Method for evaluating prebiotics and compound combination thereof based on in-vitro simulation fermentation model-co-culture cell model

Abstract

The invention provides a method for evaluating prebiotics and a compound combination thereof based on an in-vitro simulation fermentation model-co-culture cell model, and the method comprises the following steps: preparation of fecal bacteria suspension, preparation of an in-vitro fermentation culture medium, in-vitro fermentation culture and index determination of single prebiotics, and in-vitro fermentation culture and index determination of composite prebiotics. The method comprises the following steps: establishing a cell co-culture model, carrying out co-culture on composite prebiotics and cells, determining indexes, and determining optimal monomer prebiotics and composite prebiotics. By means of the method, the optimal prebiotic compound composition composed of the tea polyphenol, the fructo-oligosaccharide and the stachyose is obtained through screening for the first time, the antioxidant activity of the compound prebiotics and the synergistic anti-inflammatory effect of the short-chain fatty acid are proved, and the compound prebiotics are applied to preparation of the soft sweets.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of intersection of microbial technology and food, and specifically relates to screening and evaluation of prebiotics and related food preparation technology, and particularly discloses a method for evaluating prebiotics and their compound combinations based on an in vitro simulated fermentation model-co-cultured cell model. BACKGROUND

[0002] The intestinal microbiota is a complex and dynamic ecosystem in the human body, and its composition and activity have important influences on the host's nutrient absorption, immune regulation, metabolic balance and overall health. As a key factor for regulating the structure and function of intestinal flora, prebiotics can selectively stimulate the growth and reproduction or metabolic activity of beneficial bacteria (such as Bifidobacterium and Lactobacillus) in the intestine, and become an important dietary intervention means for improving the intestinal microecological balance.

[0003] Currently, the prebiotics that have been widely studied mainly include oligosaccharides (such as fructooligosaccharides, stachyose, inulin, etc.) and plant polyphenols, which play a health-promoting effect through regulating intestinal flora metabolism (such as promoting the production of short-chain fatty acids SCFAs), enhancing antioxidant capacity, inhibiting pathogenic bacteria colonization, etc. However, the effect of a single prebiotic is often limited: on the one hand, different prebiotics have different targeting effects on intestinal flora, and a single component is difficult to comprehensively regulate the flora structure; on the other hand, some prebiotics are easily degraded in the intestinal environment or have limited action efficiency, and their functional exertion is restricted. Therefore, the complex prebiotics formed by compounding different types of prebiotics to enhance the regulation effect through synergistic effect has become an important direction of current research.

[0004] The screening and evaluation of prebiotics need to take into account their regulation of intestinal flora and direct or indirect effects on host cells. In vitro models, due to their convenient operation, low cost and high repeatability, have become an important tool for preliminary evaluation of prebiotics. Among them, the in vitro simulated fermentation model can simulate the anaerobic environment of the intestine, study the interaction between prebiotics and intestinal flora, analyze the changes in flora composition and the generation of metabolic products (such as SCFAs and antioxidant substances); cell models (such as intestinal epithelial cell models and immune cell models) can be used to evaluate the effects of prebiotics or their metabolites on intestinal barrier function, inflammatory response, etc. at the cellular level. However, in the existing technology, the combination of in vitro fermentation models and cell models is still not systematic, especially lacking a multi-dimensional evaluation method for complex prebiotics (such as oligosaccharide and polyphenol compound systems), which is difficult to fully reflect the synergistic action mechanism and overall health effects.

[0005] In addition, with the development of the functional food market, the product forms of prebiotics are increasingly diversified. Because of good taste, easy portability, strong palatability, and the ability to stably carry functional ingredients, gummy candies have become an important carrier of prebiotic functional foods. However, there are still few studies on the application of composite prebiotics in food matrices such as gummy candies, and there is a lack of optimal composite combinations screened based on scientific evaluation, which restricts the precise development of prebiotic functional foods.

[0006] In summary, it is of great significance to establish a systematic evaluation method combining in vitro simulated fermentation and cell co-culture models for screening highly synergistic composite prebiotics and applying them to the development of functional foods, which can promote the industrial application of prebiotics in the field of intestinal health. SUMMARY

[0007] In view of the problems in the prior art, the purpose of the present application is to provide a method for evaluating prebiotics and their composite combinations based on an in vitro simulated fermentation model and a co-culture cell model. The combination of the in vitro simulated fermentation model and the co-culture cell model in the method can comprehensively analyze the overall health effects of prebiotics on microorganisms, and the method can effectively screen prebiotic combinations.

[0008] The purpose of the present application is achieved by the following technical solutions:

[0009] The first aspect of the present application provides a method for evaluating prebiotics and their composite combinations based on an in vitro simulated fermentation model and a co-culture cell model, comprising the following steps:

[0010] Step 1, preparation of fecal bacteria suspension

[0011] The feces are taken from a volunteer who is healthy, has not taken antibiotics, probiotics or prebiotics within three months, and is aged 22-26 years old, and the preparation of the fecal bacteria suspension is completed within 30 minutes after collection. The fecal sample is diluted with a phosphate buffer solution to prepare the fecal bacteria suspension, which is stored anaerobically at 4℃;

[0012] Step 2, preparation of in vitro fermentation medium

[0013] The formula of the in vitro fermentation medium is: 2 g / L peptone, 2 g / L yeast extract, 0.1 g / L NaCl, 40 mg / L K2HPO4, 40 mg / L KH2PO4, 10 mg / L MgSO4·7H2O, 10 mg / L CaCl2·6H2O, 2 g / L NaHCO3, 0.05% L-cysteine, 0.5 g / L cholate, 10 μl / L vitamin K, 2 mL / L Tween 80, and 50 mg / L hematin, adjusted to pH 7.5;

[0014] Step 3, in vitro fermentation culture of individual prebiotics and determination of indicators

[0015] The single prebiotic to be tested after filtering and sterilization is added to the sterilized fermentation medium;

[0016] The fecal bacteria suspension described in step 1 is inoculated into the in vitro fermentation medium described in step 2, and anaerobic fermentation is carried out at 37°C. After the fermentation is terminated, the fermentation broth is taken to measure the pH. In addition, after the fermentation broth is centrifuged at low temperature, the supernatant is taken for detection and analysis including antioxidant capacity, short-chain fatty acids SCFAs, etc. The bacterial cell precipitate after centrifugation is used for detection and analysis including DNA extraction and microbiota sequencing;

[0017] Step 4, in vitro fermentation culture of the composite prebiotic and determination of indicators

[0018] The in vitro fermentation medium is prepared according to the same formula as in step 2, and the fermentation medium is adjusted to pH 7.2;

[0019] The composite prebiotic combination to be tested after filtering and sterilization is added to the sterilized in vitro fermentation medium. The fecal bacteria suspension described in step 1 is inoculated into the in vitro fermentation medium, and anaerobic fermentation is carried out at 37°C. After the fermentation is terminated, the fermentation broth is taken to measure the pH. In addition, after the fermentation broth is centrifuged at low temperature, the supernatant is taken for detection and analysis including antioxidant capacity, short-chain fatty acids SCFAs, etc. The bacterial cell precipitate after centrifugation is used for detection and analysis including DNA extraction and microbiota sequencing;

[0020] Step 5, establishment of cell co-culture model

[0021] Caco-2 cells are inoculated on the upper chamber of the Transwell, and after the cells are completely differentiated, RAW 264.7 cells are inoculated into a tissue culture plate and cultured for 24 h to promote obvious adhesion of the basolateral pores, thereby constructing a cell co-culture model;

[0022] Step 6, co-culture of the composite prebiotic with cells and determination of indicators

[0023] After the cell co-culture model is established for 24 h, the upper side of the Transwell chamber is treated with the fermentation supernatant of the composite prebiotic to be tested, and after 24 h, the basal side of the culture plate is treated with LPS. After another 24 h of incubation, the culture medium and cells are collected for analysis, including determination of intestinal barrier permeability, NO, ROS, and TNF-α levels;

[0024] Step 7, determination of optimal single prebiotic and composite prebiotic

[0025] Based on the determination results of the above in vitro fermentation culture and cell co-culture, the optimal single prebiotic and the optimal composite prebiotic combination are screened according to the short-chain fatty acid yield and the regulation of the microbiota.

[0026] Further, the single prebiotics include polyphenols, oligosaccharides; the complex prebiotics are compositions formed by different single prebiotics.

[0027] Further, in steps 3 and 4, the determination of the antioxidant capacity includes determination of DPPH free radical scavenging capacity, ABTS free radical scavenging capacity and total antioxidant capacity.

[0028] Further, the specific steps of the determination of the antioxidant capacity include:

[0029] S31, determination of DPPH free radical scavenging capacity:

[0030] The experimental group is 25 μL of fermentation liquid sample added with 175 μL of 0.2 mM DPPH ethanol solution, the blank group is 25 μL of negative control fermentation liquid added with 175 μL of 0.2 mM DPPH ethanol solution; the control group is 25 μL of fermentation liquid sample added with 175 μL of anhydrous ethanol; after reaction at room temperature for 30 min, the OD value at 517 nm is determined, and the DPPH free radical scavenging rate is calculated according to the following formula:

[0031]

[0032] In the formula: A1 is the absorbance of the blank group; A2 is the absorbance of the experimental group; A3 is the absorbance of the control group;

[0033] S32, determination of ABTS free radical scavenging capacity:

[0034] The sample is diluted with distilled water to an appropriate concentration, the experimental group is 20 μL of sample added with 180 μL of ABTS working solution, the blank group is 20 μL of negative control fermentation liquid sample added with 180 μL of ABTS working solution, and the control group is 20 μL of fermentation liquid sample added with 180 μL of deionized water; after reaction at room temperature for 10 min, the OD value at 734 nm is determined, and the ABTS free radical scavenging rate is calculated according to the following formula:

[0035]

[0036] In the formula: A1 is the absorbance of the blank group; A2 is the absorbance of the experimental group; A3 is the absorbance of the control group;

[0037] S33, determination of total antioxidant capacity:

[0038] A FRAP working solution was prepared by mixing 0.3 mM sodium acetate buffer, 10 mM 2,4,6-tri-2-pyridyl-1,3,5-triazine TPTZ, and 20 mM FeCl3·6H2O at a volume ratio of 10:1:1 and incubating at 37 °C for 15 min. After diluting the sample to a suitable concentration, 20 μL of fermentation broth sample was reacted with 180 μL of FRAP working solution at 37 °C in the dark for 30 min. The absorbance was read at 593 nm and compared with 1 mM FeSO4. The FRAP value was calculated using the following formula:

[0039]

[0040] In the formula: B1 is the absorbance of the sample reacting with the FRAP working solution; B2 is the absorbance of FeSO4 reacting with the FRAP working solution; C0 is the concentration of FeSO4.

[0041] Furthermore, the specific steps for determining SCFAs in steps 3 and 4 include:

[0042] The yield of SCFAs in the fermentation supernatant was analyzed by gas chromatography: 0.4 mL of 50% v / v sulfuric acid and 2 mL of diethyl ether were added to 2 mL of fermentation supernatant, vortexed for 3 min, centrifuged, and the upper organic components were collected, filtered through a 0.22 μm organic filter membrane, and placed in a chromatographic bottle; then the contents of acetic acid, propionic acid, butyric acid, and isobutyric acid were determined by gas chromatography using the external standard curve method.

[0043] An AT-WAX column was used. The oven temperature was maintained at 90℃ for 5 min, then increased to 200℃ at a rate of 20℃ / min and maintained for 3 min. The injector temperature was set to 250℃. 1 μL of sample was injected into the instrument at a split ratio of 1:10. N2 was used as the carrier gas at a flow rate of 1 mL / min. The detector temperature was set to 250℃.

[0044] Furthermore, the specific steps for DNA extraction and microbial community sequencing in steps 3 and 4 are as follows:

[0045] Total DNA was extracted from the fermentation broth bacterial samples using the magnetic bead method soil and fecal genomic DNA extraction kit. The obtained DNA was used as a template for PCR amplification of the V3-V4 region of the bacterial 16S rRNA gene using primers 341F and 806R.

[0046] After purification, the PCR products were used The Ultra™ II FSDNA-PCR free library construction kit was used to generate sequencing libraries; after the sequencing libraries passed the testing, they were sequenced using an Illumina Novaseq 6000 PE250 sequencing instrument.

[0047] The second aspect of the present application provides the use of the method of the first aspect in evaluating and screening individual prebiotics and prebiotic combinations.

[0048] The third aspect of the present application provides a prebiotic combination screened by the method of the first aspect, which comprises, in mass percentage:

[0049] fructooligosaccharides 5%, stachyose 5%, and tea polyphenols 0.4%.

[0050] The fourth aspect of the present application provides the use of the prebiotic combination of the third aspect in the preparation of food or health food.

[0051] The fifth aspect of the present application provides the use of the prebiotic combination of the third aspect in the synergistic anti-inflammatory effect of antioxidant activity and short-chain fatty acids.

[0052] The present application has the following advantages over the prior art:

[0053] 1. Breaking through the limitations of single prebiotics and achieving synergistic effects of combinations: In the prior art, prebiotic products are mostly single oligosaccharides or similar complex oligosaccharides. Due to the differences in the metabolic enzyme systems of intestinal flora, the adaptation range and beneficial effects of single prebiotics are limited. The present application first combines polyphenols (tea polyphenols) with oligosaccharides (fructooligosaccharides, stachyose) in a specific ratio, utilizes the metabolic complementarity of different types of prebiotics, and significantly enhances the efficacy through gain effect: not only can polyphenols enhance antioxidant activity, but also can oligosaccharides promote the proliferation of intestinal flora and the generation of short-chain fatty acids (SCFAs), solving the problem of single function and limited effect of single prebiotics, and realizing the synergistic effect of "antioxidation-flora regulation-metabolic product enhancement".

[0054] 2. Building a comprehensive evaluation system to improve the scientificity and accuracy of screening: The present application innovatively combines in vitro simulated fermentation models with cell co-culture models to form a multi-dimensional evaluation system:

[0055] The in vitro fermentation model can accurately analyze the effects of prebiotics on intestinal flora structure (through 16S rRNA sequencing), metabolic products (SCFAs, antioxidant indicators), and fermentation environment (pH), revealing their regulation mechanisms on intestinal microorganisms;

[0056] The Caco-2 / RAW 264.7 cell co-culture model can further evaluate the effects of fermentation products on intestinal barrier function (permeability, TEER value), inflammatory factors (NO, TNF-α), and oxidative stress (ROS), verifying the immune regulation and anti-inflammatory efficacy from the cellular level.

[0057] The combination of the two models overcomes the defect that a single model can only reflect part of the effect, and comprehensively covers the complete chain of "microbial regulation-metabolites-cell response", providing a more scientific and accurate basis for the screening of prebiotic combinations.

[0058] 3. Clear efficacy mechanism, strengthen practical application value: through systematic experimental verification, the compound prebiotic of the present application has clear and excellent physiological activity:

[0059] Regulate intestinal flora balance: significantly increase the abundance of beneficial bacteria (such as Aggregatibacter, Bifidobacterium), reduce the proportion of harmful bacteria (such as Proteobacteria), and optimize the diversity of flora;

[0060] Enhance antioxidant capacity: after in vitro fermentation, the DPPH, ABTS free radical scavenging rate and FRAP value of the compound prebiotic maintain a high level, and the antioxidant activity is better than that of single prebiotic;

[0061] Promote the generation of beneficial metabolites: significantly increase the production of short-chain fatty acids (especially butyric acid), which is a key metabolite for maintaining intestinal health and inhibiting inflammation;

[0062] Protect the intestinal barrier and anti-inflammatory: through the cell co-culture model, it is confirmed that the fermentation supernatant can reduce the intestinal barrier permeability (increase the TEER value and reduce the FITC leakage), inhibit the secretion of NO, TNF-α and the generation of ROS induced by LPS, and effectively alleviate intestinal inflammation and oxidative stress.

[0063] 4. Expand application scenarios, improve product palatability and popularity: the efficient compound prebiotic screened by the present application is applied to the preparation of soft candy, and by optimizing the ratio of gelatin, pectin, malt syrup and other raw materials, a functional soft candy with good elasticity and chewiness is prepared. This dosage form overcomes the problem of poor palatability and difficulty in being accepted by the public of traditional prebiotic products (such as tablets, capsules), especially suitable for children, the elderly and other groups, providing a convenient and delicious new choice for daily intake of prebiotics, significantly improving the market popularization potential of functional foods. BRIEF DESCRIPTION OF DRAWINGS

[0064] The present application will be further described below in conjunction with the drawings and examples:

[0065] Figure 1 is the pH at the end of fermentation of the prebiotic substrate; wherein A: pH at the end of fermentation of plant polyphenols, B: pH at the end of fermentation of oligosaccharides;

[0066] Figure 2 is the antioxidant index of fecal bacteria fermentation broth before and after polyphenol fermentation; wherein A: DPPH scavenging rate, B: ABTS scavenging rate, C: FRAP value;

[0067] Figure 3Effect of polyphenol fermentation on SCFAs production; Wherein, A: Acetic acid, B: Propionic acid, C: Isobutyric acid, D: n-Butyric acid;

[0068] Figure 4 Effect of oligosaccharide fermentation on SCFAs production; Wherein, A: Acetic acid, B: Propionic acid, C: Isobutyric acid, D: n-Butyric acid;

[0069] Figure 5 16S rRNA sequencing results of polyphenol fermentation microbial samples added; Wherein, A: Chao1 index, B: Shannon index, C: Analysis diagram of species composition at the door level, D: Analysis diagram of species composition at the genus level;

[0070] Figure 6 16S rRNA sequencing results of oligosaccharide fermentation microbial samples added; Wherein, A: Chao1 index, B: Shannon index, C: Analysis diagram of species composition at the door level, D: Analysis diagram of species composition at the genus level;

[0071] Figure 7 Compound prebiotic in vitro fermentation end point pH;

[0072] Figure 8 Antioxidant index of fecal bacteria fermentation broth before and after compound prebiotic fermentation; Wherein, A: DPPH clearance rate, B: ABTS clearance rate, C: FRAP value;

[0073] Figure 9 Effect of compound prebiotic fermentation on SCFAs production; Wherein, A: Acetic acid, B: Propionic acid, C: Isobutyric acid, D: n-Butyric acid;

[0074] Figure 10 16S rRNA sequencing results of compound prebiotic fermentation microbial samples; Wherein, A: Chao1 index, B: Shannon index, C: Analysis diagram of species composition at the door level, D: Analysis diagram of species composition at the genus level;

[0075] Figure 11 Effect of fermentation broth treatment on the integrity of intestinal epithelial monolayer membrane; Wherein, A: TEER value, B: TEER value after LPS treatment, C: FITC value;

[0076] Figure 12 Effect of fermentation broth and LPS treatment on inflammation and oxidative stress; Wherein, A: NO secretion amount, B: TNF-α secretion amount, C: Intracellular ROS content;

[0077] Figure 13 Small-scale trial of compound prebiotic soft candy. DETAILED DESCRIPTION

[0078] The endpoints of the ranges and any values disclosed herein are not limited to the precise values recited as the exact dimensions are not critical to the invention. Any numeric range recited is intended to include all values from the lower value to the upper value, inclusive of both values, and to sub-ranges falling within the specified range. In this context, sub-ranges should be construed as having been specifically stated herein.

[0079] The application will be described in detail below with examples. It should be understood that the following examples are only used to exemplarily further explain and illustrate the content of the application, and are not used to limit the application.

[0080] Example 1

[0081] The present example provides a method for evaluating prebiotics and their combined compositions based on an in vitro simulated fermentation model-co-cultured cell model, comprising the following steps:

[0082] Step 1, preparation of fecal bacteria suspension

[0083] The feces were taken from a healthy volunteer aged 22-26 years old who had not taken antibiotics, probiotics or prebiotics within three months, and the preparation of fecal bacteria solution was completed within 30 minutes after collection; the fecal sample was diluted to 20% fecal bacteria suspension with phosphate buffer solution (0.1M, pH 7.0), and stored anaerobically at 4°C.

[0084] Step 2, preparation of in vitro fermentation medium

[0085] The formula of the in vitro fermentation medium is: 2g / L peptone, 2g / L yeast extract, 0.1g / L NaCl, 40mg / L K2HPO4, 40mg / L KH2PO4, 10mg / L MgSO4·7H2O, 10mg / L CaCl2·6H2O, 2g / L NaHCO3, 0.05% L-cysteine, 0.5g / L cholate, 10μl / L vitamin K, 2mL / L Tween 80, 50mg / L hematin chloride, adjusted to pH 7.5.

[0086] Step 3, in vitro fermentation culture of individual prebiotics and determination of indicators

[0087] The filtered and sterilized individual prebiotics to be tested were added to the sterilized fermentation medium; the individual prebiotics included: tea polyphenols (TP), apple polyphenols (AP), grape seed polyphenols (GSP), peanut skin polyphenols (PSP), pine bark polyphenols (PBP), inulin (INU), fructooligosaccharides (FOS), galactooligosaccharides (GOS), stachyose (STA), raffinose (RAF), isomaltooligosaccharides (IMO) and glucose (GLU).

[0088] Wherein, tea polyphenols (TP), apple polyphenols (AP), grape seed polyphenols (GSP), peanut skin polyphenols (PSP), pine bark polyphenols (PBP) were added to the sterilized in vitro fermentation medium at a concentration of 0.05% w / v after filtering out bacteria; inulin (INU), fructooligosaccharides (FOS), galactooligosaccharides (GOS), stachyose (STA), raffinose (RAF), isomaltooligosaccharides (IMO) and glucose (GLU) were added to the sterilized in vitro fermentation medium at a concentration of 1% w / v after filtering out bacteria, and a negative control (CK) was set by adding an equal amount of sterilized water.

[0089] The fecal bacteria suspension described in step 1 was inoculated into the in vitro fermentation medium described in step 2 and anaerobically fermented at 37°C. After 24h of fermentation, the fermentation broth was taken to measure the pH, and the results are shown in Figure 1 As shown in the figure, before in vitro fermentation, the pH of the fermentation broth of each group was 7.52±0.03. After 24h of fermentation, the pH of the CK group decreased to about 7.0, and the pH of the tea polyphenol, apple polyphenol, peanut skin polyphenol and pine bark polyphenol groups were significantly lower than that of the CK group. The pH level of the inulin and galactooligosaccharide groups after fermentation was similar to that of the glucose group, and the pH of the fructooligosaccharide (pH 3.99), stachyose (pH 3.97) and raffinose (pH 4.04) groups was slightly higher.

[0090] In addition, 6mL of fermentation broth was centrifuged at 4000xg for 10min at 4°C, and the supernatant was used for detection and analysis of antioxidant capacity and short-chain fatty acids (SCFAs); the bacterial cell pellet after centrifugation was used for DNA extraction and microbiota sequencing analysis.

[0091] The results of antioxidant capacity are shown in Figure 2 The results of DPPH radical scavenging rate showed that the antioxidant activity of the negative control group was significantly reduced after fecal bacteria fermentation; in the fermentation groups with the addition of plant polyphenols, except for the grape seed polyphenol group and the peanut skin polyphenol group, the DPPH radical scavenging capacity of the tea polyphenol group, the apple polyphenol group and the pine bark polyphenol group was significantly improved. The results of ABTS radical scavenging rate showed that there was no significant difference in antioxidant activity before and after in vitro fermentation in the negative control group, the tea polyphenol group, the apple polyphenol group and the pine bark polyphenol group. The change trend of FRAP before and after the fermentation of plant polyphenols was similar to that of ABTS scavenging rate. After fecal bacteria fermentation, the FRAP value of the tea polyphenol group and the apple polyphenol group did not change significantly. Overall, among the five plant polyphenols, the in vitro antioxidant capacity of tea polyphenol was the highest, and the antioxidant capacity was basically unchanged in the in vitro fermentation model, and even the DPPH radical scavenging capacity was improved under the action of fecal bacteria.

[0092] The SCFAs yield of in vitro fermentation for 24h under the treatment of different polyphenol substrates is shown in Figure 3As shown in Figure 2, the acetic acid, propionic acid and n-butyric acid production of tea polyphenol group had no significant difference compared with the negative control group. The acetic acid production of apple polyphenol, grape seed polyphenol, peanut skin polyphenol and pine bark polyphenol groups had a rising trend, but also had no significant difference compared with the negative control group. But the propionic acid and n-butyric acid production of the fermentation groups with the addition of the four polyphenols had a significant increase compared with the negative control group. Under the treatment of different oligosaccharide substrates, the SCFAs production of SCFAs after 24h in vitro fermentation was as shown in Figure 3. The acetic acid production after the fermentation of galactooligosaccharide and stachyose was slightly higher than that of glucose fermentation. The acetic acid and propionic acid production after the fermentation of raffinose was the lowest. The propionic acid production of inulin group was slightly higher than that of glucose group, but its isobutyric acid concentration was significantly lower than that after glucose fermentation. Compared with the SCFAs fermentation of glucose, fructooligosaccharide produced a higher level of isobutyric acid and significantly increased the production of n-butyric acid. Figure 4

[0093] The bacterial sediment after the treatment of each polyphenol was collected for 16S rRNA sequencing analysis, and the results were as shown in Figure 4. Compared with the CK group, the five kinds of plant polyphenols all reduced the community richness. According to the Shannon index, tea polyphenol significantly improved the diversity of the community. At the level of phylum, the relative abundance of Proteobacteria in tea polyphenol, apple polyphenol and grape seed polyphenol groups was significantly lower than that in the CK group, while the relative abundance of Proteobacteria in peanut skin polyphenol and pine bark polyphenol groups was significantly higher. At the level of genus, tea polyphenol could increase the relative abundance of anti-inflammatory microorganisms such as Agathobacter and Christensenellaceae. The bacterial sediment after the treatment of each oligosaccharide was collected for 16S rRNA sequencing analysis, and the results were as shown in Figure 5. The addition of oligosaccharide fermentation had a tendency to reduce the community richness, especially the fermentation of fructooligosaccharide and galactooligosaccharide significantly reduced the Chao1 index, and the bacterial community richness after the fermentation of stachyose and glucose was similar. According to the Shannon index, compared with the negative control group, oligosaccharide significantly reduced the diversity of intestinal flora, but the diversity of the bacterial community after the fermentation of the three kinds of oligosaccharides tested was higher than that of the positive control glucose group. At the level of phylum, compared with CK, fructooligosaccharide and galactooligosaccharide significantly reduced the abundance of Proteobacteria, and the addition of fructooligosaccharide had a positive effect on the growth and reproduction of Firmicutes bacteria. Figure 5 Figure 6 Step 4, in vitro fermentation culture of complex prebiotics and determination of indexes

[0094] The in vitro fermentation culture medium formula was the same as step 2, except that the fermentation culture medium was adjusted to pH 7.2 in this step.

[0095] The in vitro fermentation culture medium formula was the same as step 2, except that the fermentation culture medium was adjusted to pH 7.2 in this step.

[0096] Table 1: Complex prebiotic treatment number and addition ratio

[0097] ​​

[0098] The low oligosaccharides, stachyose and tea polyphenols filtered and sterilized were added to the in vitro fermentation medium after sterilization according to the group and proportion shown in Table 1 at the concentration of oligosaccharides 1% w / v and polyphenols 0.05% w / v; inoculate the fecal bacteria suspension described in step 1 into the in vitro fermentation medium, anaerobic fermentation at 37°C; after 24h fermentation, take the fermentation broth to measure pH, the results are shown in Figure 7 The addition of oligosaccharides can significantly reduce the pH of the fermentation environment, and the pH value of group H with only tea polyphenols added is significantly higher than that of other treatment groups, but lower than that of the negative control group without the addition of prebiotics.

[0099] In addition, 6mL of fermentation broth was centrifuged at 4000xg at 4°C for 10min, and the supernatant was used for antioxidant capacity, short-chain fatty acid SCFAs detection and analysis; the bacterial cell pellet after centrifugation was used for DNA extraction and flora sequencing analysis.

[0100] The results of antioxidant capacity determination are shown in Figure 8 Among the 7 kinds of complex prebiotics, groups B and C have the highest in vitro antioxidant capacity, and the antioxidant capacity is basically maintained stable in the in vitro fermentation model, and even the free radical scavenging capacity is improved under the action of intestinal microorganisms.

[0101] The SCFAs concentration of the culture medium after 24h fermentation is shown in Figure 9 The fermentation of tea polyphenols reduces the SCFAs yield, the addition of oligosaccharides can increase the concentration of n-butyric acid and reduce the level of propionic acid and isobutyric acid, and different proportions of oligosaccharides have different effects on SCFAs.

[0102] After comprehensive evaluation, we selected groups B, C, D, E, F, H and L (CK) to enter the subsequent intestinal microorganism regulation and cell experiment research.

[0103] The alpha diversity analysis in the in vitro fermentation model is shown in Figure 10 Compared with the CK group, the community richness of the 6 experimental groups is improved, and the community richness after the fermentation of complex prebiotics is higher than that of the flora structure level after the fermentation of tea polyphenols, and group B has the highest community richness. According to the Shannon index, the community of tea polyphenol group and CK group is more. At the door level, compared with CK and tea H, complex prebiotics (groups B, C, D, E and F) significantly reduce the abundance of Proteobacteria, and increase the abundance of Firmicutes and Actinobacteria. Compared with the CK group, group H increases the amount of Streptococcus and Bifidobacterium.

[0104] Step 5, establishment of cell co-culture model

[0105] Caco-2 cells were seeded at 1×10 5Cells were seeded at a density of [number] cells / mL in the Transwell chamber. The medium was changed every other day for the first 14 days, and daily for the next 7 days, until complete differentiation (21 days, TEER ≥ 500 Ω·cm). 2 RAW 264.7 cells were fed at a rate of 4 × 10⁻⁶. 5 Cells were seeded at a density of 1 / mL into tissue culture plates and cultured for 24 h to promote significant adhesion of cells to the outer pores of the substrate, thus constructing a cell co-culture model.

[0106] Step 6: Co-culture of compound prebiotics with cells and determination of indicators

[0107] Twenty-four hours after the cell co-culture model was established, the upper side (Apical, AP side) of the Transwell chamber was treated with fermentation supernatant of different test prebiotic combinations. Twenty-four hours later, the basal side (Basolateral, BL side) of the culture plate was treated with LPS. After another 24 hours of incubation, the culture medium and cells were collected for analysis, including the determination of intestinal barrier permeability, NO, ROS and TNF-α levels.

[0108] Table 2 Experimental design for evaluating the anti-inflammatory effects of complex prebiotics using an inflammatory cell model.

[0109]

[0110] The integrity of the intestinal epithelial monolayer was assessed using TEER and FITC, and the results were as follows: Figure 11 As shown, under normal conditions without inflammation, the fermentation broths of groups B and E increased the TEER level of the monolayer membrane. After LPS treatment induced inflammation, only the pretreatment of the fermentation broth of group E increased the TEER level of the monolayer membrane. Compared with the control group (CK), the FITC value increased after LPS treatment, while the pretreatment of the fermentation broth of group E decreased the FITC value and increased the integrity of the intestinal epithelium.

[0111] The results of the index measurement are as follows Figure 12 As shown, all fermentation broth pretreatment significantly reduced LPS-induced cellular NO secretion, inhibited the secretion of inflammatory factor TNF-α, and reduced intracellular ROS concentration. The compound prebiotics effectively inhibited LPS-induced intestinal inflammation and oxidative stress.

[0112] Step 7: Determining the optimal single prebiotic and compound prebiotic

[0113] Based on the results of the above in vitro fermentation culture and cell co-culture measurements, the optimal compound prebiotic combination was selected according to the production of short-chain fatty acids and their regulatory effect on the microbial community: 5% fructooligosaccharide, 5% stachyose and 0.4% tea polyphenols.

[0114] Example 2

[0115] The optimal composite prebiotic combination screened in Example 1 is applied to the preparation of food, and a composite prebiotic soft candy is provided, and the preparation method thereof comprises the following steps:

[0116] Step 1, laboratory trial production

[0117] Gelatin and purified water are mixed in a certain proportion, fully swollen, and dissolved in a water bath at 70°C for standby. An appropriate amount of malt syrup is weighed, and the water bath is heated before the glue liquid is added and mixed evenly. After adding an appropriate amount of citric acid, essence and an appropriate amount of pigment, stirring evenly, 60°C water bath preservation. The mold is oiled, and the prepared liquid is poured into the mold and cooled to shape for 12h before demolding.

[0118] The effects of different amounts of maltitol, gelatin addition, and different amounts of water on the sensory quality of gelatin soft candy are shown in Table 3. The optimal gelatin soft candy formula is: 70% maltitol liquid, 10% gelatin content, gelatin: water = 1:3, 0.15% citric acid, 3% essence, and an appropriate amount of pigment. Under this formula, the gelatin soft candy has the best hardness, elasticity, cohesiveness, color and flavor.

[0119] Table 3 Sensory evaluation results of soft candy

[0120]

[0121] Step 2, small-scale trial production in the factory

[0122] Gelatin, pectin and purified water are mixed in a certain proportion, fully swollen, and dissolved in a water bath at 70°C for standby. An appropriate amount of malt syrup is weighed, and the water bath is heated before the glue liquid is added and mixed evenly. After adding a composite prebiotic (5% w / w fructooligosaccharide, 5% w / w stachyose and 0.4% w / w tea polyphenol), an appropriate amount of citric acid, essence and an appropriate amount of pigment, stirring evenly, 60°C water bath preservation. The mold is oiled, and the prepared liquid is poured into the mold and cooled to shape for 12h before demolding. The product is as shown in Figure 13 .

[0123] Finally, it should be noted that the above is only used to illustrate the technical solutions of the present application and is not limiting. Although the present application has been described in detail with reference to the preferred arrangement, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application.

Claims

1. A method for evaluating prebiotics and their formulated combinations based on an in vitro simulated fermentation model - co-cultured cell model, characterized in that, The method comprises the following steps: Step 1, preparation of fecal bacteria suspension The feces are taken from a healthy volunteer aged 22-26 years old who has not taken antibiotics, probiotics or prebiotics within three months, and the preparation of the fecal bacteria suspension is completed within 30 minutes after collection; the fecal sample is diluted with a phosphate buffer solution to obtain the fecal bacteria suspension, which is stored anaerobically at 4°C; Step 2, preparation of in vitro fermentation medium The in vitro fermentation medium is prepared according to the following formula: 2 g / L peptone, 2 g / L yeast extract, 0.1 g / L NaCl, 40 mg / L K2HPO4, 40 mg / L KH2PO4, 10 mg / L MgSO4·7H2O, 10 mg / L CaCl2·6H2O, 2 g / L NaHCO3, 0.05% L-cysteine, 0.5 g / L cholate, 10 μl / L vitamin K, 2 mL / L Tween 80, and 50 mg / L hematin, and the pH is adjusted to 7.5; Step 3, in vitro fermentation culture of single prebiotic and determination of indexes The filtered and sterilized single prebiotic to be tested is added to the sterilized fermentation medium; The fecal bacteria suspension of step 1 is inoculated into the in vitro fermentation medium of step 2, and anaerobic fermentation is carried out at 37°C; after the fermentation is terminated, the fermentation broth is taken to determine the pH; in addition, the fermentation broth is centrifuged at low temperature, and the supernatant is used for detection and analysis including antioxidant capacity and short-chain fatty acids (SCFAs); the bacterial cell pellet after centrifugation is used for detection and analysis including DNA extraction and microbiota sequencing; Step 4, in vitro fermentation culture of complex prebiotic and determination of indexes The in vitro fermentation medium is prepared according to the same formula as step 2, and the fermentation medium is adjusted to pH 7.2; The filtered and sterilized complex prebiotic combination to be tested is added to the sterilized in vitro fermentation medium; the fecal bacteria suspension of step 1 is inoculated into the in vitro fermentation medium, and anaerobic fermentation is carried out at 37°C; after the fermentation is terminated, the fermentation broth is taken to determine the pH; in addition, the fermentation broth is centrifuged at low temperature, and the supernatant is used for detection and analysis including antioxidant capacity and short-chain fatty acids (SCFAs); the bacterial cell pellet after centrifugation is used for detection and analysis including DNA extraction and microbiota sequencing; Step 5, establishment of cell co-culture model Caco-2 cells are inoculated in the upper chamber of a Transwell, and after the cells are completely differentiated, RAW 264.7 cells are inoculated into a tissue culture plate and cultured for 24 h to promote the obvious adhesion of the basolateral pores, thereby constructing a cell co-culture model; Step 6, co-culture of complex prebiotic and cells and determination of indexes After the cell co-culture model is established for 24 h, the Transwell upper chamber is treated with the complex prebiotic combination fermentation supernatant to be tested, the culture plate basal side is treated with LPS after 24 h, and after another 24 h of incubation, the culture medium and cells are collected for analysis, including determination of intestinal barrier permeability, NO, ROS and TNF-α levels; Step 7, determination of optimal single prebiotic and complex prebiotic Based on the determination results of the in vitro fermentation culture and cell co-culture described above, the optimal single prebiotic and the optimal complex prebiotic combination are screened according to the short-chain fatty acid yield and the regulation of microbiota.

2. The method of claim 1, wherein, The single prebiotics include polyphenols and oligosaccharides; and the complex prebiotics are compositions formed by different single prebiotics.

3. The method of claim 1, wherein, In steps 3 and 4, the determination of the antioxidant capacity includes determination of DPPH free radical scavenging capacity, ABTS free radical scavenging capacity and total antioxidant capacity.

4. The method of claim 3, wherein, The specific steps of the determination of the antioxidant capacity include: S31, determination of DPPH free radical scavenging capacity: The experimental group is 25 μL of the fermentation liquid sample added with 175 μL of 0.2 mM DPPH ethanol solution, the blank group is 25 μL of the negative control fermentation liquid added with 175 μL of 0.2 mM DPPH ethanol solution; the control group is 25 μL of the fermentation liquid sample added with 175 μL of anhydrous ethanol; after reaction at room temperature for 30 min, the OD value at 517 nm is determined, and the DPPH free radical scavenging rate is calculated according to the following formula: In the formula, A1 is the absorbance of the blank group; A2 is the absorbance of the experimental group; and A3 is the absorbance of the control group; S32, determination of ABTS free radical scavenging capacity: The sample is diluted with distilled water to an appropriate concentration, the experimental group is 20 μL of the sample added with 180 μL of ABTS working solution, the blank group is 20 μL of the negative control fermentation liquid sample added with 180 μL of ABTS working solution, and the control group is 20 μL of the fermentation liquid sample added with 180 μL of deionized water; after reaction at room temperature for 10 min, the OD value at 734 nm is determined, and the ABTS free radical scavenging rate is calculated according to the following formula: In the formula, A1 is the absorbance of the blank group; A2 is the absorbance of the experimental group; and A3 is the absorbance of the control group; S33, determination of total antioxidant capacity: 0.3 mM sodium acetate buffer solution, 10 mM 2,4,6-tri-2-pyridyl-1,3,5-triazine TPTZ and 20 mM FeCl3·6H2O are mixed at a volume ratio of 10:1:1, incubated at 37℃ for 15 min to prepare FRAP working solution, and then the sample is diluted to an appropriate concentration; 20 μL of the fermentation liquid sample is reacted with 180 μL of the FRAP working solution at 37℃ for 30 min in the dark, the absorbance is read at 593 nm, and the FRAP value is calculated according to the following formula with 1 mM FeSO4 as a reference: In the formula, B1 is the absorbance of the sample and the FRAP working solution; B2 is the absorbance of FeSO4 and the FRAP working solution; and C0 is the concentration of FeSO4.

5. The method of claim 1, wherein, The specific steps of the determination of SCFAs in steps 3 and 4 include: The SCFA yield in the fermentation supernatant is analyzed by using a gas chromatograph: 0.4 mL of 50% v / v sulfuric acid and 2 mL of ether are added to 2 mL of the fermentation supernatant, vortexed for 3 min and then centrifuged, the upper organic component is collected, filtered through a 0.22 μm organic filter membrane and then placed in a chromatography bottle; then the contents of acetic acid, propionic acid, butyric acid and isobutyric acid are determined by using a gas chromatograph external standard curve method; An AT-WAX column was used, the oven temperature was kept at 90°C for 5 min, and then increased to 200°C at a rate of 20°C / min, and kept for 3 min; the injector temperature was set at 250°C, 1 μL sample was injected into the instrument at a split ratio of 1:10, N2 was used as the carrier gas at a flow rate of 1 mL / min, and the detector temperature was set at 250°C.

6. The method of claim 1, wherein, The specific steps of DNA extraction and flora sequencing in steps 3 and 4 are as follows: The fermentation broth flora sample was subjected to total DNA extraction according to the experimental operation of the soil and fecal genomic DNA extraction kit, and the obtained DNA was used as a template to perform PCR amplification of the V3-V4 region of the bacterial 16S rRNA gene using primers 341F and 806R; After purification of the PCR products, the following steps were performed: Ultra™II FS DNA-PCR free library construction kit was used to generate sequencing library; after the sequencing library passed the detection, Illumina Novaseq 6000 was used for PE250 on-machine sequencing.

7. Use of the method according to any one of claims 1 to 6 for evaluating and screening individual prebiotics and prebiotic combinations.

8. A prebiotic composition of matter selected by the method of any one of claims 1 to 6, characterized in that, The prebiotic combination composition comprises, in mass percentage: fructooligosaccharides 5%, stachyose 5%, and tea polyphenols 0.4%.

9. Use of the prebiotic combination composition according to claim 8 for preparing food or health food.

10. Use of the prebiotic combination composition according to claim 8 for antioxidation activity and synergistic anti-inflammatory effect of short-chain fatty acids.

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