Use of lactic acid bacteria fermentation product for regulating gastrointestinal tract

Abstract

This invention proposes the use of a lactic acid bacteria ferment for preparing a composition for gastrointestinal conditioning, wherein the lactic acid bacteria ferment comprises a postbiotic obtained by fermenting a matrix containing yam extract, hericium erinaceus extract, tremella extract, and okra extract using Lactobacillus paracasei strain ET-66 and Lactobacillus plantarum strain LPL28.

Description

Technical Field

[0001] This invention relates to the use of lactic acid bacteria ferments, and particularly to the use of lactic acid bacteria ferments for gastrointestinal conditioning. Prior Technology

[0002] The human body possesses multiple layers of mucosal epithelium, forming a direct barrier between the intestinal environment and the host's internal environment. The gastrointestinal tract, as one of the largest luminal regions within this barrier, plays a crucial role in the immune system, thus influencing health. The gastrointestinal mucosa has complex functions, acting as a semi-permeable barrier to allow the absorption of nutrients and immune sensing, while restricting the transport of potentially harmful antigens and microorganisms. This seemingly "conflicting" function is primarily achieved through the interplay of structural composition and molecular mechanisms within the intestinal mucosa, which operates dynamically to maintain intestinal integrity and immune stability. The function of the intestinal barrier can be impaired by severe structural damage to the mucosa or subtle changes in its regulatory composition.

[0003] Defects in the intestinal barrier are associated with many diseases, including gastrointestinal diseases (such as celiac disease, inflammatory bowel disease, and colorectal cancer) and extraintestinal diseases (such as chronic hepatitis, type 1 diabetes, and obesity). For these diseases, it is generally assumed that impaired intestinal barrier function and uncontrolled flow of antigens across the intestinal epithelium may challenge the immune system of susceptible individuals and affect the balance between the host and microbes, thus triggering inflammatory mechanisms in the intestine or organs further afield.

[0004] In conclusion, the development of compositions that can improve the gastrointestinal barrier is indeed of great benefit to those skilled in the art. Summary of the Invention

[0005] This invention is based on the discovery that the postbiotic obtained by fermenting a matrix containing yam extract, hericium erinaceus extract, tremella extract and okra extract with lactic acid bacteria can improve the gastrointestinal barrier, and therefore the obtained postbiotic can be used for gastrointestinal conditioning.

[0006] Accordingly, the present invention aims to provide the use of a lactic acid bacteria ferment for preparing a composition for gastrointestinal conditioning, wherein the lactic acid bacteria ferment comprises a postbiotic obtained by fermenting a matrix containing yam extract, lion's mane mushroom extract, tremella extract, and okra extract using Lactobacillus paracasei ET-66 strain (accession number: BCRC 910753) and Lactobacillus plantarum LPL28 strain (accession number: BCRC 910536).

[0007] In the example implementation, the gastrointestinal conditioning composition is used to improve the structure and function of the gastrointestinal mucosal epithelium, prevent pathogen infection, promote the colonization of probiotics, or regulate the gastrointestinal flora.

[0008] In the example implementation, the gastrointestinal conditioning composition is used to treat or prevent celiac disease, inflammatory bowel disease, colorectal cancer, diarrhea, gastroenteritis, irritable bowel syndrome, typhoid fever, short bowel syndrome, or small intestinal bacterial overgrowth.

[0009] In the example implementation, based on the total weight of the yam extract, the yam extract contains more than 16 wt% diosgenin; based on the total weight of the hericium erinaceus extract, the hericium erinaceus extract contains more than 10 wt% polysaccharides and more than 15 ppm erinacines A; based on the total weight of the tremella extract, the tremella extract contains more than 80 wt% polysaccharides; and based on the total weight of the okra extract, the okra extract contains more than 0.5 wt% isoquercitrin.

[0010] In the example implementation, the method for preparing lactic acid bacteria fermentation products includes: inoculating *Lactobacillus paracasei* ET-66 strain and *Lactobacillus plantarum* LPL28 strain into a substrate containing yam extract, *Hericium erinaceus* extract, *Tremella fuciformis* extract, and okra extract and culturing them, wherein the colony-forming unit (CFU) ratio between *Lactobacillus paracasei* ET-66 strain and *Lactobacillus plantarum* LPL28 strain is 1:(0.5 to 2).

[0011] In the example implementation, the total weight of yam extract, hericium erinaceus extract, tremella extract and okra extract in each liter of matrix is ​​30g to 120g.

[0012] In the example implementation, the weight ratio of yam extract, hericium erinaceus extract, tremella extract, and okra extract is 1:(0.5 to 2):(0.5 to 2):(0.5 to 2).

[0013] In the example implementation, the incubation time is 8 to 48 hours.

[0014] In the example implementation, the inoculation colony-forming unit ratio between *Lactobacillus paracasei* strain ET-66 and *Lactobacillus plantarum* strain LPL28 was 1:1; the total weight of yam extract, *Hericium erinaceus* extract, *Tremella fuciformis* extract, and okra extract per liter of substrate was 60 g; the weight ratio of yam extract, *Hericium erinaceus* extract, *Tremella fuciformis* extract, and okra extract was 1:1:0.5:0.5; and the incubation time was 16 hours.

[0015] In the example implementation, the method for preparing lactic acid bacteria fermentation product further includes: performing solid-liquid separation on the cultured substrate to obtain fermentation broth. Simple Explanation of the Diagram

[0016] Figure 1 is a curve graph showing the changes in substrate pH after different strains were cultured in single or multiple culture formats; Figure 2 is a graph showing the changes in substrate pH after culturing Lactobacillus paracasei ET-66 and Lactobacillus plantarum LPL28 strains at different colony-forming unit ratios. "1:1" indicates a colony-forming unit ratio of 1:1 between ET-66 and LPL28 strains, "1:2" indicates a ratio of 1:2 between ET-66 and LPL28 strains, and "2:1" indicates a ratio of 2:1 between ET-66 and LPL28 strains. Figure 3 is a curve graph showing the effect of substrate fermentation versus non-fermentation on the growth of Lactobacillus rhamnosus; Figure 4 is a curve graph showing the effect of fermented and unfermented substrates on the growth of Bifidobacterium animalis; Figure 5 is a bar graph showing the effect of fermented and unfermented substrates on the rate of change in transepithelial electrical resistance of AGS-treated human gastric epithelial cells, where *** indicates P < 0.001 and ## indicates P < 0.01; Figure 6 is a bar graph showing the effect of different doses of fermented and unfermented substrate on the rate of change of transepithelial electrical resistance in AGS human gastric epithelial cells, where *** indicates P < 0.001 and ## indicates P < 0.01; Figure 7 is a bar graph showing the effect of fermented and unfermented substrates on the rate of change in transepithelial electrical resistance of Caco-2 human intestinal epithelial cells, where *** indicates P < 0.001 and ### indicates P < 0.001; Figure 8 is a bar graph showing the effect of different doses of fermented and unfermented substrate on the rate of change of transepithelial electrical resistance in Caco-2 human intestinal epithelial cells, where *** indicates P < 0.001 and ## indicates P < 0.01; Figure 9 is a bar graph showing the effect of fermented and unfermented substrates on ZO-1 expression levels in AGS-treated human gastric epithelial cells, where * indicates P < 0.05 and # indicates P < 0.05; Figure 10 is a bar graph showing the effect of different doses of fermented and unfermented substrate on ZO-1 expression in AGS-treated human gastric epithelial cells. * indicates P < 0.05, and # indicates P < 0.05. Figure 11 is a bar graph showing the effect of fermented and unfermented substrates on JAM-A expression levels in AGS-treated human gastric epithelial cells, where * indicates P < 0.05 and ## indicates P < 0.01; Figure 12 is a bar graph showing the effect of different doses of fermented and unfermented substrate on JAM-A expression levels in AGS-treated human gastric epithelial cells, where *** indicates P < 0.001 and ## indicates P < 0.01; Figure 13 is a bar chart showing the effect of fermented and unfermented substrates on the expression level of occludin in Caco-2 human intestinal epithelial cells, where * indicates P < 0.05 and ### indicates P < 0.001; Figure 14 is a bar graph showing the effect of fermented and unfermented substrates on the expression level of occludin in Caco-2 human intestinal epithelial cells, where * indicates P<0.05 and # indicates P<0.05. Implementation

[0017] To make the above and / or other objects, effects, and features of the present invention more apparent and understandable, preferred embodiments are described in detail below:

[0018] The present invention found that the obtained metagenes can increase the transepithelial resistance of human gastric epithelial cells and intestinal epithelial cells, and increase the gene expression of tight junction-related proteins. Therefore, the obtained metagenes can improve the gastrointestinal barrier and can be applied to gastrointestinal conditioning.

[0019] One embodiment of the present invention provides a lactic acid bacteria ferment, which can be prepared into a gastrointestinal conditioning composition. The lactic acid bacteria ferment comprises a postbiotic obtained by fermenting a matrix containing yam extract, lion's mane mushroom extract, tremella extract, and okra extract using *Lactobacillus paracasei* strain ET-66 and *Lactobacillus plantarum* strain LPL28. The resulting gastrointestinal conditioning composition can be a pharmaceutical composition or a food composition. Furthermore, the resulting gastrointestinal conditioning composition can improve the structure and function of the gastrointestinal mucosal epithelium, prevent pathogen infection, promote probiotic colonization, or regulate the gastrointestinal flora. It can also treat or prevent celiac disease, inflammatory bowel disease, colorectal cancer, diarrhea, gastroenteritis, irritable bowel syndrome, typhoid fever, short bowel syndrome, or small intestinal bacterial overgrowth.

[0020] Yam extract can be prepared using food-grade extraction solvents such as water, ethanol, vegetable oil, glycerol, propylene glycol, or supercritical carbon dioxide; however, regardless of the extraction solvent used, the yam extract preferably contains at least 16 wt% diosgenin based on the total weight of the yam extract. Hericium erinaceus extract can be prepared using food-grade extraction solvents such as water, ethanol, vegetable oil, glycerol, propylene glycol, or supercritical carbon dioxide; however, regardless of the extraction solvent used, the Hericium erinaceus extract preferably contains at least 10 wt% polysaccharides and at least 15 ppm hericin A based on the total weight of the Hericium erinaceus extract. Tremella fuciformis extract can be prepared using food-grade extraction solvents such as water, ethanol, vegetable oil, glycerol, propylene glycol, or supercritical carbon dioxide; however, regardless of the extraction solvent used, the Tremella fuciformis extract preferably contains at least 80 wt% polysaccharides based on the total weight of the Tremella fuciformis extract. Okra extract can be prepared using food-grade extraction solvents such as water, ethanol, vegetable oil, glycerol, propylene glycol, or supercritical carbon dioxide; however, regardless of the extraction solvent used, the okra extract preferably contains at least 0.5 wt% isoquercitrin based on the total weight of the okra extract. Information regarding the presence of *Lactobacillus paracasei* strain ET-66 and *Lactobacillus plantarum* strain LPL28 is listed in Table 1. Table 1. Registered Information strain Deposit number Deposit Date Lactobacillus paracasei (Lactobacillus paracasei) ET-66 strain BCRC 910753 November 3, 2016 CGMCC 13514 December 29, 2016 Lactobacillus plantarum (Lactobacillus plantarum) LPL28 strain BCRC 910536 December 27, 2011 CGMCC 17954 June 18, 2019

[0021] A method for preparing lactic acid bacteria fermentation products may include the following steps: inoculating *Lactobacillus paracasei* ET-66 strain and *Lactobacillus plantarum* LPL28 strain into a substrate containing yam extract, *Hericium erinaceus* extract, *Tremella fuciformis* extract, and okra extract, and culturing them, wherein the inoculation colony-forming unit ratio between *Lactobacillus paracasei* ET-66 strain and *Lactobacillus plantarum* LPL28 strain is 1:(0.5 to 2), preferably 1:(0.5, 1, 1.5, or 2). Furthermore, the preparation method may further include: performing solid-liquid separation on the cultured substrate to obtain a fermentation broth, thereby using the fermentation broth as the lactic acid bacteria fermentation product. Additionally, the preparation method may further include: drying the obtained fermentation broth to obtain a fermentation powder, thereby using the fermentation powder as the lactic acid bacteria fermentation product.

[0022] In each liter of matrix, the total weight of yam extract, hericium erinaceus extract, tremella extract, and okra extract can be from 30g to 120g, preferably 30g, 60g, 90g, or 120g. Moreover, the weight ratio between yam extract, hericium erinaceus extract, tremella extract, and okra extract can be 1:(0.5 to 2):(0.5 to 2):(0.5 to 2), preferably 1:(0.5, 1, 1.5, or 2):(0.5, 1, 1.5, or 2):(0.5, 1, 1.5, or 2).

[0023] The incubation time can be from 8 hours to 48 hours, preferably 8 hours, 16 hours, 24 hours, 32 hours, 40 hours, or 48 hours. Moreover, the incubation temperature can be from 30°C to 42°C, preferably 30°C, 32°C, 34°C, 36°C, 37°C, 38°C, 40°C, or 42°C.

[0024] The pH of the matrix can be from 5.0 to 6.5, preferably 5.0, 5.5, 6, or 6.5. Furthermore, in addition to the above-mentioned extracts, the matrix may contain 2 wt% to 15 wt% skim milk powder, 3 wt% to 10 wt% soy protein isolate, 1 wt% to 5 wt% yeast extract, 1 wt% to 5 wt% glucose, 0.05 wt% to 0.2 wt% potassium dihydrogen phosphate, 0 wt% to 0.1 wt% sodium citrate, 0.05 wt% to 0.2 wt% polysorbate 80, 0 wt% to 0.05 wt% magnesium sulfate, 0 wt% to 0.1 wt% manganese sulfate, and the balance being water.

[0025] The following specific examples further illustrate the implementation of the present invention:

[0026] Example 1: Optimization of fermentation conditions

[0027] The fermentation substrate formula is as follows: 2wt% to 15wt% skim milk powder, 3wt% to 10wt% soy protein isolate, 1wt% to 5wt% yeast extract, 1wt% to 5wt% glucose, 0.05wt% to 0.2wt% potassium dihydrogen phosphate, 0wt% to 0.1wt% sodium citrate, 0.05wt% to 0.2wt% polysorbate 80, 0wt% to 0.05wt% magnesium sulfate, 0wt% to 0.1wt% manganese sulfate, yam extract (active ingredient: more than 16wt% diosgenin), hericium erinaceus extract (active ingredient: more than 10wt% polysaccharides and more than 15ppm hericin A), tremella extract (active ingredient: more than 80wt% polysaccharides), okra extract (active ingredient: more than 0.5wt% isoquercitrin), and the balance water.

[0028] First, different bacterial strains were cultured in the fermentation substrate at 37℃ in single or multi-strain mode, and the pH changes of the fermentation substrate were observed at different time points. The total colony-forming units (CFU) were the same in both single and multi-strain modes, and the CFU of all strains in the multi-strain mode were also the same. The fermentation endpoint was set at pH 5.0, representing a strong fermentation capacity of the strains in the fermentation substrate. As shown in Figure 1, culturing *Lactobacillus paracasei* ET-66 and *Lactobacillus plantarum* LPL28 in the multi-strain mode for 16 hours was the most suitable fermentation condition. As shown in Figure 2, culturing *Lactobacillus paracasei* ET-66 and *Lactobacillus plantarum* LPL28 in a 1:1 CFU ratio for 16 hours was the most suitable fermentation condition.

[0029] Next, *Lactobacillus paracasei* ET-66 and *Lactobacillus plantarum* LPL28 were cultured in a fermentation substrate at 37°C for 16 hours at a colony-forming unit ratio of 1:1. The fermentation broth was then obtained by centrifugation and dried to obtain fermentation powder. Unless otherwise defined, the "low-dose substrate," "medium-dose substrate," and "high-dose substrate" used in this paper have roughly the same composition, except that the "low-dose substrate" contains 10 g / L of yam extract, 10 g / L of Hericium erinaceus extract, 5 g / L of Tremella fuciformis extract, and 5 g / L of okra extract; the "medium-dose substrate" contains 20 g / L of yam extract, 20 g / L of Hericium erinaceus extract, 10 g / L of Tremella fuciformis extract, and 10 g / L of okra extract; and the "high-dose substrate" contains 40 g / L of yam extract, 40 g / L of Hericium erinaceus extract, 20 g / L of Tremella fuciformis extract, and 20 g / L of okra extract.

[0030] Then, *Lactobacillus rhamnosus* and *Bifidobacterium animalis* were cultured in a substrate supplemented with fermentation powder. As shown in Figures 3 and 4, the fermentation powder obtained from a medium dose of substrate was beneficial to the growth of probiotics.

[0031] Example 2: Transepithelial Resistance Analysis

[0032] Transepithelial electrical resistance (TEER) analysis can assess the impact of fermentation powder on epithelial barrier function.

[0033] AGS Human Gastric Epithelial Cells

[0034] AGS cells were seeded at 2 x 10⁵ cells per well in 6-well transwell inserts and cultured for 6 days, with the culture medium changed every 3 days. After removing the culture medium, fresh RPMI-1640 medium was added, with 3 mL for the upper layer and 2 mL for the lower layer, and the initial transmembrane resistance was measured. After removing the culture medium, RPMI-1640 medium containing fermentation powder was added, with 3 mL for the upper layer and 2 mL for the lower layer, and the cells were co-cultured with the AGS cells for 5 hours. After the culture was completed, the culture medium was removed and the cells were washed twice with PBS. Finally, fresh RPMI-1640 medium was added, and the final transmembrane resistance was measured. The formula for the rate of change of transepithelial resistance is as follows: Transepithelial resistivity change rate = x100%.

[0035] As shown in Figure 5, compared with the unfermented powder obtained from the medium-dose matrix, the unfermented powder obtained from the matrix containing 60 g / L yam extract, the unfermented powder obtained from the matrix containing 60 g / L Hericium erinaceus extract, the unfermented powder obtained from the matrix containing 60 g / L Tremella fuciformis extract, and the unfermented powder obtained from the matrix containing 60 g / L okra extract, the fermented powder obtained from the medium-dose matrix can increase the transmembrane resistance of AGS cells, indicating that the fermented matrix can strengthen the gastrointestinal epithelial barrier function.

[0036] As shown in Figure 6, compared with the unfermented powder obtained from the corresponding dose matrix and the control group, the fermented powder obtained from the medium dose matrix can increase the transmembrane resistance of AGS cells, indicating that it can enhance the gastrointestinal epithelial barrier function.

[0037] Caco-2 Human Intestinal Epithelial Cells

[0038] Caco-2 cells were seeded at 3 x 10⁵ cells per well in 6-well transwell inserts and cultured for 6 days, with the culture medium changed every 3 days. After removing the culture medium, fresh DMEM culture medium was added (3 mL for the upper layer and 2 mL for the lower layer), and the initial transmembrane resistance was measured using electrodes. After removing the culture medium, DMEM culture medium containing fermentation powder was added (3 mL for the upper layer and 2 mL for the lower layer), and the Caco-2 cells were co-cultured for 5 hours. After culture, the culture medium was removed, and the cells were washed twice with PBS. Finally, fresh DMEM culture medium was added, and the final transmembrane resistance was measured using electrodes. The formula for the rate of change of transepithelial resistance is as follows: Transepithelial resistivity change rate = x100%.

[0039] As shown in Figure 7, compared with the unfermented powder obtained from the medium-dose matrix, the unfermented powder obtained from the matrix containing 60 g / L yam extract, the unfermented powder obtained from the matrix containing 60 g / L Hericium erinaceus extract, the unfermented powder obtained from the matrix containing 60 g / L Tremella fuciformis extract, and the unfermented powder obtained from the matrix containing 60 g / L okra extract, the fermented powder obtained from the medium-dose matrix can increase the transmembrane resistance of Caco-2 cells, indicating that the fermented matrix can strengthen the intestinal epithelial barrier function.

[0040] As shown in Figure 8, compared with the unfermented powder obtained from the corresponding dose matrix and the control group, the fermented powder obtained from the medium dose matrix can increase the transmembrane resistance of Caco-2 cells, indicating that it can strengthen the intestinal epithelial barrier function.

[0041] Example 3: Gene expression analysis of tight junction-related proteins

[0042] Real-time quantitative PCR can analyze whether fermentation powder positively regulates the gene expression of tight junction-related proteins (such as ZO-1, occludin, and JAM-A), and confirm whether it can promote the formation or stability of intercellular tight junctions, thus supporting its potential application in gastrointestinal mucosal protection.

[0043] AGS Human Gastric Epithelial Cells

[0044] AGS cells were seeded at 2 x 10⁵ cells per well in 6-well cell culture dishes and cultured for 6 days, with the culture medium changed every 3 days. After removing the culture medium, the cells were washed twice with PBS, and then RPMI-1640 culture medium containing fermentation powder was added and co-cultured with the AGS cells for 5 hours. After the culture was completed, the culture medium was removed and the cells were washed twice with PBS. Cells were collected by adding 500 μL of RX buffer to each well, and RNA was extracted using a VIOGENE Total RNA Mini kit (model: GR1001). The RNA was reverse transcribed into cDNA using Promega GoScript™ reverse transcriptase. Finally, real-time quantitative PCR was performed, and the relative expression level of the target gene was calculated using the 2-ΔΔCt method.

[0045] As shown in Figure 9, compared with the unfermented powder obtained from the medium-dose matrix, the unfermented powder obtained from the matrix containing 60 g / L yam extract, the unfermented powder obtained from the matrix containing 60 g / L Hericium erinaceus extract, the unfermented powder obtained from the matrix containing 60 g / L Tremella fuciformis extract, and the unfermented powder obtained from the matrix containing 60 g / L okra extract, the fermented powder obtained from the medium-dose matrix can increase the ZO-1 expression level of AGS cells, indicating that the fermented matrix can strengthen the tight junctions of the gastrointestinal epithelium.

[0046] As shown in Figure 10, compared with the unfermented powder obtained from the corresponding dose matrix and the control group, the fermented powder obtained from the medium dose matrix can increase the ZO-1 expression level of AGS cells, indicating that it can strengthen the tight junctions of the gastric epithelium.

[0047] As shown in Figure 11, compared with the unfermented powder obtained from the medium-dose matrix, the unfermented powder obtained from the matrix containing 60 g / L yam extract, the unfermented powder obtained from the matrix containing 60 g / L Hericium erinaceus extract, the unfermented powder obtained from the matrix containing 60 g / L Tremella fuciformis extract, and the unfermented powder obtained from the matrix containing 60 g / L okra extract, the fermented powder obtained from the medium-dose matrix can increase the JAM-A expression level of AGS cells, indicating that the fermented matrix can strengthen the tight junctions of the gastrointestinal epithelium.

[0048] As shown in Figure 12, compared with the unfermented powder obtained from the corresponding dose matrix and the control group, the fermented powder obtained from the medium dose matrix can increase the JAM-A expression level of AGS cells, indicating that it can strengthen the tight junctions of the gastric epithelium.

[0049] Caco-2 Human Intestinal Epithelial Cells

[0050] Caco-2 cells were seeded at 2 x 10⁵ cells per well in 6-well cell culture dishes and cultured for 6 days, with the culture medium changed every 3 days. After removing the culture medium, the cells were washed twice with PBS, then DMEM medium containing fermentation powder was added, and the cells were co-cultured with the Caco-2 cells for 5 hours. After the culture was completed, the culture medium was removed, and the cells were washed twice with PBS. Cells were collected by adding 500 μL of RX buffer to each well, and RNA was extracted using a VIOGENE Total RNA Mini kit (model: GR1001). The RNA was reverse transcribed into cDNA using Promega GoScript™ reverse transcriptase. Finally, real-time quantitative PCR was performed, and the relative expression level of the target gene was calculated using the 2-ΔΔCt method.

[0051] As shown in Figure 13, compared with the unfermented powder obtained from the medium-dose matrix, the unfermented powder obtained from the matrix containing 60 g / L yam extract, the unfermented powder obtained from the matrix containing 60 g / L Hericium erinaceus extract, the unfermented powder obtained from the matrix containing 60 g / L Tremella fuciformis extract, and the unfermented powder obtained from the matrix containing 60 g / L okra extract, the fermented powder obtained from the medium-dose matrix can increase the occludin expression level in Caco-2 cells, indicating that the fermented matrix can strengthen the tight junctions of the intestinal epithelium.

[0052] As shown in Figure 14, compared with the unfermented powder obtained from the corresponding dose matrix and the control group, the fermented powder obtained from the medium dose matrix can increase the occludin expression level in Caco-2 cells, indicating that it can strengthen the tight junctions of the intestinal epithelium.

[0053] However, the above description is only a preferred embodiment of the present invention, and should not be construed as limiting the scope of the present invention. Therefore, any simple equivalent changes and modifications made in accordance with the scope of the patent application and the contents of the specification of the present invention shall still fall within the scope of the patent of the present invention.

[0054] 1. Biological Resource Conservation and Research Center, Food Industry Development Research Institute, Republic of China, Taiwan, 2016 / 11 / 03, BCRC 910753; 2. Biological Resource Conservation and Research Center, Food Industry Development Research Institute, Republic of China, Taiwan, 2011 / 12 / 27, BCRC 910536.

Claims

1. The use of a lactic acid bacteria ferment for preparing a composition for gastrointestinal conditioning, wherein the lactic acid bacteria ferment comprises a postbiotic obtained by fermenting a matrix containing yam extract, hericium erinaceus extract, tremella fuciformis extract, and okra extract with Lactobacillus paracasei ET-66 strain (accession number: BCRC 910753) and Lactobacillus plantarum LPL28 strain (accession number: BCRC 910536); wherein the inoculum colony-forming unit ratio between Lactobacillus paracasei ET-66 strain and Lactobacillus plantarum LPL28 strain is 1:1; the total weight of the yam extract, hericium erinaceus extract, tremella fuciformis extract, and okra extract per liter of the matrix is ​​60g; and the weight ratio of the yam extract, hericium erinaceus extract, tremella fuciformis extract, and okra extract is 1:1:0.5:0.

5.

2. The use as described in claim 1, wherein the gastrointestinal conditioning composition is used to improve the structure and function of the gastrointestinal mucosal epithelium, prevent pathogen infection, promote the colonization of probiotics, or regulate the gastrointestinal flora.

3. The use as described in claim 1, wherein, based on the total weight of the yam extract, the yam extract contains more than 16 wt% diosgenin; based on the total weight of the Hericium erinaceus extract, the Hericium erinaceus extract contains more than 10 wt% polysaccharides and more than 15 ppm erinacines A; based on the total weight of the Tremella fuciformis extract, the Tremella fuciformis extract contains more than 80 wt% polysaccharides; and based on the total weight of the okra extract, the okra extract contains more than 0.5 wt% isoquercitrin.

4. The use as described in claim 1, wherein the method for preparing the lactic acid bacteria ferment includes: The *Lactobacillus paracasei* strain ET-66 and the *Lactobacillus plantarum* strain LPL28 were inoculated into a substrate containing yam extract, *Hericium erinaceus* extract, *Tremella fuciformis* extract, and okra extract and cultured for 8 to 48 hours.

5. The use as described in claim 4, wherein the incubation time is 16 hours.

6. The use as described in claim 4, wherein the method for preparing the lactic acid bacteria ferment further comprises: The cultured substrate was subjected to solid-liquid separation to obtain the fermentation broth.

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