Preparation method and application of shiitake mushroom soluble dietary fiber combined phenol

Abstract

The invention discloses a preparation method and application of mushroom soluble dietary fiber combined phenol. Comprising the following steps: sieving shiitake mushrooms, and dissolving the sieved shiitake mushrooms in water; adding high-temperature-resistant alpha-amylase to carry out amylase reaction; after the amylase reaction is finished, protease is added for a protease reaction; after the protease reaction is finished, adding saccharifying enzyme to carry out saccharifying enzyme reaction; and after the saccharifying enzyme reaction is finished, centrifuging to take a supernatant, precipitating the supernatant in an ethanol solution, centrifuging to take a precipitate, and sequentially washing and freeze-drying the precipitate to obtain the shiitake mushroom soluble dietary fiber bound phenol. Animal experiments prove that the shiitake mushroom soluble dietary fiber combined phenol has a remarkable effect of relieving colitis, is derived from medicinal and edible shiitake mushrooms, is simple and convenient in process and easy to industrialize, realizes high-value utilization of shiitake mushroom resources, and can be used for preparing functional foods, health-care foods or biological medicine products for adjuvant therapy for relieving colitis.

Description

Technical Field

[0001] This invention relates to the fields of functional food ingredients and biomedicine, specifically to a method for preparing and applying soluble dietary fiber bound to phenols from shiitake mushrooms. Background Technology

[0002] Colitis is a chronic intestinal disease characterized by inflammation and damage to the colonic mucosa. Its pathogenesis is closely related to intestinal flora imbalance, excessive oxidative stress, abnormal release of inflammatory factors, and impaired intestinal barrier function. Clinical manifestations include diarrhea, abdominal pain, and bloody, mucus-containing stools, severely impacting patients' quality of life. Furthermore, long-term recurrent attacks may increase the risk of colorectal cancer. Current clinical treatment primarily relies on anti-inflammatory drugs and immunosuppressants, but these have significant side effects, are prone to drug resistance, and are not curative. Therefore, developing safe and effective dietary intervention products has become a research hotspot in the prevention and treatment of colitis.

[0003] The active ingredients in functional foods exhibit unique advantages in maintaining gut health due to their high safety and mild effects. Among them, dietary fiber and phenolic compounds are two widely studied gut health promoting factors: dietary fiber can exert its probiotic effects by regulating the gut microbiota structure, promoting the production of short-chain fatty acids (SCFAs), and enhancing the intestinal barrier function; while phenolic compounds, with their significant antioxidant and anti-inflammatory activities, can scavenge free radicals, inhibit the expression of pro-inflammatory cytokines, and reduce intestinal inflammatory damage.

[0004] Bound phenols in dietary fiber refer to complexes formed by the covalent bonding of phenolic substances with dietary fiber through ester, ether, or glycosidic bonds. Compared to free phenols, they exhibit stronger gastrointestinal stability and colon-targeting properties. Studies have shown that these complexes can be metabolized and broken down by gut microbiota in the colon, slowly releasing the active phenolic components while retaining the probiotic properties of dietary fiber, thus achieving a synergistic effect between dietary fiber and phenols.

[0005] As a fungus used in both medicine and food, the physiological functions of its active ingredients have been widely confirmed. The soluble dietary fiber abundant in shiitake mushrooms has significant immunomodulatory effects, activating macrophages and NK cells, enhancing the body's immune response, regulating gut microbiota balance, reducing lipid absorption, and providing a fundamental guarantee for gut health. Studies have also confirmed that when shiitake mushroom soluble dietary fiber is used in combination with probiotics, it can reduce disease activity in a mouse model of ulcerative colitis by regulating gut microbiota, increasing butyrate synthesis, and reducing the production of pro-inflammatory cytokines.

[0006] Existing research on dietary fiber-bound phenols largely focuses on byproducts of grain or fruit and vegetable processing, such as buckwheat, wheat bran, prickly pear pomace, and white kidney bean husks. No research has yet explored the use of shiitake mushrooms as a raw material to prepare soluble dietary fiber-bound phenols and systematically investigated their efficacy in alleviating colitis. Current research on shiitake mushroom-derived components for colitis intervention focuses only on extracting single active ingredients such as polysaccharides, failing to explore the synergistic anti-inflammatory potential of shiitake mushroom's soluble dietary fiber (SDF) and natural phenols, resulting in limited anti-inflammatory effects. Furthermore, when shiitake mushroom phenols are used alone, they are easily degraded by gastrointestinal digestive fluids, leading to insufficient effective concentrations and low bioavailability at the colonic lesion site. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for preparing and applying phenolic compounds bound to lentinan soluble dietary fiber. This invention utilizes the inherent binding properties of lentinan soluble dietary fiber (SDF) with natural phenols to achieve a synergistic effect without the introduction of exogenous substances: SDF acts as a natural carrier, protecting phenols from gastrointestinal degradation and enabling colon-targeted delivery, thus improving bioavailability; simultaneously, the two form a synergistic system, significantly enhancing anti-inflammatory effects. Furthermore, the preparation process is simple and safe, providing an efficient solution for natural intervention in colitis.

[0008] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides a method for preparing soluble dietary fiber bound phenols from shiitake mushrooms, comprising the following steps: (1) Dissolve the shiitake mushrooms in water after sieving them; (2) Add heat-resistant α-amylase to carry out amylase reaction; (3) After the amylase reaction is completed, add protease to carry out the protease reaction; (4) After the protease reaction is completed, add saccharifying enzyme to carry out the saccharifying enzyme reaction; (5) After the saccharifying enzyme reaction is completed, the supernatant is centrifuged and precipitated in ethanol solution. The precipitate is centrifuged and the precipitate is washed and freeze-dried in sequence to obtain soluble dietary fiber bound phenols from shiitake mushrooms.

[0009] Furthermore, in step (1), the mesh size of the sieve is 80~100 mesh; In step (2), the activity of the thermoresistant α-amylase is 20,000~25,000 U / ml, the reaction temperature of the amylase is 95~100℃, and the reaction time of the amylase is 30~35 min. The mass-to-volume ratio of the shiitake mushroom and the thermoresistant α-amylase is 1:0.05~0.08 g / mL.

[0010] Furthermore, the sieve mesh size is 80 mesh; The thermoresistant α-amylase has an enzyme activity of 20,000 U / ml, the amylase reaction temperature is 95℃, and the amylase reaction time is 35 min. The mass-to-volume ratio of the shiitake mushroom and the heat-resistant α-amylase was 1:0.05 g / mL.

[0011] Furthermore, in step (3), the enzyme activity of the protease is 100~150 U / mg, the temperature of the protease reaction is 40~45℃, and the reaction time is 30~35 min; The mass ratio of shiitake mushroom to protease is 1:0.01~0.015.

[0012] Furthermore, the enzyme activity of the protease is 100 U / mg, the reaction temperature of the protease is 40℃, and the reaction time of the protease is 30 min; The mass ratio of shiitake mushroom to protease is 1:0.01.

[0013] Furthermore, in step (4), the enzyme activity of the saccharifying enzyme is 100,000~110,000 U / mL, the temperature of the saccharifying enzyme reaction is 40~45℃, and the reaction time is 30~35 min; The mass-to-volume ratio of shiitake mushroom and saccharifying enzyme is 1:0.3~0.35 g / mL; In step (5), the concentration of the ethanol solution is 90-95%.

[0014] Furthermore, the activity of the saccharifying enzyme is 100,000 U / ml, the reaction temperature is 40℃, and the reaction time is 30 min; The mass-to-volume ratio of shiitake mushroom and saccharifying enzyme is 1:0.3 g / mL; the concentration of the ethanol solution is 95%.

[0015] The present invention also provides a soluble dietary fiber conjugated phenol of shiitake mushroom prepared by the preparation method described above.

[0016] The present invention also provides the application of the soluble dietary fiber bound phenols of shiitake mushrooms in the preparation of functional foods, health foods or drugs for relieving colitis.

[0017] The present invention also provides a functional food for relieving colitis, the functional food comprising the soluble dietary fiber bound phenols of shiitake mushrooms.

[0018] The beneficial effects of this invention are: 1. Significant relief of inflammatory symptoms and improvement of core physical signs: The disease activity index (DAI) score of model mice after intervention with soluble dietary fiber from shiitake mushrooms combined with phenol was lower than that of the model group, the colon length was significantly increased compared with the model group, and the spleen index (core indicator of inflammatory status) was significantly reduced. This indicates that the intervention can effectively alleviate clinical symptoms such as diarrhea and hematochezia caused by ulcerative colitis (UC), alleviate inflammatory-related signs such as colonic atrophy and splenomegaly, and effectively control the overall severity of inflammation.

[0019] 2. Precise repair of colonic pathological damage and reconstruction of barrier structure integrity: HE staining results showed that the continuity of colonic mucosal epithelium in SDF-BP group mice was restored, the degree of glandular disorder was significantly improved, and the number of inflammatory cell infiltration was significantly reduced compared with the model group; Alixin blue staining confirmed that the number of goblet cells in SDF-BP group was significantly increased, the thickness of the mucus layer was increased and the continuity was reconstructed, indicating that this measure can simultaneously repair the pathological damage of the intestinal physical barrier and the mucus chemical barrier, and reverse the UC-related colonic tissue morphological abnormalities.

[0020] 3. Highly efficient regulation of inflammatory molecular pathways and blockade of inflammatory cascade reactions: The levels of three pro-inflammatory factors, IL-6, IL-1β, and TNF-α, in the colonic tissue of the SDF-BP group were significantly lower than those of the M group, and the levels of IL-6 and TNF-α tended to normalize. The level of the anti-inflammatory factor IL-10 in the colonic tissue of the SDF-BP group was significantly higher than that of the M group. Moreover, the regulatory effect of SDF-BP on inflammatory factors was superior to that of the SDF-PR, BP, and SDF-PR+BP groups, indicating that SDF-BP can effectively restore the balance of inflammatory factors in colonic tissue and exert a significant anti-inflammatory effect.

[0021] 4. Superior intervention effect compared to single components, with synergistic and stable mechanism of action: Compared with dephenolized dietary fiber, free phenols, and physical mixtures of the two, the intervention of soluble dietary fiber of shiitake mushroom combined with phenols is more effective in improving DAI, restoring colon length, repairing pathological damage, and downregulating inflammatory factors. This confirms that the combination of dietary fiber and phenols can achieve synergistic effects, solving the problems of limited intervention effect and single target of action of single components, and significantly improving the stability and effectiveness of the effect. Attached Figure Description

[0022] Figure 1 The results of the core vital signs of mice are shown in the figure. In the figure, A represents the Disease Activity Index (DAI); B represents the spleen index; C represents the colon length; and D represents the colon image. Figure 2 This is a diagram showing the pathological morphological examination results of mice. In the figure, A shows H&E staining of mouse colon tissue; B shows alcian blue staining of mouse colon tissue. Figure 3The graph shows the results of inflammation-related marker detection in mice. In the figure, A represents IL-6; B represents IL-1β; C represents IL-10; and D represents TNF-α. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0024] Example 1 Preparation of soluble dietary fiber conjugated phenol (SDF-BP) from shiitake mushrooms 1. Raw material pretreatment: Select shiitake mushrooms as dietary fiber raw material, crush them and pass them through an 80-mesh sieve for later use.

[0025] 2. Take 100 g of sieved shiitake mushrooms, dissolve them in distilled water (solid-to-liquid ratio 1:30 g / mL), add 5 mL of 20000 U / mL heat-resistant α-amylase (pH 6, Shanghai Yuanye, product number: S10005) in sequence, and react in a 95℃ water bath for 35 min.

[0026] 3. After the reaction is complete, add 1g of 100 U / mg protease (pH 7, Shanghai Yuanye, product number: S10051) to the system in step 2, and react in a 40℃ water bath for 30 min.

[0027] 4. After the reaction is complete, add 30 mL of 100,000 U / ml saccharifying enzyme (pH 4.6, Shanghai Yuanye, product number: S10018) to the system in step 3, and react in a 40℃ water bath for 30 min. The above reaction removes starch and protein from the shiitake mushrooms.

[0028] 5. After the reaction was completed, the supernatant was collected by centrifugation (5000 rpm / min, 10 min). The supernatant was precipitated in 4 times the volume of 95% ethanol solution for 12 h. The precipitate was collected by centrifugation (5000 rpm / min, 10 min), washed with 95% ethanol and then freeze-dried to obtain lentinan soluble dietary fiber bound phenol (SDF-BP).

[0029] Comparative Example 1 Preparation of dephenolized soluble dietary fiber (SDF-PR) The SDF-BP prepared in Example 1 was treated with 2 mol / L NaOH for 4 h, the pH was adjusted to 2, and the precipitate was precipitated in 4 times the volume of anhydrous ethanol. After centrifugation, the precipitate was dialyzed to obtain dephenolized soluble dietary fiber (SDF-PR).

[0030] Comparative Example 2 Preparation of free phenol (BP) SDF-PR from Comparative Example 1 was concentrated by vacuum rotary evaporation at 40°C, then extracted with ethyl acetate, and concentrated again by empty rotary evaporation to obtain free phenol (BP).

[0031] Comparative Example 3 Preparation of physical mixtures Based on the actual phenol content (2%) in SDF-BP of Example 1, SDF-PR of Comparative Example 1 and BP of Comparative Example 2 were mixed (mixing ratio of SDF-PR∶BP=98∶2) to obtain a physical mixture (SDF-PR+BP).

[0032] Example 2 UC Model Construction and Intervention Six-week-old SPF-grade C57BL / 6 mice were randomly divided into six groups (n=12): blank control group (C group), model group (M group), SDF-BP group, SDF-PR group, BP group, and SDF-PR+BP group.

[0033] 1. UC model construction: Except for group C, the other groups drank 2.5% (w / v) sodium dextran sulfate DSS aqueous solution for 7 days to construct the UC model; 2. Intervention treatment: After constructing the UC model, the mice in each group were treated as follows: (1) Group C and Group M: Oral administration of physiological saline (dose 10 mL / kg); (2) SDF-BP group: SDF-BP of Example 1 was administered by gavage daily (dose 500 mg / kg). (3) SDF-PR group: SDF-PR (dose 500 mg / kg) was administered by gavage to Comparative Example 1 daily. (4) BP group: BP (10 mg / kg, consistent with the phenol content in SDF-BP) was administered by gavage daily to the control group 2. (5) SDF-PR+BP group: SDF-PR+BP (500 mg / kg) was administered by gavage to the control group 3 daily. The patient was given gavage once a day for 8 consecutive days.

[0034] 3. After the intervention began, the weight, fecal characteristics and blood in the stool of each group of mice were recorded daily, and the disease activity index (DAI) was calculated according to the standard. After the intervention ended, each group of mice was sacrificed, the colon was separated and its length was measured, the spleen was separated and weighed, and the spleen index was calculated according to the formula "spleen index = spleen weight (mg) / mouse weight (g)".

[0035] 4. Colon tissues from mice in each group were collected, fixed with 4% paraformaldehyde for 24 h, routinely embedded in paraffin, sectioned (5 μm thick), and stained with HE and alicin blue.

[0036] 5. Take colon tissue from each group of mice and add pre-cooled physiological saline at a material-to-liquid ratio of 1:10 (g:mL) to prepare homogenate. Centrifuge (10000 rpm, 4℃, 10 min) and collect the supernatant. Use an ELISA kit to detect the content of IL-6, IL-1β, IL-10 and TNF-α in the supernatant.

[0037] Comparative Analysis of Effects Compared with the SDF-BP, BP, SDF-PR+BP intervention groups and the model group M, the SDF-BP intervention group has the following significant advantages: 1) Better relief of inflammatory symptoms: such as Figure 1 As shown, the DAI score of mice in the SDF-BP intervention group was lower than that in the M group, the colon length was significantly increased compared with the M group, and the spleen index was significantly decreased compared with the M group. This indicates that SDF-BP has a better effect on improving diarrhea, hematochezia, colonic atrophy and splenomegaly caused by UC than single components SDF-PR, BP and physical mixtures.

[0038] 2) More thorough repair of colonic pathological damage: such as Figure 2 As shown, HE staining results indicated that the continuity of the colonic mucosal epithelium in the SDF-BP intervention group mice was restored to a certain extent, and the glands were arranged neatly. Alixin blue staining showed that the number of goblet cells in the SDF-BP group increased compared with the M group, the thickness of the mucus layer was restored, and the reconstruction effect of the intestinal barrier structure integrity was better than that of other intervention groups.

[0039] 3) Stronger regulatory effect of inflammatory molecules: such as Figure 3 As shown, the levels of three pro-inflammatory factors, IL-6, IL-1β, and TNF-α, in the colonic tissue of the SDF-BP group were significantly lower than those of the M group, and the levels of IL-6 and TNF-α tended to normalize. The level of the anti-inflammatory factor IL-10 in the colonic tissue of the SDF-BP group was significantly higher than that of the M group. Furthermore, the regulatory effect of SDF-BP on inflammatory factors was superior to that of the SDF-PR, BP, and SDF-PR+BP groups, indicating that SDF-BP can effectively restore the balance of inflammatory factors in colonic tissue and exert a significant anti-inflammatory effect.

[0040] 4) Significant synergistic effect: Compared with the SDF-PR+BP intervention group, the SDF-BP group's SDF-BP solves the problem of free phenols being easily degraded by gastric acid and having low bioavailability through the natural binding of phenols and dietary fiber. At the same time, it enhances the inflammatory regulation activity of dietary fiber, achieving a synergistic effect of dietary fiber + phenols. The intervention effect is better than simple physical mixing.

[0041] In summary, the soluble dietary fiber bound phenol (SDF-BP) prepared by this invention has a significantly better intervention effect on ulcerative colitis than single dephenolized dietary fiber, single free phenol, and physical mixtures of the two. Moreover, the preparation process is simple, green and environmentally friendly, and suitable for large-scale production and functional food development.

[0042] Example 3 A functional food for relieving colitis, wherein the functional food of this embodiment includes the lentinan from shiitake mushroom soluble dietary fiber in Example 1.

[0043] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing phenolic soluble dietary fiber from shiitake mushrooms, characterized in that: Includes the following steps: (1) Dissolve the shiitake mushrooms in water after sieving them; (2) Add heat-resistant α-amylase to carry out amylase reaction; (3) After the amylase reaction is completed, add protease to carry out the protease reaction; (4) After the protease reaction is completed, add saccharifying enzyme to carry out the saccharifying enzyme reaction; (5) After the saccharifying enzyme reaction is completed, the supernatant is centrifuged and precipitated in ethanol solution. The precipitate is centrifuged and the precipitate is washed and freeze-dried in sequence to obtain soluble dietary fiber bound phenols from shiitake mushrooms.

2. The preparation method according to claim 1, characterized in that: In step (1), the mesh size of the sieve is 80~100 mesh; In step (2), the activity of the thermoresistant α-amylase is 20,000~25,000 U / ml, the reaction temperature of the amylase is 95~100℃, and the reaction time of the amylase is 30~35 min. The mass-to-volume ratio of the shiitake mushroom and the thermoresistant α-amylase is 1:0.05~0.08 g / mL.

3. The preparation method according to claim 2, characterized in that: The sieve mesh size is 80 mesh; The thermoresistant α-amylase has an enzyme activity of 20,000 U / ml, the amylase reaction temperature is 95℃, and the amylase reaction time is 35 min. The mass-to-volume ratio of the shiitake mushroom and the heat-resistant α-amylase was 1:0.05 g / mL.

4. The preparation method according to claim 1, characterized in that: In step (3), the enzyme activity of the protease is 100~150 U / mg, the temperature of the protease reaction is 40~45℃, and the reaction time is 30~35 min. The mass ratio of shiitake mushroom to protease is 1:0.01~0.

015.

5. The preparation method according to claim 4, characterized in that: The enzyme activity of the protease is 100 U / mg, the reaction temperature is 40℃, and the reaction time is 30 min. The mass ratio of shiitake mushroom to protease is 1:0.

01.

6. The preparation method according to claim 1, characterized in that: In step (4), the enzyme activity of the saccharifying enzyme is 100,000~110,000 U / mL, the temperature of the saccharifying enzyme reaction is 40~45℃, and the reaction time is 30~35 min. The mass-to-volume ratio of shiitake mushroom and saccharifying enzyme is 1:0.3~0.35 g / mL; In step (5), the concentration of the ethanol solution is 90-95%.

7. The preparation method according to claim 6, characterized in that: The saccharifying enzyme has an activity of 100,000 U / ml, the reaction temperature is 40℃, and the reaction time is 30 min. The mass-to-volume ratio of shiitake mushroom and saccharifying enzyme is 1:0.3 g / mL; the concentration of the ethanol solution is 95%.

8. A soluble dietary fiber conjugated phenol of shiitake mushroom prepared by the preparation method according to any one of claims 1 to 7.

9. The use of the lentinan soluble dietary fiber bound phenol as described in claim 8 in the preparation of functional foods, health foods, or drugs for relieving colitis.

10. A functional food for relieving colitis, characterized in that: The functional food contains the lentinan of shiitake mushroom soluble dietary fiber as described in claim 8.

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