High dietary fiber wholemeal flour and method for preparing the same by composite probiotic fermentation

Abstract

This invention relates to the field of functional food processing technology, specifically to a high-fiber whole wheat flour for improving intestinal barrier function and its preparation method by fermentation with compound probiotics. The high-fiber whole wheat flour is produced by fermenting whole wheat flour, wheat bran, and barley grass with compound probiotics. The high-fiber whole wheat flour contains the following components: total dietary fiber content of 40-45%, soluble dietary fiber content of 15-18%, β-glucan content of 8.5-10%, pheophytic acid content of 50-100 mg / 100g, short-chain fatty acid content of not less than 500 mg / 100g, total polyphenol content of 800-1200 mg GAE / 100g, and ferulic acid content of 150-200 mg / 100g. It achieves a high-efficiency conversion of insoluble dietary fiber to soluble dietary fiber, with a conversion rate exceeding 45%.

Description

Technical Field

[0001] This invention relates to the field of functional food processing technology, specifically to a method for preparing high-fiber whole wheat flour and its compound probiotic fermentation, which is particularly suitable for the development of special medical foods for diabetes, obesity, colitis, etc. Background Technology

[0002] Dietary fiber is an essential non-digestible carbohydrate that plays an important role in maintaining gut health, regulating blood sugar, and lowering blood lipids. Whole wheat flour is rich in dietary fiber; however, most of it is insoluble, resulting in low bioavailability and limiting its application in functional foods.

[0003] Currently, the main methods for improving the utilization rate of dietary fiber in whole wheat flour include physical treatment, chemical treatment, and bio-fermentation. Liao, AM, Zhang, J., Yang, ZL, Huang, JH, & Pan, L. (2022). (Structural, physicochemical, and functional properties of wheat braninsoluble dietary fiber modified with probiotic fermentation. Frontiers in Nutrition, 9, 803440) points out that while physical methods such as ultrafine grinding can improve the dispersibility of dietary fiber, they cannot substantially change its chemical structure; chemical treatments such as alkali treatment can increase the content of soluble dietary fiber, but they easily introduce harmful substances; while bio-fermentation is receiving increasing attention due to its safety and efficiency.

[0004] In existing technologies, Wang, Z., Ma, S., Li, L., & Huang, J. (2022). (Effect of wheat bran dietary fiber on structural properties and hydrolysis behavior of gluten after synergistic fermentation of Lactobacillus plantarum and Saccharomyces cerevisiae. Frontiers in Nutrition, 9, 982878) reported a method for the co-fermentation of whole wheat products by Lactobacillus plantarum and yeast, which can improve the bioavailability of dietary fiber to some extent. However, this study mainly focuses on improving protein digestibility, and there is insufficient systematic research on dietary fiber conversion. Moreover, the conversion rate of soluble dietary fiber is only about 35%.

[0005] Zhao, HM, Guo, XN, & Zhu, KX (2017). (Impact of solid statefermentation on nutritional, physical and flavor properties of wheat bran. Food Chemistry, 217, 409–414) used solid-state fermentation to process wheat bran, which improved its sensory properties and increased the content of some bioactive substances. However, the fermentation time was as long as 96 hours, resulting in low process efficiency, and the improvement effect and mechanism on intestinal barrier function were not yet investigated.

[0006] To address the issue of increasing β-glucan content, Prins, A., Shewry, P., & Lovegrove, A. (2023). (Analysis of mixed linkage β-glucan content and structure indifferent wheat flour milling fractions. Journal of Cereal Science, 110, 103745) analyzed the content and structure of β-glucan in different wheat flour fractions. The results showed that the β-glucan content in natural wheat is generally low (0.5-1.0%), far lower than that in oats and barley, which limits its functional applications.

[0007] Bertsch, A., Roy, D., & LaPointe, G. (2020). (Fermentation of wheatbran and whey permeate by mono-cultures of Lacticaseibacillus rhamnosus strains and co-culture with yeast enhances bioactive properties. Frontiers in Bioengineering and Biotechnology, 8, 956) attempted to increase the content of bioactive substances in wheat bran through co-fermentation of Lactobacillus and yeast, but their main focus was on polyphenols and antioxidant activity, failing to simultaneously achieve efficient conversion of dietary fiber and a significant increase in β-glucan content.

[0008] Regarding the mechanisms by which intestinal barrier function is improved, Wang, J., Ji, H., Wang, S., Liu, H., Zhang, W., & Zhang, D. (2018). (Probiotic Lactobacillus plantarum promotes intestinal barrier function by strengthening the epithelium and modulating gut microbiota. Frontiers in Microbiology, 9, 1953.) demonstrated that specific strains of Lactobacillus plantarum can improve intestinal barrier function by enhancing the expression of tight junction proteins and regulating the gut microbiota. However, the regulatory role of specific Lactobacillus plantarum in conjunction with plant components such as chlorophyll on intestinal barrier function has not yet been investigated. Furthermore, the role of plant fermentation products such as pheophytin in this process remains to be explored. Byndloss, MX, Olsan, EE, Rivera-Chávez, F., Tiffany, CR, et al. (2017). (Microbiota-activated PPAR-γ signaling inhibits dysbiotic Enterobacteriaceae expansion. Science, 357(6351), 570–575.) Studies have shown that short-chain fatty acids, especially butyrate, can improve intestinal barrier function by activating the PPAR-γ signaling pathway to inhibit the proliferation of harmful bacteria in the gut. However, systematic research on integrating this mechanism with the function of whole-wheat fermented products is still lacking.

[0009] In summary, current technologies lack a method for preparing whole wheat flour that can simultaneously achieve high dietary fiber conversion rates, high β-glucan content, and targeted improvement of intestinal barrier function. In particular, existing technologies fail to elucidate how to optimize process parameters through compound probiotic fermentation to achieve synergistic regulation of a triple mechanism involving chlorophyll bioconversion, specific gut microbiota proliferation, and PPAR signaling pathway regulation, thereby comprehensively enhancing the intestinal barrier function-improving effect of whole wheat flour. Summary of the Invention

[0010] To address the problems existing in the prior art, this invention provides a method for preparing high-fiber whole wheat flour and its compound probiotic fermentation. By screening specific Lactobacillus plantarum and Lactobacillus acidophilus compound strains and combining them with an optimized two-stage solid-state fermentation process, the method achieves efficient conversion of insoluble dietary fiber into soluble dietary fiber, while significantly increasing the β-glucan content, so that the final product has a clear function of improving the intestinal barrier.

[0011] The first objective of this invention is to provide whole wheat flour with high dietary fiber.

[0012] A second objective of this invention is to provide a method for preparing the above-mentioned high-fiber whole wheat flour.

[0013] To achieve the above objectives, the present invention adopts the following technical solution:

[0014] High-fiber whole wheat flour, characterized in that it is made from whole wheat flour, wheat bran and barley grass through fermentation with compound probiotics, and contains the following components: total dietary fiber content of 40-45%, soluble dietary fiber content of 15-18%, β-glucan content of 8.5-10%, pheophytic acid content of 50-100mg / 100g, short-chain fatty acid content of not less than 500mg / 100g, total polyphenol content of 800-1200mg GAE / 100g, and ferulic acid content of 150-200mg / 100g.

[0015] Preferably, the high-fiber whole wheat flour has a moisture content of 8-10%, a pH value of 5.5-6.5, a water solubility index of not less than 25%, a water holding capacity of not less than 5g / g, and an oil holding capacity of not less than 2g / g.

[0016] Preferably, the high-fiber whole wheat flour contains at least 1×10⁻⁶ active probiotics. 8 The content of harmful bacteria is less than 10 CFU / g, and the content of mold and yeast is less than 100 CFU / g.

[0017] Preferably, the high-fiber whole wheat flour has a predicted glycemic index of less than 55, an ORAC antioxidant value of not less than 15,000 μmol TE / 100g, and an in vitro SCFAs production capacity of not less than 50 mmol / L.

[0018] A method for preparing high-fiber whole wheat flour, characterized by comprising the following steps:

[0019] (1) Prepare the fermentation substrate: Mix whole wheat flour, wheat bran and barley grass in a mass ratio of 60:20:20, add 5% by mass of yeast β-glucan concentrate, 2% by mass of ...

[0020] (2) Inoculation with compound probiotics: Lactobacillus plantarum and Lactobacillus acidophilus were mixed at a ratio of 2:1 and inoculated into the fermentation substrate. The total inoculation amount was 1×10⁻⁶. 9 CFU / g substrate;

[0021] (3) Two-stage fermentation was carried out: the first stage was fermented for 36 hours at 37°C±1°C, humidity 55-60%, initial pH 6.5, and aeration rate of 0.5 vvm; the second stage was fermented for 36 hours at 30°C±1°C, humidity 55%, pH 4.5-5.0, and aeration rate of 0.3 vvm, while tannic acid at 0.05% of the substrate mass was added at the beginning of the second stage.

[0022] (4) Post-fermentation treatment: The fermented material is dried in a fluidized bed at 45°C to a moisture content of 8-10%, then ground to a particle size of 150-250μm, and finally maltodextrin is added for standardization.

[0023] (5) Packaging and storage: The standardized products are packaged in nitrogen and stored away from light.

[0024] Preferably, the *Lactobacillus plantarum* comprises a mixed strain of *Lactobacillus plantarum* ZLP001 and *Lactobacillus plantarum* WCFS1, and the *Lactobacillus acidophilus* is *Lactobacillus acidophilus* CGMCC1.1878.

[0025] Preferably, the pH value naturally decreases during the fermentation process, with the initial pH value dropping from 6.5 to 5.0-5.5 in the first stage and the pH value remaining at 4.5-5.0 in the second stage.

[0026] Preferably, the whole wheat flour has a protein content of not less than 13%, the wheat bran has a particle size of 100-200 μm, and the yeast β-glucan concentrate has a purity of 20%.

[0027] Preferably, the second stage of fermentation promotes the conversion of chlorophyll to pheophytic chlorophyll, with a conversion rate of 60-70%, achieved through a dual mechanism of pH-driven and microbial enzyme system.

[0028] Preferably, the dietary fiber content is determined by AOAC 2011.25 method, the β-glucan content is determined by AOAC 995.16 method, the short-chain fatty acid and its precursor content is determined by GC-MS method, the polyphenol and chlorophyll metabolite content is determined by HPLC-DAD method, and the probiotic composition is verified by 16S rRNA sequencing.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. This invention establishes a highly efficient fermentation system by selecting a compound strain of Lactobacillus plantarum and Lactobacillus acidophilus, achieving a high-efficiency conversion of insoluble dietary fiber to soluble dietary fiber with a conversion rate exceeding 45%, which is significantly higher than the conversion rate of no more than 40% in existing technologies.

[0031] 2. The two-stage fermentation process adopted in this invention innovatively solves the contradiction between dietary fiber conversion and functional component preservation. The first stage of high temperature promotes enzyme activity and dietary fiber conversion, while the second stage of low temperature optimizes the accumulation of functional components, so that the β-glucan content of the product reaches 8.5-10%, which is much higher than the content level of about 0.8% in natural wheat.

[0032] 3. This invention is the first to systematically elucidate the complete molecular mechanism by which fermented whole wheat flour improves intestinal barrier function through a triple action of "chlorophyll biotransformation, specific intestinal flora proliferation, and PPAR signaling pathway regulation," providing a theoretical basis for the targeted development of functional foods.

[0033] 4. The high dietary fiber whole wheat flour prepared by this invention not only has good processing suitability, but also has a clear function of improving the intestinal barrier and regulating blood sugar. It is particularly suitable for developing special medical foods for diabetes, obesity, colitis and other conditions, and has broad market application prospects. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0035] This invention addresses the problem of high insoluble dietary fiber and low soluble dietary fiber content in traditional whole wheat flour. By screening specific compound probiotic strains and optimizing the fermentation process, it achieves efficient conversion of insoluble dietary fiber into soluble dietary fiber, while significantly increasing β-glucan content, thus producing high-fiber whole wheat flour with a clear effect on improving intestinal barrier function.

[0036] In this invention, the extracellular polysaccharides and low-molecular-weight organic acids produced by *Lactobacillus plantarum* during fermentation can disrupt the cell wall structure of wheat bran, causing partial hydrolysis of hemicellulose and cellulose in insoluble dietary fiber into soluble oligosaccharides. Simultaneously, the specific enzyme system produced by *Lactobacillus acidophilus* promotes the release of phenolic acids such as ferulic acid, enhancing the product's antioxidant activity. Furthermore, the innovative two-stage fermentation process enables the efficient conversion of chlorophyll in barley grass into pheophytic acid and increases the content of short-chain fatty acids, which have been shown to improve intestinal barrier function by regulating the PPAR signaling pathway.

[0037] Example 1

[0038] High-fiber whole wheat flour, wherein the high-fiber whole wheat flour is produced by fermentation of whole wheat flour, wheat bran and barley grass powder with compound probiotics, and its preparation method includes the following steps:

[0039] (1) Preparation of fermentation substrate: Whole wheat flour with a protein content of 13% was mixed with wheat bran with a particle size of 100μm and barley grass in a mass ratio of 60:20:20. 5% by mass of yeast β-glucan concentrate (purity 20%, purchased from Angel Yeast Co., Ltd.), 2% by mass of oligofructose (purity ≥95%, purchased from Quantum Hi-Tech (China) Biotechnology Co., Ltd.), and 0.5% by mass of mineral premix (containing 2000mg / kg zinc and 50mg / kg selenium, purchased from Anhui Tiger Biotechnology Co., Ltd.) were added. After mixing evenly, an appropriate amount of sterile water was added to adjust the moisture content to 55%.

[0040] (2) Inoculation with compound probiotics: Lactobacillus plantarum ZLP001 (purchased from Qingdao Blue Ocean Biotechnology Co., Ltd.) and Lactobacillus plantarum WCFS1 (ATCC BAA-793, purchased from the American Center for Type Culture Collection) were mixed at a 1:1 ratio, and then mixed with Lactobacillus acidophilus CGMCC 1.1878 (purchased from the China General Microbiological Culture Collection Center) at a 2:1 ratio before being inoculated into the fermentation substrate. The total inoculation amount was 1×10⁻⁶. 9 CFU / g substrate;

[0041] (3) Two-stage fermentation was carried out: the first stage was carried out at 37°C, 55% humidity, initial pH 6.5, and aeration rate of 0.5 vvm for 36 hours, during which the pH value naturally dropped to 5.0; the second stage was carried out at 30°C, 55% humidity, pH 4.5, and aeration rate of 0.3 vvm for 36 hours, and at the beginning of the second stage, 0.05% by mass of tannic acid (analytical grade, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) was added.

[0042] (4) Post-fermentation treatment: The fermented material was dried in a fluidized bed at 45°C to a moisture content of 8%, then ground to a particle size of 150μm, and finally an appropriate amount of maltodextrin (DE value 12-15, purchased from Shandong Longli Biotechnology Co., Ltd.) was added for standardization treatment.

[0043] (5) Packaging and storage: The standardized products are packaged in nitrogen and stored away from light.

[0044] The high-fiber whole wheat flour prepared by the above method has the following characteristics: total dietary fiber content is 42%, soluble dietary fiber content is 16%, β-glucan content is 9.0%, pheophoric acid content is 75 mg / 100g, short-chain fatty acid content is 550 mg / 100g, total polyphenol content is 950 mg GAE / 100g, and ferulic acid content is 175 mg / 100g. Moisture content is 8%, pH value is 6.0, water solubility index is 28%, water holding capacity is 5.5 g / g, and oil holding capacity is 2.3 g / g. The number of active probiotics is 3.2 × 10⁻⁶. 8 The concentration of harmful bacteria is less than 5 CFU / g, and the concentration of mold and yeast is less than 50 CFU / g. The predicted glycemic index is 48, the ORAC antioxidant value is 17,500 μmol TE / 100g, and the in vitro SCFAs production capacity is 62 mmol / L.

[0045] Example 2

[0046] High-fiber whole wheat flour, wherein the high-fiber whole wheat flour is produced by fermentation of whole wheat flour, wheat bran and barley grass powder with compound probiotics, and its preparation method includes the following steps:

[0047] (1) Prepare the fermentation substrate: Mix whole wheat flour with a protein content of 14%, wheat bran with a particle size of 150μm, and barley grass in a mass ratio of 60:20:20. Add 5% by mass of yeast β-glucan concentrate (purity of 20%), 2% by mass of ...

[0048] (2) Inoculation with compound probiotics: Lactobacillus plantarum ZLP001 and Lactobacillus plantarum WCFS1 were mixed at a ratio of 1:1, and then mixed with Lactobacillus acidophilus CGMCC 1.1878 at a ratio of 2:1 before being inoculated into the fermentation substrate. The total inoculation amount was 1.2 × 10⁻⁶. 9 CFU / g substrate;

[0049] (3) Two-stage fermentation was carried out: the first stage was fermented for 36 hours at 37°C, 58% humidity, initial pH 6.5, and aeration rate of 0.5 vvm, during which the pH value naturally dropped to 5.2; the second stage was fermented for 36 hours at 30°C, 55% humidity, pH 4.7, and aeration rate of 0.3 vvm, while tannic acid at 0.05% of the substrate mass was added at the beginning of the second stage.

[0050] (4) Post-fermentation treatment: The fermented material is dried in a fluidized bed at 45°C to a moisture content of 9%, then ground to a particle size of 200μm, and finally an appropriate amount of maltodextrin is added for standardization treatment.

[0051] (5) Packaging and storage: The standardized products are packaged in nitrogen and stored away from light.

[0052] The high-fiber whole wheat flour prepared by the above method has the following characteristics: total dietary fiber content is 43%, soluble dietary fiber content is 17%, β-glucan content is 9.5%, pheophoric acid content is 85 mg / 100g, short-chain fatty acid content is 580 mg / 100g, total polyphenol content is 1000 mg GAE / 100g, and ferulic acid content is 185 mg / 100g. Moisture content is 9%, pH value is 6.2, water solubility index is 30%, water holding capacity is 5.8 g / g, and oil holding capacity is 2.5 g / g. The number of active probiotics is 4.5 × 10⁻⁶. 8 The content of harmful bacteria is less than 5 CFU / g, and the content of mold and yeast is less than 40 CFU / g. The predicted glycemic index is 46, the ORAC antioxidant value is 18,200 μmol TE / 100g, and the in vitro SCFAs production capacity is 65 mmol / L.

[0053] Example 3

[0054] High-fiber whole wheat flour, wherein the high-fiber whole wheat flour is produced by fermentation of whole wheat flour, wheat bran and barley grass powder with compound probiotics, and its preparation method includes the following steps:

[0055] (1) Prepare the fermentation substrate: Mix whole wheat flour with a protein content of 15%, wheat bran with a particle size of 200μm, and barley grass in a mass ratio of 60:20:20. Add 5% by mass of yeast β-glucan concentrate (purity of 20%), 2% by mass of ...

[0056] (2) Inoculation with compound probiotics: Lactobacillus plantarum ZLP001 and Lactobacillus plantarum WCFS1 were mixed at a ratio of 1:1, and then mixed with Lactobacillus acidophilus CGMCC 1.1878 at a ratio of 2:1 before being inoculated into the fermentation substrate. The total inoculation amount was 1.5 × 10⁻⁶. 9 CFU / g substrate;

[0057] (3) Two-stage fermentation was carried out: the first stage was fermented for 36 hours at 37°C, 60% humidity, initial pH 6.5, and aeration rate of 0.5 vvm, during which the pH value naturally dropped to 5.5; the second stage was fermented for 36 hours at 30°C, 55% humidity, pH 5.0, and aeration rate of 0.3 vvm, while tannic acid at 0.05% of the substrate mass was added at the beginning of the second stage.

[0058] (4) Post-fermentation treatment: The fermented material is dried in a fluidized bed at 45°C to a moisture content of 10%, then ground to a particle size of 250μm, and finally an appropriate amount of maltodextrin is added for standardization treatment.

[0059] (5) Packaging and storage: The standardized products are packaged in nitrogen and stored away from light.

[0060] The high-fiber whole wheat flour prepared by the above method has the following characteristics: total dietary fiber content is 45%, soluble dietary fiber content is 18%, β-glucan content is 10.0%, pheophoric acid content is 100 mg / 100g, short-chain fatty acid content is 600 mg / 100g, total polyphenol content is 1200 mg GAE / 100g, and ferulic acid content is 200 mg / 100g. Moisture content is 10%, pH value is 6.5, water solubility index is 32%, water holding capacity is 6.0 g / g, and oil holding capacity is 2.8 g / g. The number of active probiotics is 5.0 × 10⁻⁶. 8 The concentration of harmful bacteria is less than 3 CFU / g, and the concentration of mold and yeast is less than 30 CFU / g. The predicted glycemic index is 45, the ORAC antioxidant value is 20,000 μmol TE / 100g, and the in vitro SCFAs production capacity is 70 mmol / L.

[0061] Example 4

[0062] High-fiber whole wheat flour, wherein the high-fiber whole wheat flour is produced by fermentation of whole wheat flour, wheat bran and barley grass powder with compound probiotics, and its preparation method includes the following steps:

[0063] (1) Preparation of fermentation substrate: Mix whole wheat flour with a protein content of 13.5% with wheat bran with a particle size of 125μm and barley grass in a mass ratio of 60:20:20. Add 5% by mass of yeast β-glucan concentrate (purity of 20%), 2% by mass of fructooligosaccharides, and 0.5% by mass of mineral premix. After mixing evenly, add an appropriate amount of sterile water to adjust the moisture content to 57%.

[0064] (2) Inoculation with compound probiotics: Lactobacillus plantarum ZLP001 and Lactobacillus plantarum WCFS1 were mixed at a ratio of 1:1, and then mixed with Lactobacillus acidophilus CGMCC 1.1878 at a ratio of 2:1 before being inoculated into the fermentation substrate. The total inoculation amount was 1.3 × 10⁻⁶. 9 CFU / g substrate;

[0065] (3) Two-stage fermentation was carried out: the first stage was fermented for 36 hours at 38°C, 57% humidity, initial pH 6.5 and aeration rate of 0.5 vvm, during which the pH value naturally dropped to 5.3; the second stage was fermented for 36 hours at 31°C, 55% humidity, pH 4.8 and aeration rate of 0.3 vvm, while tannic acid at 0.05% of the substrate mass was added at the beginning of the second stage.

[0066] (4) Post-fermentation treatment: The fermented material is dried in a fluidized bed at 45°C to a moisture content of 9.5%, then ground to a particle size of 200μm, and finally an appropriate amount of maltodextrin is added for standardization treatment;

[0067] (5) Packaging and storage: The standardized products are packaged in nitrogen and stored away from light.

[0068] The high-fiber whole wheat flour prepared by the above method has the following characteristics: total dietary fiber content is 44%, soluble dietary fiber content is 17.5%, β-glucan content is 9.8%, pheophoric acid content is 90 mg / 100g, short-chain fatty acid content is 590 mg / 100g, total polyphenol content is 1100 mg GAE / 100g, and ferulic acid content is 190 mg / 100g. Moisture content is 9.5%, pH value is 6.3, water solubility index is 31%, water holding capacity is 5.9 g / g, and oil holding capacity is 2.6 g / g. The number of active probiotics is 4.8 × 10⁻⁶. 8 The concentration of harmful bacteria was less than 4 CFU / g, and the concentration of mold and yeast was less than 35 CFU / g. The predicted glycemic index was 45.5, the ORAC antioxidant value was 19,000 μmol TE / 100g, and the in vitro SCFAs production capacity was 68 mmol / L.

[0069] Example 5

[0070] High-fiber whole wheat flour, wherein the high-fiber whole wheat flour is produced by fermentation of whole wheat flour, wheat bran and barley grass powder with compound probiotics, and its preparation method includes the following steps:

[0071] (1) Prepare the fermentation substrate: Mix whole wheat flour with a protein content of 13%, wheat bran with a particle size of 100-200μm, and barley grass in a mass ratio of 60:20:20. Add 4% by mass of yeast β-glucan concentrate (purity of 20%), 1.5% by mass of fructooligosaccharides, and 0.4% by mass of mineral premix. After mixing evenly, add an appropriate amount of sterile water to adjust the moisture content to 56%.

[0072] (2) Inoculation with compound probiotics: Lactobacillus plantarum ZLP001 and Lactobacillus plantarum WCFS1 were mixed at a ratio of 2:1, and then mixed with Lactobacillus acidophilus CGMCC 1.1878 at a ratio of 2:1 before being inoculated into the fermentation substrate. The total inoculation amount was 1.1 × 10⁻⁶. 9 CFU / g substrate;

[0073] (3) Two-stage fermentation was carried out: the first stage was fermented for 36 hours at 36°C, 56% humidity, initial pH 6.5, and aeration rate of 0.5 vvm, during which the pH value naturally dropped to 5.1; the second stage was fermented for 36 hours at 29°C, 55% humidity, pH 4.6, and aeration rate of 0.3 vvm, while tannic acid at 0.04% of the substrate mass was added at the beginning of the second stage.

[0074] (4) Post-fermentation treatment: The fermented material is dried in a fluidized bed at 44°C to a moisture content of 8.5%, then ground to a particle size of 175μm, and finally an appropriate amount of maltodextrin is added for standardization treatment;

[0075] (5) Packaging and storage: The standardized products are packaged in nitrogen and stored away from light.

[0076] The high-fiber whole wheat flour prepared by the above method has the following characteristics: total dietary fiber content is 41%, soluble dietary fiber content is 15.5%, β-glucan content is 8.8%, pheophoric acid content is 65 mg / 100g, short-chain fatty acid content is 530 mg / 100g, total polyphenol content is 900 mg GAE / 100g, and ferulic acid content is 160 mg / 100g. Moisture content is 8.5%, pH value is 5.8, water solubility index is 27%, water holding capacity is 5.3 g / g, and oil holding capacity is 2.2 g / g. The number of active probiotics is 3.0 × 10⁻⁶. 8 The content of harmful bacteria is less than 6 CFU / g, and the content of mold and yeast is less than 60 CFU / g. The predicted glycemic index is 50, the ORAC antioxidant value is 16,500 μmol TE / 100g, and the in vitro SCFAs production capacity is 58 mmol / L.

[0077] Example 6

[0078] High-fiber whole wheat flour, wherein the high-fiber whole wheat flour is produced by fermentation of whole wheat flour, wheat bran and barley grass powder with compound probiotics, and its preparation method includes the following steps:

[0079] (1) Preparation of fermentation substrate: Mix whole wheat flour with a protein content of 14.5%, wheat bran with a particle size of 180μm, and barley grass in a mass ratio of 60:20:20. Add 6% by mass of yeast β-glucan concentrate (purity of 20%), 2.5% by mass of fructooligosaccharides, and 0.6% by mass of mineral premix. After mixing evenly, add an appropriate amount of sterile water to adjust the moisture content to 59%.

[0080] (2) Inoculation with compound probiotics: Lactobacillus plantarum ZLP001 and Lactobacillus plantarum WCFS1 were mixed at a ratio of 1.5:1, and then mixed with Lactobacillus acidophilus CGMCC 1.1878 at a ratio of 2:1 before being inoculated into the fermentation substrate. The total inoculation amount was 1.4 × 10⁻⁶. 9 CFU / g substrate;

[0081] (3) Two-stage fermentation was carried out: the first stage was fermented for 36 hours at 37.5°C, 59% humidity, initial pH 6.5, and aeration rate of 0.5 vvm, during which the pH value naturally dropped to 5.4; the second stage was fermented for 36 hours at 30.5°C, 55% humidity, pH 4.9, and aeration rate of 0.3 vvm, while tannic acid at 0.06% of the substrate mass was added at the beginning of the second stage.

[0082] (4) Post-fermentation treatment: The fermented material is dried in a fluidized bed at 46°C to a moisture content of 9.8%, then ground to a particle size of 225μm, and finally an appropriate amount of maltodextrin is added for standardization treatment;

[0083] (5) Packaging and storage: The standardized products are packaged in nitrogen and stored away from light.

[0084] The high-fiber whole wheat flour prepared by the above method has the following characteristics: total dietary fiber content is 44.5%, soluble dietary fiber content is 17.8%, β-glucan content is 9.7%, pheophoric acid content is 95 mg / 100g, short-chain fatty acid content is 595 mg / 100g, total polyphenol content is 1150 mg GAE / 100g, and ferulic acid content is 195 mg / 100g. Moisture content is 9.8%, pH value is 6.4, water solubility index is 31.5%, water holding capacity is 5.9 g / g, and oil holding capacity is 2.7 g / g. The number of active probiotics is 4.9 × 10⁻⁶. 8 The concentration of harmful bacteria was less than 3 CFU / g, and the concentration of mold and yeast was less than 32 CFU / g. The predicted glycemic index was 45.2, the ORAC antioxidant value was 19,500 μmol TE / 100g, and the in vitro SCFAs production capacity was 69 mmol / L.

[0085] Comparative Example 1 (without using compound probiotics)

[0086] The same raw materials and process conditions as in Example 1 were used, but only Lactobacillus plantarum ZLP001 was used for fermentation, without the addition of Lactobacillus plantarum WCFS1 and Lactobacillus acidophilus CGMCC 1.1878.

[0087] The whole wheat flour product prepared by this method has the following characteristics: total dietary fiber content of 40%, soluble dietary fiber content of 12%, β-glucan content of 7.5%, pheophoric acid content of 35 mg / 100g, short-chain fatty acid content of 350 mg / 100g, total polyphenol content of 650 mg GAE / 100g, and ferulic acid content of 120 mg / 100g. Moisture content is 8%, pH value is 5.6, water solubility index is 22%, water holding capacity is 4.2 g / g, and oil holding capacity is 1.8 g / g. The number of active probiotics is 1.8 × 10⁻⁶. 8 The concentration of harmful bacteria is less than 8 CFU / g, and the concentration of mold and yeast is less than 80 CFU / g. The predicted glycemic index is 58, the ORAC antioxidant value is 12,500 μmol TE / 100g, and the in vitro SCFAs production capacity is 42 mmol / L.

[0088] Comparative Example 2 (without using a two-stage fermentation process)

[0089] The same raw materials and strains as in Example 1 were used, but only a single-stage fermentation was carried out. The fermentation was carried out continuously for 72 hours at 37°C, 55% humidity, initial pH 6.5, and aeration rate of 0.5 vvm. The temperature and aeration rate were not adjusted during the process, and no tannic acid was added.

[0090] The whole wheat flour product prepared by this method has the following characteristics: total dietary fiber content of 41%, soluble dietary fiber content of 15%, β-glucan content of 8.0%, pheophoric acid content of 30 mg / 100g, short-chain fatty acid content of 480 mg / 100g, total polyphenol content of 820 mg GAE / 100g, and ferulic acid content of 150 mg / 100g. Moisture content is 8%, pH value is 5.2, water solubility index is 25%, water holding capacity is 5.0 g / g, and oil holding capacity is 2.0 g / g. The number of active probiotics is 2.5 × 10⁻⁶. 8 The concentration of harmful bacteria is less than 7 CFU / g, and the concentration of mold and yeast is less than 70 CFU / g. The predicted glycemic index is 52, the ORAC antioxidant value is 15,000 μmol TE / 100g, and the in vitro SCFAs production capacity is 55 mmol / L.

[0091] Comparative Example 3 (without added yeast β-glucan concentrate and tannins)

[0092] The same raw materials, strains and process conditions as in Example 1 were used, but yeast β-glucan concentrate and tannic acid were not added.

[0093] The whole wheat flour product prepared by this method has the following characteristics: total dietary fiber content of 38%, soluble dietary fiber content of 15%, β-glucan content of 1.2%, pheophoric acid content of 70 mg / 100g, short-chain fatty acid content of 520 mg / 100g, total polyphenol content of 900 mg GAE / 100g, and ferulic acid content of 170 mg / 100g. Moisture content is 8%, pH value is 6.0, water solubility index is 27%, water holding capacity is 5.3 g / g, and oil holding capacity is 2.2 g / g. The number of active probiotics is 3.0 × 10⁻⁶. 8 The content of harmful bacteria is less than 5 CFU / g, and the content of mold and yeast is less than 50 CFU / g. The predicted glycemic index is 54, the ORAC antioxidant value is 16,500 μmol TE / 100g, and the in vitro SCFAs production capacity is 60 mmol / L.

[0094] To verify the effect of the product of this invention on improving intestinal barrier function, the following experiments were conducted:

[0095] Experiment 1: Evaluation of an in vitro intestinal epithelial cell model

[0096] Experimental methods: LPS-stimulated Caco-2 cells were used as a model of impaired intestinal barrier function. The extracts (5 mg / mL) of the samples from Examples 1-6 and Comparative Examples 1-3 were co-cultured with Caco-2 cells for 24 hours. Transcellular monolayer resistance (TEER), tight junction protein expression level and cell permeability were detected.

[0097] The experimental results are shown in Table 1:

[0098] Table 1. Effects of different samples on the Caco-2 cell model

[0099] Example 1 45.2 1.68 1.72 42.5 Example 2 48.5 1.75 1.8 45.2 Example 3 52.3 1.82 1.85 48.7 Example 4 50.1 1.78 1.82 46.8 Example 5 43.8 1.65 1.7 41.3 Example 6 51.2 1.8 1.83 47.5 Comparative Example 1 26.4 1.3 1.35 25.6 Comparative Example 2 38.7 1.52 1.6 36.4 Comparative Example 3 42.3 1.63 1.68 40.2 control group 0 1 1 0

[0100] As shown in Table 1, Examples 1-6 of this invention significantly increased the TEER value of Caco-2 cells after LPS stimulation, enhanced the expression of tight junction proteins ZO-1 and Occludin, and reduced the permeability of the cell monolayer, indicating that Examples 1-6 can effectively improve intestinal epithelial barrier function. In contrast, the effects of Comparative Examples 1-3 were significantly weaker, especially Comparative Example 1, which had the worst effect on improving intestinal barrier function due to the lack of synergistic effect of the compound probiotics.

[0101] Experiment 2: Evaluation of Short-Chain Fatty Acid Production Capacity

[0102] Experimental methods: An in vitro intestinal flora fermentation model was used. The samples (1g) of Examples 1-6 and Comparative Examples 1-3 were co-cultured with fecal flora (10%) of healthy people under anaerobic conditions for 24 hours, and the content of short-chain fatty acids in the fermentation broth was detected.

[0103] The experimental results are shown in Table 2:

[0104] Table 2 Short-chain fatty acid production capacity of different samples

[0105] Example 1 62.3 35.6 13.2 13.5 21.7 Example 2 65.2 36.8 14 14.4 22.1 Example 3 70.5 39.5 15.2 15.8 22.4 Example 4 68.1 38.2 14.7 15.2 22.3 Example 5 58.4 33.3 12.6 12.5 21.4 Example 6 69.2 38.7 14.9 15.6 22.5 Comparative Example 1 42.8 26.5 9.5 6.8 15.9 Comparative Example 2 55.3 32.2 12 11.1 20.1 Comparative Example 3 60.5 34.8 13 12.7 21 Reference substrate (inulin) 65.4 37.5 14.2 13.7 21

[0106] As shown in Table 2, Examples 1-6 of this invention all exhibited good short-chain fatty acid production capacity, especially high butyric acid production, with a butyric acid / total SCFAs ratio between 21.4% and 22.5%, which is superior to traditional prebiotic inulin. Comparative Example 1, due to the lack of synergistic effect from the compound probiotics, showed lower short-chain fatty acid production capacity, particularly butyric acid. This result indicates that the product of this invention helps improve intestinal barrier function by increasing the production of short-chain fatty acids, especially butyric acid.

[0107] Experiment 3: Evaluation of the regulatory role of the PPAR signaling pathway

[0108] Experimental methods: Using an LPS-stimulated HT-29 cell model, extracts (5 mg / mL) from the samples of Examples 1-6 and Comparative Examples 1-3 were co-cultured with HT-29 cells for 24 hours. The activity of PPAR-γ and the expression levels of its downstream proteins EHHADH, FABP5, SCD1, etc. were detected.

[0109] The experimental results are shown in Table 3:

[0110] Table 3. Regulatory effects of different samples on the PPAR signaling pathway

[0111] Example 1 1.75 38.5 42.3 35.6 Example 2 1.82 41.2 45 38.2 Example 3 1.9 45.3 48.7 42.5 Example 4 1.85 43.1 46.8 40.3 Example 5 1.7 36.8 40.5 33.7 Example 6 1.88 44.2 47.5 41.8 Comparative Example 1 1.32 22.5 25.8 20.6 Comparative Example 2 1.65 34.7 38.2 31.5 Comparative Example 3 1.72 37.2 41 34.2 control group 1 0 0 0

[0112] As shown in Table 3, Examples 1-6 of this invention significantly activated PPAR-γ and downregulated the expression of its downstream proteins EHHADH, FABP5, and SCD1, with Example 3 showing the most significant effect. In contrast, the effects of Comparative Examples 1-3 were weaker, especially Comparative Example 1, where the PPAR-γ activity was only 1.32, and the rate of reduction in downstream protein expression was much lower than that of the Examples of this invention. This result indicates that the product of this invention can improve intestinal barrier function and metabolic disorders by regulating the PPAR signaling pathway.

[0113] Experiment 4: Evaluation of the role of gut microbiota regulation

[0114] Experimental methods: Samples (1g) from Examples 1-6 and Comparative Examples 1-3 were co-cultured with fecal microbiota (10%) from healthy individuals under anaerobic conditions for 48 hours. Changes in microbiota composition were analyzed by 16S rRNA gene sequencing.

[0115] The experimental results are shown in Table 4:

[0116] Table 4. Regulatory effects of different samples on gut microbiota composition

[0117] Example 1 2.6 2.2 48.5 1.25 Example 2 2.8 2.5 52.3 1.28 Example 3 3.2 2.9 58.7 1.32 Example 4 3 2.7 55.2 1.3 Example 5 2.5 2 45.8 1.22 Example 6 3.1 2.8 56.5 1.31 Comparative Example 1 1.5 1.3 25.6 1.05 Comparative Example 2 2.2 1.8 42.3 1.18 Comparative Example 3 2.4 2.1 46.5 1.2 control group 1 1 0 0.98

[0118] As shown in Table 4, Examples 1-6 of this invention significantly increased the abundance of beneficial bacteria Blautia and Romboutsia, decreased the abundance of harmful bacteria Desulfovibrio, and optimized the Firmicutes / Bacteroidetes ratio. In contrast, Comparative Example 1 showed the worst gut microbiota regulation effect, further demonstrating the importance of the synergistic effect of compound probiotics in gut microbiota regulation.

[0119] The above experimental results demonstrate that the product of this invention effectively improves intestinal barrier function through a synergistic effect of multiple mechanisms, including increasing TEER value, enhancing tight junction protein expression, promoting the production of short-chain fatty acids, especially butyrate, activating the PPAR-γ signaling pathway, and regulating gut microbiota. Example 3 showed the most significant effect and can be considered the optimal implementation scheme of this invention.

[0120] By comparing the results of different embodiments and comparative examples, it can be seen that the present invention has the following innovative points and technical effects:

[0121] 1. A highly efficient fermentation system was established by screening specific Lactobacillus plantarum and Lactobacillus acidophilus co-cultures in an optimal 2:1 ratio. Comparative results from Example 1 showed that a single strain could not achieve the same results as the co-culture, particularly in butyrate production and PPAR-γ activation.

[0122] 2. The innovative design of the two-stage fermentation process resolved the contradiction between fiber conversion and the preservation of functional components. The results of Comparative Example 2 showed that while single-stage fermentation could also achieve a certain dietary fiber conversion rate, it was significantly less effective than the two-stage fermentation process in improving pheophytic chlorophyll content and intestinal barrier function.

[0123] 3. By adding yeast β-glucan concentrate and tannic acid, the limitation of low natural β-glucan content in wheat was successfully overcome. The results of Comparative Example 3 show that although its soluble dietary fiber content is comparable to that of the embodiments of the present invention, its β-glucan content is only 1.2%, far lower than the 8.5-10% of the embodiments of the present invention.

[0124] 4. The product of this invention achieves comprehensive improvement of intestinal barrier function through the triple synergistic effect of "chlorophyll biotransformation, specific intestinal flora proliferation, and PPAR signaling pathway regulation", and has a clear mechanism of action and significant functional advantages.

[0125] In summary, the high dietary fiber whole wheat flour and its compound probiotic fermentation preparation method provided by this invention have overcome the technical bottlenecks in the prior art, such as low dietary fiber utilization rate, low β-glucan content, and unclear mechanism for improving intestinal barrier function. It has important innovative and application value, and is particularly suitable for the development of special medical foods for people with diabetes, obesity, colitis, etc.

[0126] The above description is merely an embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention should be included within the scope of the claims of the present invention.

Claims

1. High-fiber whole wheat flour, characterized in that, The high-fiber whole wheat flour is made from whole wheat flour, wheat bran, and barley grass through fermentation with compound probiotics. The high-fiber whole wheat flour contains the following components: total dietary fiber content of 40-45%, soluble dietary fiber content of 15-18%, β-glucan content of 8.5-10%, pheophytic acid content of 50-100 mg / 100g, short-chain fatty acid content of not less than 500 mg / 100g, total polyphenol content of 800-1200 mg GAE / 100g, and ferulic acid content of 150-200 mg / 100g. The high-fiber whole wheat flour is prepared using the following method: (1) Prepare fermentation substrate: Mix whole wheat flour, wheat bran and barley grass in a mass ratio of 60:20:20, add 5% by mass of yeast β-glucan concentrate, 2% by mass of ... (2) Inoculation of complex probiotics: Lactobacillus plantarum and Lactobacillus acidophilus are mixed in a ratio of 2:1 and inoculated into the fermentation substrate, with a total inoculation amount of 1 x 10 9 CFU / g substrate; (3) Two-stage fermentation was carried out: the first stage was fermented for 36 hours at 37°C±1°C, humidity 55-60%, initial pH 6.5, and aeration rate 0.5 vvm; the second stage was fermented for 36 hours at 30°C±1°C, humidity 55%, pH 4.5-5.0, and aeration rate 0.3 vvm, while tannic acid at 0.05% of the substrate mass was added at the beginning of the second stage. (4) Post-fermentation treatment: The fermented material is dried in a fluidized bed at 45°C to a moisture content of 8-10%, then ground to a particle size of 150-250μm, and finally maltodextrin is added for standardization treatment; (5) Packaging and storage: The standardized products are packaged with nitrogen and stored away from light.

2. The high-fiber whole wheat flour according to claim 1, characterized in that, The high-fiber whole wheat flour has a moisture content of 8-10%, a pH value of 5.5-6.5, a water solubility index of not less than 25%, a water holding capacity of not less than 5g / g, and an oil holding capacity of not less than 2g / g.

3. The high-fiber whole wheat flour according to claim 1, characterized in that, The high-fiber whole wheat flour contains no less than 1×10⁻⁶ active probiotics. 8 The content of harmful bacteria is less than 10 CFU / g, and the content of mold and yeast is less than 100 CFU / g.

4. The high-fiber whole wheat flour according to claim 1, characterized in that, The high-fiber whole wheat flour has a predicted glycemic index of less than 55, an ORAC antioxidant value of not less than 15,000 μmol TE / 100g, and an in vitro SCFAs production capacity of not less than 50 mmol / L.

5. The high-fiber whole wheat flour according to claim 1, characterized in that, The plantarum lactobacillus includes a mixed strain of L. plantarum ZLP001 and L. plantarum WCFS1, and the acidophilus lactobacillus is L. acidophilus CGMCC 1.1878.

6. The high-fiber whole wheat flour according to claim 1, characterized in that, During the fermentation process, the pH value naturally decreases. In the first stage, the pH value drops from the initial pH value of 6.5 to 5.0-5.5, and in the second stage, the pH value is maintained at 4.5-5.

0.

7. The high-fiber whole wheat flour according to claim 1, characterized in that, The whole wheat flour has a protein content of not less than 13%, the wheat bran has a particle size of 100-200 μm, and the yeast β-glucan concentrate has a purity of 20%.

8. The high-fiber whole wheat flour according to claim 1, characterized in that, The second stage of fermentation promotes the conversion of chlorophyll to pheophytic acid, with a conversion rate of 60-70%, achieved through a dual mechanism of pH-driven and microbial enzyme system.

9. The high-fiber whole wheat flour according to claim 1, characterized in that, Dietary fiber content was determined using AOAC 2011.25 method, β-glucan content was determined using AOAC 995.16 method, short-chain fatty acid content was determined using GC-MS, polyphenol and chlorophyll metabolite content was determined using HPLC-DAD method, and probiotic composition was verified by 16S rRNA sequencing.