , Faecalibacterium spp . and Prevotella spp. are beneficial bacteria.

Therefore, the beneficial bacteria of the present invention are specifically Oscillospira spp ., Collinsella spp ., Bacteroides spp ., Bifidobacterium spp ., Phascolarctobacterium spp ., Anaerostipes spp ., Ruminococcus bromii , Faecalibacterium spp . And Prevotella spp . It may include any one or more selected from the group consisting of.

In addition, the harmful bacteria of the present invention are specifically Bilophila spp . or Sutterella spp. may include

In the above, it was confirmed that the species of microorganisms having high abundance in the fermented product was changed differently depending on the target food.

Therefore, in the functional food evaluation method of the present invention, when the abundance of beneficial microorganisms analyzed in the fermented product of the target food is the same as or higher than the abundance of the beneficial bacteria analyzed in the fermented product of the standard food , or when the abundance of harmful bacteria of microorganisms analyzed in the fermentation product is the same as or lower than the abundance of the index harmful bacteria analyzed in the fermentation product of standard food, evaluating the target food as a functional food. can do.

In an embodiment of the present invention, fermented products were prepared using galactooligosaccharide (GOS), a type of prebiotics, and cheonggukjang, a type of fermented food, which is a health functional food. and changes in the content of short-chain fatty acids were analyzed.

In another embodiment of the present invention, fecal microorganisms and cheonggukjang isolated from 8 subjects were used for fecal fermentation in a test tube for 2 hours, and changes in the content of short-chain fatty acids and intestinal microorganisms were analyzed.

That is, it was confirmed that even if the same functional food (Chungkukjang) was consumed, the effect on changes in the intestinal environment, specifically short-chain fatty acids (Acetate, Propionate, Butyrate) production change, was different depending on the subject. did Therefore, it is possible to predict changes in the intestinal environment (change in short-chain fatty acid production) for each individual when ingesting functional foods using a simulated fermentation system using fecal microorganisms.

In addition, the intestinal microflora was analyzed and the production route of SCFAs was predicted by linking it with the Picrust result, and the amount of SCFAs (Acetate, Propanoate, Butyrate) produced by cheonggukjang according to each subject and the amount of intestinal microbes that increased or decreased according to it varied. It was confirmed that

Therefore, fecal fermentation of fecal microorganisms separated from the analysis target (individual or individual) is performed, and the change in the intestinal environment (content of intestinal microorganisms and short-chain fatty acids) after food intake is analyzed in a test tube, and each individual (or individual) consumes functional health food. It was confirmed that changes in the market environment can be predicted.

Through prediction of such changes in the intestinal environment, the effects of health functional foods for each individual or individual can be predicted and evaluated.

In addition, it can be used to prescribe health functional food that promotes the production of SCFAs (Acetate, Propanoate, Butyrate) lacking in the intestines of the target object by predicting the production path of SCFAs through microbial analysis.

In an embodiment of the present invention, as a result of separation of feces from each subject and microbial analysis after fecal fermentation, L-Lactate ([S]-Lactate, Lactate, D-Lactate, [R]-Lactate) was produced in all subjects. Collinsella aerofaciens (OTU0034), Lactobacillus spp . (Otu0047), Lactobacillus ruminis (Otu0045), Dorea spp. (Otu0041) and Coprococcus spp . (Otu0008, OTU0012) intestinal microflora increased.

The L-Lactate is converted to Acetate by pyruvate metabolism (metabolism), Megasphaera spp consuming the L-Lactate. (Otu0022) and Bacteroides fragilis (Otu0011) microorganisms were decreased in subjects S7 or S4 in which acetate was not significantly increased.

In addition, referring to the results of FIG. 13 , pyruvate metabolism (metabolism) increased in most of the subjects in which Acetate was significantly increased in the Picrust results (except for S3), and there was no significant difference or decreased in the subjects in which Acetate did not increase. .

In addition, it is thought that pyruvate metabolism was increased in subjects S1, S2, S5 and S8 because L-Lactate produced by intestinal microbes was produced as a succinate material through the process of pyruvate metabolism. . In addition, in S1 and S2 of these subjects, the c5-branched dibasic acid metabolism process was decreased, and as a result, propionate was added to cheonggukjang only in S1 and S2 subjects by propanoate (Propionate) metabolism, which converts propanoyl-CoA to propionate. It was confirmed that there was a significant increase by

Therefore, the indicator beneficial bacteria of the present invention may further include a strain that produces L-Lactate.

The strain producing the L-Lactate is Collinsella aerofaciens (OTU0034), Lactobacillus spp . (Otu0047), Lactobacillus ruminis (Otu0045), Dorea spp . (Otu0041) and Coprococcus spp . (Otu0008, OTU0012) may be at least one selected from the group consisting of.

In addition, the indicator beneficial bacteria may further include strains involved in pyruvate metabolism using L-Lactate, and the strains involved in pyruvate metabolism using L-Lactate are specifically Megasphaera spp . (Otu0022) or Bacteroides fragilis (Otu0011).

As a result of comparison of the indicator beneficial bacteria in the present invention, when the strain producing L-Lactate and the strain involved in pyruvate metabolism using the L-Lactate increase at the same time, it is predicted that the production of short-chain fatty acids in the intestine will increase when the target food is ingested. Or it can be evaluated, specifically, it can be predicted or evaluated by increasing acetate (Acetate) or propionate (Propionate).

In the present invention, "standard food" is a health functional food, and the health functional food may be specifically prebiotics, probiotics, or fermented food. The standard food may specifically include galactooligosaccharide (GOS) or cheonggukjang.

In the present invention, "health functional food" refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients useful for the human body. Here, the term "functionality" refers to obtaining a useful effect for health purposes such as regulating nutrients or physiological action with respect to the structure and function of the human body.

In the present invention, "prebiotics (prebiotics)" refers to substances that serve as a nutrient source for probiotics as an indigestible component that helps the growth of beneficial bacteria in the intestine and helps to improve the intestinal environment. Prebiotics are often composed of carbohydrates like oligosaccharides, and most of them exist in the form of dietary fiber.

In the present invention, "probiotics" refers to bacteria that are beneficial to our body as a generic term for live bacteria that are beneficial to the human body when consumed in an appropriate amount. Most probiotics known to date are lactic acid bacteria. Lactobacillus or beneficial bacteria, which are probiotics, survive in stomach acid and bile acid in the body, reach the small intestine, and multiply and settle in the intestine. To have a beneficial effect on health in the settled intestine, these probiotics must be non-toxic and non-pathogenic.

In the present invention, "fermented food" is a food with improved digestibility and flavor as a result of the physiological activity of lactic acid bacteria, and the production and proliferation of harmful bacterial species (decaying bacteria) is prevented when beneficial bacterial species are dominant in the food, and the storage period is increased. The fermented food may contain a strain that produces a substance helpful for health promotion. Kimchi, plum, sauerkraut (German food fermented with cabbage), soy sauce (soy sauce, soybean paste, gochujang, cheonggukjang), as well as various alcoholic beverages such as sake, beer, wine, soju, vinegar, natto, bread, cheese, yogurt, salted fish etc.

In a temporary embodiment of the present invention, by changing the change of metabolites according to the type of food to 2, 4, 6, 8, 12, 24 hours for fermentation time, pH change and short-chain fatty acids (acetate, butyrate, propionate) ) was measured, and the increase rate of short-chain fatty acids was analyzed compared to before fermentation.

As a negative control (blank), a fermentation product of fecal microorganisms was used, and as a positive control, galactooligosaccharide (GOS), a prebiotic, was used. Fermentation products of fecal microorganisms had a pH of 7.5 to 8 during the fermentation time, and the SCFA increase rate was confirmed that the increase rate of SCFA for 24 hours was 0 or less in the case of propionate and butyrate. and acetate increased from 0 at 12 hours to about 5 at 24 hours.

On the other hand, in the cheonggukjang samples and fermented products of galactooligosaccharide (GOS), it was confirmed that the pH decreased and the amount of acetate, butyrate, and propionate production significantly increased compared to the negative control group.

It is known that as the number of beneficial bacteria increases, the production of acetate, butyrate, and propionate increases, and as the pH decreases, the growth of harmful bacteria is inhibited. Therefore, it is possible to evaluate the effect of improving the intestinal environment of the target food by using the generation of short-chain fatty acids or the change in pH.

Therefore, the indicator beneficial bacteria of the present invention can specifically increase the production of short-chain fatty acids.

The short chain fatty acid (SCFA) may include acetate, butyrate or propionate.

The functional food evaluation method according to the present invention may further include analyzing the content of short-chain fatty acids in the fermented product.

In addition, the step of measuring the pH of the fermentation product and when the measured pH is the same as or lower than the pH value measured in the standard food, it may further include the step of selecting the target food as a functional food.

As another aspect for achieving the above object, the present invention provides a kit for evaluation of functional food.

The functional food evaluation kit of the present invention includes an artificial digestive juice, an intestinal composition medium, and fecal microorganisms.

In the present invention, "functional food evaluation" refers to sequentially digesting target food using artificial digestive juices of the oral cavity, stomach, and small intestine to produce a digested product, and fermenting the digested product in an anaerobic chamber using fecal microorganisms and an intestinal composition medium. By reproducing the digestion and fecal fermentation of functional foods in a test tube, it means simulating the effect on changes in the intestinal environment through digestion and fermentation of food by comparing changes in the composition of microorganisms caused by the target food. The changes in the intestinal environment include changes in microbial ecology, changes in pH, and changes in the amount of short-chain fatty acids produced.

When the proportion of beneficial bacteria increases or the proportion of harmful bacteria decreases in the microbial ecology analyzed in the fermented product of the target food, or when the proportion of bacteria that promotes the production of short-chain fatty acids increases, the target food can be evaluated as a functional food have.

In addition, when the content of short-chain fatty acids in the fermented product of the target food is increased, or in the fermented product, a food whose pH is changed the same as or lower than that of a standard food can be evaluated as a functional food.

The artificial digestive fluid of the present invention includes simulated saliva, simulated gastrointestinal fluid, and simulated small intestine fluid.

The simulated saliva contains digestive enzymes secreted from the oral cavity, the simulated gastrointestinal fluid contains digestive enzymes secreted from the stomach, and the simulated small intestine contains digestive enzymes secreted from the small intestine. Specifically, the simulated saliva may include amylase, the simulated gastrointestinal fluid may include pepsin, and the simulated small intestine fluid may include pancreatin.

The intestinal composition medium of the present invention is characterized in that it is used by removing nutrients such as peptone and yeast extract in order to induce only fermentation caused by food. In addition, the intestinal composition medium is characterized in that oxygen is removed from the anaerobic chamber.

In an embodiment of the present invention, when peptone and yeast extract were included in the intestinal composition medium, it was confirmed that the short-chain fatty acid content was significantly changed from 2 hours of fermentation due to these peptone and yeast extract. Therefore, in order to evaluate the effect of functional food, the intestinal composition medium preferably does not contain peptone and yeast extract, which are energy sources.

Fecal microorganisms are microorganisms collected from fresh feces, and may be obtained by suspending feces in PBS from which oxygen has been removed to remove suspended matter, but is not limited thereto.

The functional food evaluation kit of the present invention may further include an agent for measuring the beneficial indicator bacteria or the indicator harmful bacteria.

The description of the indicator beneficial bacteria and the indicator harmful bacteria of the present invention is the same as described above.

In the present invention, the "measurement agent" includes a primer consisting of a nucleotide sequence complementary to the 16S rDNA gene of the indicator beneficial bacteria or the indicator harmful bacterium. The gene base sequence of the 16S rDNA of the indicator beneficial bacteria or the indicator harmful bacterium is a known base sequence can be used.

The functional food evaluation system of the present invention is a technology that compares and analyzes changes in the microbial ecology of individual fecal microorganisms according to food in vitro, and measures the change in the content of short-chain fatty acids (SCFA). can be evaluated in a short time. Therefore, it is possible to reduce the time and cost required for the development and evaluation of health functional food by replacing the time-consuming and costly animal experimentation. In addition, it is possible to track beneficial microorganisms that are not present in individuals, so it can be used to develop personalized probiotic products.

1 is a schematic diagram of the simulated digestion process. Specifically, A is the oral cavity, B is the stomach, and C is the simulated digestive process of the small intestine.
2 is a result of analyzing ChaoI and Shannon according to fermentation time in Comparative Example 1-1.
3 is a result of analyzing the cluster change of fecal microorganisms according to fermentation time in Comparative Example 1-1.
4 is a comparison result of microbial genus appearing in abundance (abundance) in fecal microorganisms cultured for 0 to 6 hours and 8 to 24 hours.
5 is a result of comparative analysis of changes in fecal microbial ecology according to time according to incubation time in Example 1-2 and Comparative Example 1-1.
6 shows microorganisms appearing in abundance after 0 hours and 2 hours of fermentation in Comparative Example 1-1.
7 is a result of analyzing the microbial ecology of the fermented product according to the type of food after fecal fermentation for 2 hours.
8 is a result of analyzing the pH and short-chain fatty acid increase rate in fermented products according to the type of food for each time.
9 shows the change in the short-chain fatty acid (SCFAs; acetate, propionate, and butyrate) content over time during fecal fermentation according to the energy source contained in the intestinal composition medium. In the bilateral T-test, *p <0.05 vs. 0 hours; § p <0.0.5 before sampling time for each group in a two-sided T-test.
10 is an analysis of SCFAs content changed by cheonggukjang for each subject in vitro. The mean value ± SD was expressed, and the control group (B) for 2 hours (2h) and the cheonggukjang-treated group (T) for 2 hours (2h) were compared with the same subject using one-way ANOVA (* p <0.001).
11 is a result of analyzing the intestinal microorganisms changed by cheonggukjang for each subject through NMDS. NMDS was prepared based on Bray-Curtis. ● Symbols indicate the microbiota of control (B) 0 h and control (B) 2 h fecal fermentation. □ The × symbol in the box means the microorganism total of 2 hours fecal fermentation treated with cheonggukjang.
12 is a comparison of intestinal microbial ecology for each subject using an OTUs-based tree.
Fig. 13 is a linear discriminant analysis effect size (LefSe) including the distinct abundance in metabolism of PICRUSt ((hylogenetic Investigation of Communities by econstruction of Unobserved States)) after fecal fermentation with cheonggukjang. Fecal fermentation for 2 hours is the predicted KEGG functional pathway difference between the control group (blue) and the cheonggukjang-treated group (red) in white, and the white color does not mean significantly different pathways between them.

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

<Example 1> Preparation of fermented products

In imitation of the human digestive system, food decomposition, that is, artificial digestion, was performed in vitro using simulated digestive juices. Foods were digested in turn by adding simulated digestive juices secreted from each digestive system in each artificially created oral, gastrointestinal, and small intestine environment. Thereafter, microorganisms isolated from feces were added to the digestion product and cultured to prepare a fermentation product.

Preparation of simulated digestive juices

The simulated digestive juices were prepared with the compositions shown in Tables 1 to 3.

Table 1 shows the composition of simulated saliva (SSF).

Table 2 shows the composition of simulated gastric fluid (SGF).

Table 3 shows the composition of simulated small intestine fluid.

Artificial digestion (oral)

The digestive process in the oral cavity is shown in Fig. 1A. First, 0.1 (w/v)% food and 5 ml PBS were put into a brown test tube blocked from light, based on 100 ml of the fecal fermentation process, and then 3.5 ml of simulated salivary fluid (SSF) was added, and 12U/mg alpha -Amylase (α-amylase) was dissolved in 0.5 ml SSF and then added. 25 μl of 0.3M CaCl ₂ and 975 μl of sterile water were finally added, and oral digestion was performed for 2 minutes at 100 rpm in a shaking incubator at 37°C.

Artificial digestion (above)

Next, the gastric digestion process was performed using the oral digestion product. The digestion process in the stomach is shown in FIG. 1B. The following substances were sequentially added to the digested product after oral digestion to go through the digestion process in the stomach. 7.5ml of simulated gastric fluid (Simulated Gastric Fluid, SGF) was added, and 727 U/mg of pepsin was dissolved in 1.6ml SGF and then added. In addition, 0.3M CaCl ₂ 5 μl, 1M HCl 0.2 ml, and sterile water 695 μl were added, and then 1M HCl was added to adjust the pH to 2. Thereafter, digestion was performed in the stomach at 100 rpm for 2 hours at 37° C. in a shaking incubator.

Artificial digestion (small intestine)

Next, the digestion process of the small intestine was performed using the gastrointestinal digestion product as shown in FIG. 1C. After putting 11 ml of Simulated Intestinal Fluid (SIF) in the gastric digestion product, 7.776 TAME μM Unit/mg of pancreatin was dissolved in 5 ml of SIF and added, 160 mM bile acid 2.5 ml, 0.3M CaCl2 40 μl, 0.15 ml of 1M NaOH, and 1.31 ml of sterile water were added. Thereafter, digestion of the small intestine was performed in a shaking incubator at 37° C. for 2 hours at 100 rpm. After completing all digestion processes, the digestion products were immediately cooled with liquid nitrogen, lyophilized, and stored at -20°C.

Fecal Fermentation (FF)

Microorganisms were first isolated from fresh feces for fecal fermentation. For fecal fermentation, microorganisms isolated from human feces collected on the same day were used.

To separate fecal microorganisms, 1:10 (v/v) PBS was added to the feces and mixed. Thereafter, the pore size was filtered through a sieve of 250 μm, 150 μm, and 53 μm in order to remove suspended matter in the feces, and finally only fecal microorganisms were collected. At this time, the PBS was put into an anaerobic chamber the day before the experiment to remove all oxygen was used.

Intestinal composition medium, freeze-dried digestion products and fecal microorganisms were added to the anaerobic chamber. Fecal microorganisms and intestinal composition medium were added in a ratio of 1:10 (w/v). After preparation for fermentation, anaerobic conditions were maintained, light was blocked in a shaking incubator, and fermentation was performed at 37° C. at 100 rpm.

In general, yeast extract is known as a major source of vitamin B complex in bacterial culture. It makes nitrogen readily assimilated and is used as a carbon and mineral source for microorganisms.

In order to establish experimental conditions in the intestinal composition medium of fecal fermentation, the effect of energy sources (peptone, yeast extract) on changes in short-chain fatty acid production during fecal fermentation was confirmed in FIG. 9 .

The intestinal composition medium was prepared according to the composition of Table 4, and in the case of the energy source addition group (group E), peptone (2g) and yeast extract (2g) were additionally added to the medium of Table 4.

The intestinal composition medium was prepared with the composition shown in Table 4.

Table 4 is the composition of the intestinal composition medium.

After autoclaving the prepared intestinal composition medium with autoclave, 50 mg/L of hemin and 10 μl/L of vitamin K) were added when the medium cooled.

After artificial digestion of galactooligosaccharide (GOS), the experiment was conducted by dividing the feces into four groups (B, E, G1 and G5) using the feces of one subject.

Group B:

group without GOS, peptone and yeast extract,

Group E:

Group with no added GOS and added peptone and yeast extract

G1 group:

The group that added 0.1 g of GOS after GID without adding peptone and yeast extract

G5 group:

The group that added 0.5 g of GOS without adding peptone and yeast extract after GID

The contents of short-chain fatty acids (acetate, propionate and butyrate) at 0 hours, 2 hours, 4 hours, 8 hours, 12 hours, 18 hours and 24 hours of fermentation of each group are shown in FIG. 9 .

In group B, three short-chain fatty acids (SCFAs) were statistically increased after 18 h of fermentation (P<0.05).

In other groups, acetate and propionate increased significantly compared to 0 h after 2 hours of fermentation (P<0.05), and in the case of G1 group, butyrate was 0 after 4 hours of fermentation It increased significantly when compared with time (P<0.05). That is, it was confirmed that the energy sources (peptone and yeast extract) included in the intestinal composition had an effect on the change in the production of short-chain fatty acids from 2 hours after fermentation.

Therefore, in the case of the intestinal composition medium, nutrients such as peptone and yeast extract were removed to evaluate the effect of fermentation due to food, and it was used for gastric digestion-fecal fermentation (In vitro GID-FF) in vitro.

Example 1-1

By using boiled soybeans as the food, a digestion product was prepared through the oral, stomach, and small intestine digestion process in sequence, and fecal fermentation (FF) was performed by adding fecal microorganisms to the digestion product to prepare a fermented product.

Example 1-2

A fermented product was prepared in the same manner as in Example 1-1, except that commercially available cheonggukjang was used. The commercially available cheonggukjang was purchased and used as unsalted cheonggukjang from Suncheonjang Liu Company.

Examples 1-3

A fermented product was prepared in the same manner as in Example 1-1, except that Cheonggukjang (SRCM 101336) prepared with a strain ( Bacillus subtilis ) isolated from Cheonggukjang cookies was used.

Examples 1-4

A fermentation product was prepared in the same manner as in Example 1-1, except that Cheonggukjang (SRCM 100169) prepared with a strain ( Bacillus subtilis ) isolated from red pepper paste was used.

Examples 1-5

A fermentation product was prepared in the same manner as in Example 1-1, except that Cheonggukjang (SRCM 100730) prepared with a strain ( Bacillus amyloliquefaciens ) isolated from red pepper paste was used.

Examples 1-6

A digestion product was prepared in the same manner as in Example 1-1, except that Cheonggukjang (SRCM 100731) prepared with a strain ( Bacillus amyloliquefaciens ) isolated from red pepper paste was used.

Comparative Example 1-1

As a negative control (blank), fecal microorganisms and intestinal composition medium were added to an anaerobic chamber in a ratio of 1: 10 (w/v), light was blocked in a shaker incubator, and reacted at 37 ° C. at 100 rpm to prepare a fermentation product.

Comparative Example 1-2

A fermented product was prepared in the same manner as in Example 1-1, except that galactooligosaccharide (GOS) represented by Chemical Formula 1 was used as a food as a positive control.

<Example 2> Analysis of content and pH of short chain fatty acid (SCFA)

To analyze the effect of food on changes in the intestinal environment, fermented products fermented for 0, 2, 4, 6, 8, 12, and 24 hours were sampled and analyzed using GC-MS to obtain short-chain fatty acids (acetate, propionate, butyrate) were analyzed, and the increase rate of each short-chain fatty acid was analyzed based on 0 hours. In addition, the pH change of the fermentation product according to the fermentation time was measured.

<Example 3> Microbial ecology analysis

Microbial DNA was extracted from the fermentation product and the microbial ecology was analyzed based on the V4 variable region of the 16S rDNA gene. Mothur was used for the analysis, and sequencing errors were removed, species diversity analysis (Chao, Shanon index), comparison of microbial ecological association between OTU level groups (ANOVA), and a non-metric multidimensional scale method that expressed association between groups as distance Comparison of microbial ecology by statistical analysis such as scaling, NMDS) and tree analysis, comparative analysis of significant abundance at Genus and OTU level (metastats, lefse), and estimation of metabolic pathway based on 16S rDNA gene (PICRUSt) analyzed.

<Experimental Example 1> Confirmation of changes in microbial ecology according to fermentation time of fecal microorganisms

To check the fermentation completion time of fecal microorganisms, fecal microorganisms were cultured in a shaking incubator by blocking light in an anaerobic state at 37°C for 0, 2, 4, 6, 8, 12, and 24 hours in the intestinal composition medium. and a fermentation product was prepared, and DNA was extracted from the fermentation product. The extracted DNA was analyzed for microbial ecology based on the 16S rDNA gene.

2, it was confirmed that Chao 1 decreased during fermentation for more than 6 hours, thereby reducing species diversity, and Shannon also decreased after 6 hours, confirming that microbial evenness was reduced. Therefore, it was confirmed that the microbial fermentation time is preferably carried out for 6 hours or less.

Next, using Comparative Example 1-1, the change of the microbial ecology at fermentation time was compared. 3 is a result of analyzing the microbial ecology according to time. In FIG. 3, it was confirmed that the microbial ecology of less than 6 hours and more than 8 hours was different. It was confirmed that the microbial ecology of 24 hours, 12 hours, and 8 hours was classified into the same cluster.

To confirm this in detail, in FIG. 4 , microbial species present in abundance in the fecal microbial fermentation product for 0 to 6 hours and 8 to 24 hours were compared. Green color in FIG. 4 indicates microorganism species that appear abundantly for 0 to 6 hours, and red color indicates microorganisms that appear abundantly during fermentation for 8 to 24 hours. As a result, at 0 to 6 hours, microorganisms of the genus Dorea , L. Ruminococcus , R. Ruminococcus , Ruminococcaceae , Roseburia , Blautia , Coprococcus , Bfidobacterium , Ruminococcaceae , dialister , and faecalibaterium were abundant. (abundance), and during fermentation for 8 to 24 hours, bacteroides genus, Bilophila genus, Parabacteroides genus, Clostridiales family, Lachnospiraceae family. Oscillospira , Akkermansia , Desulfovibrio and Enlerobacteriaceae were abundantly present, confirming that the microbial ecology was different depending on the fermentation time.

<Experimental Example 2> Analysis of microbial ecology change according to food fermentation time

After artificial digestion of commercial cheonggukjang (Example 1-2) and fermentation with fecal microorganisms, the microbial ecology change according to fermentation time was analyzed and compared with Comparative Example 1-1.

5 is a result of analyzing the microbial ecology change according to the fermentation time of food. DNA was extracted from each fermentation product and 16S rDNA gene was analyzed, and the result was subjected to non-metric multidimensional scaling (NMDS).

In FIG. 5 , it was confirmed that the microbial ecology was distributed differently from 2 hours after fermentation time compared to before fermentation (0 hours). It was confirmed that the distribution shifted to the right as the fermentation time increased for both the commercial fermented product of cheonggukjang (Example 1-2) and the fermented product fermented only with fecal microorganisms (Comparative Example 1-1). This is considered to be the result of in vitro digestion and fermentation effects.

In addition, it was confirmed that the distribution of microorganisms in Example 1-2 moved in the direction of the red arrow compared to Comparative Example 1-1. This is considered to be a change caused by food.

Through the above experimental results, it was confirmed that food affects the microbial ecology 2 hours before fermentation. To confirm this in more detail, the microorganisms abundant in the fermentation product were comparatively analyzed.

In FIG. 6a, in Comparative Example 1-1, changes in microbial ecology were compared and analyzed before fermentation and after fermentation for 2 hours. When only feces were fermented for 2 hours, Parabacteroides spp ., Bilophila spp ., Sutterella spp ., Oscillospira spp .

Next, the microorganisms abundant in the fermentation products of Comparative Example 1-1 and Example 1-2 fermented for 2 hours were comparatively analyzed. In Example 1-2, Collinsella spp ., Bacteroides spp ., Lachnospiraceae family , Bifidobacterium spp ., Ruminococcus family, Phascolarctobacterium spp. of microorganisms were significantly increased, and relatively Anaerostipes spp. , Peptostreptococcaceae , Ruminococcus bromii , Oscillospira spp ., Rikenellaceae , Sutterella spp ., Faecalibacterium spp. , Prevotella spp . was significantly decreased.

In the microbial strain, Bilophila spp . and Sutterella spp . are harmful bacteria, and the Oscillospira spp ., Collinsella spp ., Bacteroides spp ., Bifidobacterium spp ., Phascolarctobacterium spp . , Anaerostipes spp . and Prevotella spp . are beneficial bacteria.

<Experimental Example 3> Analysis of changes in microbial ecology of fermented products according to food types

Next, the microbial ecology was analyzed in the fermented products according to the type of food. 7 is a result of analyzing the microbial ecology in the fermentation products of Examples 1-1 to 1-6, Comparative Example 1-1 and Comparative Example 1-2. In FIG. 7 , it was confirmed that the microbial ecology was different depending on the type of food at 2 hours of fermentation. Through the above experimental results, it was confirmed that food had an effect on the microbial ecology 2 hours before fermentation. Therefore, it was confirmed that the fermentation time using fecal microorganisms is preferably carried out for less than 2 hours.

<Experimental Example 4> SCFA and pH analysis of fermented products according to the type of food

It was confirmed that the microbial ecology of the fermented product was changed according to the type of food, and it was confirmed that the intestinal microbial environment was changed according to the food. Next, by varying the change of metabolites according to the type of food to 2, 4, 6, 8, 12, and 24 hours of fermentation, the pH change and the content of SCFA (acetate, propionate, butyrate) were measured, and fermentation The increase rate of SCFA compared to before was analyzed.

It is known that as the number of beneficial bacteria increases, the production of acetate, butyrate, and propionate increases, and as the pH decreases, the growth of harmful bacteria is inhibited. Therefore, it is possible to evaluate the effect of improving the intestinal environment of food by using the production of short-chain fatty acids and/or changes in pH.

Comparative Example 1-1 was used as a negative control group (blank), and Comparative Example 1-2 well known as prebiotics was used as a positive control group. In the case of Comparative Example 1-1, the pH was measured between 7.5 and 8 during the fermentation time, and the SCFA increase rate was propionate, butyrate, the increase rate of SCFA for 24 hours was 0 or less. It was confirmed, and acetate increased from 0 at 12 hours to about 5 at 24 hours.

In the case of galactooligosaccharide (GOS) of Comparative Example 1-2, which is a positive control, the increase rate of acetate, butyrate, and proionate was high for 2 to 24 hours of fermentation, and short-chain fatty acids were observed until 6 hours of fermentation. It was confirmed that the increase rate of

In Examples 1-1 to 1-6, it was confirmed that the pH decreased according to fermentation, and the amount of acetate, butyrate, and propionate production increased compared to the negative control group.

<Experimental Example 5> Application of health functional food evaluation system - Functional evaluation of cheonggukjang

Next, in order to evaluate the effect of cheonggukjang, a functional food, on the intestinal microbial ecology, fecal microorganisms were isolated from 8 subjects (S1 to S8), and fecal fermentation was performed for 2 hours using the separated fecal microorganism samples from each subject. and analyzed changes in the production of short-chain fatty acids (Acetate, Propionate, Butyrate) and changes in microbial ecology by Cheonggukjang. Figure 10 shows the change in the short-chain fatty acid content of the control group (Blank, B) and the cheonggukjang treatment group (Treatment, T) after 2 hours of fecal fermentation.

Referring to Figure 10, when the subject S2 added cheonggukjang, acetate, propionate, and butyrate all significantly increased, and in the case of subjects S4, S6 and S7, acetate (Acetate), It was confirmed that propionate (Propionate), butyrate (Butyrate) does not increase significantly. In subject S1, acetate and propionate increased significantly, in subjects S3 and S5, only acetate increased, and in subject S8, only acetate and butyrate significantly increased.

That is, it was confirmed that even if the same functional food (Chungkukjang) was consumed, the effect on changes in the intestinal environment, specifically short-chain fatty acids (Acetate, Propionate, Butyrate) production change, was different depending on the subject. did Therefore, it is possible to predict changes in the intestinal environment (change in short-chain fatty acid production) for each individual when ingesting functional foods using a simulated fermentation system using fecal microorganisms.

This difference in short-chain fatty acid production is predicted as a result of individual differences in the ecology of intestinal microbes. Therefore, the intestinal microbial ecology according to each subject was analyzed, and the intestinal microbial ecology according to each subject group on the non-quantitative multidimensional scale method (NMDS) is shown in FIG. All subjects, except for subject S3, were able to confirm that the direction after fermentation of the control group (Blank) for 2 hours and the cheonggukjang treatment group for 2 hours was different based on the fermentation time of the control group (Blank). It was possible to confirm that the microbial ecology was different.

The results of the OTUs-based tree are shown in FIG. 12 . As a result, small groups were clustered for each subject, and the intestinal microbial ecology of the subjects was clustered into three groups. Subjects S8, S5, and S4 were clustered in Cluster I, subjects S7 and S6 were clustered in Cluster II-a, and subjects S3, S2, and S1 were clustered in Cluster II-b.

In addition, in all subjects, the control (Blank) group 0 hour fermentation and the control (Blank) 2 hour fermentation sample were located in one cluster, and the sample to which cheonggukjang was added was not clustered with the control group (Blank). When looking at the sound, it can be seen that changes in intestinal microbial ecology occurred in all people after sample processing through an in-vitro experiment.

According to the linear discriminant analysis effect size (LEfSe) at the OTUs level, when comparing the 2-hour fermentation in the blank group and the 2-hour fermentation in the cheonggukjang-treated group, the intestinal microorganisms increased or decreased, and Sung, Jaeyun Based on , et al. (2017), it is shown in Table 5 whether intestinal microbes produce short-chain fatty acids (Acetate (A), Propionate (P), Butyrate (B)) at the species level (LDA> 3.0).

Table 5 shows the intestinal microorganisms changed by Cheonggukjang during in vitro fermentation by OTUs level per subject.

All OTUs were selected to exhibit p values at LDA > 3.0 and T. Test <0.05 compares the gap ratio of RRA (Relative Abundance) and RA >1.0% between B 2h and T 2h. Among them, decreased OUT was denoted by D, and increased OUT was denoted by I. - and + mean reduction or increase in OTUs representing at least 0.5% of the RA interval ratio. Blank means that no OTU is detected.

In Table 5, Faecalibacterium prausnitzii (Otu0005) decreased in all subjects, but Faecalibacterium prausnitzii (Otu0023) increased in subjects S1, S2, S6 and S7. Bifidobacterium spp. is known to increase when fermented soybean natto is consumed by Bacillus subtilis similar to cheonggukjang, and in the case of Bifidobacterium adolescentis (Otu0001), a type of Bifidobacterium spp ., it was increased in all subjects except for subject S7.

Acetate-producing intestinal microflora ( Bifidobacterium adolescentis (Otu0001), Alistipes putredinis (Otu0033), Bacteroides caccae (Otu0052), Bacteroides fragilis (Otu0011), Bacteroides ovatus (Otu0069), Bactero000ides uniformis (Otu0020) ), Clostridium perfringens (Otu0089), Faecalibacterium prausnitzii (Otu0005, Otu0023), Lactobacillus ruminis (Otu0045), Ruminococcus bromii (Otu0018), [ Ruminococcus] gnavus (Otu0013)), increased or decreased.

Specifically, Bifidobacterium adolescentis (Otu0001) increased in S1, S2, S3, S4, S5, S6 and S8, Alistipes putredinis (Otu0033) increased only in S1, Bacteroides caccae (Otu0052) decreased in S5, and Bacteroides fragilis ( Otu0011) decreased only in S4. Bacteroides ovatus (Otu0069) decreased only in S8, and Bacteroides uniformis (Otu0020) decreased in S5 and increased in S1. Prevotella copri (Otu0002) was decreased in S1, S2, S3 and S6, Clostridium perfringens (Otu0089) was increased in S6, Faecalibacterium prausnitzii (Otu0005) was decreased in all of S1 to S8, and Faecalibacterium prausnitzii (Otu0023) was decreased in S1, S8. increased in S2, S6 and S7, and Lactobacillus ruminis (Otu0045) increased in S3 and S8. Also, Ruminococcus bromii (Otu0018) decreased in S3, [ Ruminococcus] gnavus (Otu0013) decreased in S4 and increased in S5 and S8, and [Ruminococcus] torques (Otu0043) decreased in S3 and S4.

However, referring to FIG. 10, acetate was significantly increased only in subjects of S1, S2, S3, S5 and S8.

In addition, Collinsella aerofaciens (OTU0034), Lactobacillus spp , which produce L-Lactate ([S]-Lactate, Lactate, D-Lactate, [R]-Lactate) in each subject from the results of this experiment. (Otu0047), Lactobacillus ruminis (Otu0045), Dorea spp . (Otu0041) and Coprococcus spp . (Otu0008, OTU0012) and other intestinal microorganisms increased.

This substance is converted to Acetate by pyruvate metabolism (metabolism), and Megasphaera spp consuming this substance in the results of this experiment. (Otu0022) and Bacteroides fragilis (Otu0011) microorganisms were decreased in subjects S7 or S4 in which acetate was not significantly increased.

In addition, referring to the results of FIG. 13 , pyruvate metabolism (metabolism) increased in most of the subjects in which Acetate was significantly increased in the Picrust results (except for S3), and there was no significant difference or decreased in the subjects in which Acetate did not increase. .

Based on these results, it is considered that Acetate increases or decreases due to the increase or decrease of Pyruvate metabolism by the microorganisms consuming L-Lactate depending on the subjects during In vitro-FF with Cheonggukjang.

In Table 5, Propanoate-producing intestinal microorganism Alistipes putredinis (Otu0033) increased in S1, Bacteroides uniformis (Otu0020) increased in S1, Clostridium perfringens (Otu0089) increased only in S6, Faecalibacterium prausnitzii (Otu0005) increased in S1 to S8 were all decreased, and Faecalibacterium prausnitzii (Otu0023)) was increased in S1, S2, S6 and S7. However, referring to Figure 10, only the S1 and S2 subjects showed a significant increase in propinoate.

As described above, as described above, L-Lactate produced by intestinal microbes is succinate through the process of pyruvate metabolism, resulting in pyruvate in subjects S1, S2, S5 and S8. It is thought that metabolism was increased. In addition, among these subjects, S1 and S2 were only found in 2 subjects (S1, S2) due to reduced c5-branched dibasic acid metabolism and consequently propanoate (Propionate) metabolism, which converts propanoyl-CoA to propionate. Propionate was significantly increased by Cheonggukjang.

In the results of this experiment, by analyzing the intestinal microbes increased or decreased by Cheonggukjang, the Acetate and Propanoate substances were linked with the Picrust result to predict the production route, and the SCFAs (Acetate, Propanoate, Butyrate) generated by Cheonggukjang according to each subject It was confirmed that the amount and increase or decrease of intestinal microflora were diverse.

Claims (11)

preparing a digestive product by adding an artificial digestive juice to the target food;
preparing a fermentation product by adding the fecal microorganism and intestinal composition medium of the test subject to the digestion product;
checking the microbial ecology by analyzing the nucleotide sequence of the 16S rDNA gene in the fermentation product; and
Comprising the step of comparing the microorganism of the target food fermented product and the indicator beneficial bacteria or indicator harmful bacteria of the standard food fermented product,
The comparing step is
When the abundance of beneficial bacteria confirmed in the fermented product of the target food is the same as or higher than that of the index beneficial bacteria confirmed in the fermented product of the standard food,
Or, if the abundance of harmful bacteria confirmed in the fermented product of the target food is the same as or lower than the abundance of the index harmful bacteria confirmed in the fermented product of the standard food, the target food is evaluated as a functional food.
Evaluation method of functional food. delete The method of claim 1,
The standard food is characterized in that it is a prebiotic or probiotic food,
Evaluation method of functional food. The method of claim 1,
The indicator beneficial bacteria is characterized in that it increases the production of short-chain fatty acids,
Evaluation method of functional food. 5. The method of claim 4,
The short chain fatty acid (SCFA) is characterized in that it comprises acetate, butyrate or propionate,
Evaluation method of functional food. The method of claim 1,
Further comprising the step of analyzing the content of short-chain fatty acids in the fermentation product,
Evaluation method of functional food. The method of claim 1,
measuring the pH of the fermentation product; and
When the pH measured above is the same as or lower than the pH value measured in the standard food, further comprising the step of selecting the target food as a functional food,
Evaluation method of functional food. The method of claim 1,
The indicator beneficial bacteria are Oscillospira spp ., Collinsella spp ., Bacteroides spp ., Bifidobacterium spp ., Phascolarctobacterium spp ., Anaerostipes spp ., Ruminococcus bromii , Faecalibacterium spp . And Prevotella spp . Any one or more selected from the group consisting of,
Evaluation method of functional food. The method of claim 1,
The indicator harmful bacteria is Bilophila spp . Or Sutterella spp . Any one or more selected from the group consisting of,
Evaluation method of functional food. delete delete

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