An α-glucosidase inhibitor, its preparation method and application

Abstract

This invention belongs to the field of fermentation engineering technology, specifically relating to an α-glucosidase inhibitor, its preparation method, and its application. The α-glucosidase inhibitor is prepared by mixing *Saccharomyces cerevisiae*, *Lactobacillus plantarum*, and papaya substrate at a ratio of 0.5 × 10⁻⁶. 6 CFU: (1×10) 6 CFU~5×10 6 CFU): The α-glucosidase inhibitor is obtained by anaerobic fermentation after mixing at a ratio of 1 mL. Compared with α-glucosidase inhibitors from single Lactobacillus plantarum fermentation and other single lactic acid bacteria fermentation, the α-glucosidase inhibitor has a higher α-glucosidase activity inhibition effect and can be used to prepare health products that help maintain healthy blood sugar levels, while also enhancing the fermented flavor.

Description

Technical Field

[0001] This invention belongs to the field of fermentation engineering technology, specifically relating to an α-glucosidase inhibitor, its preparation method, and its application. Background Technology

[0002] Alpha-glucosidase inhibitors can delay the digestion and absorption of carbohydrates and are important functional substances for regulating postprandial blood glucose, widely used in the food, health product, and pharmaceutical fields. Clinically available alpha-glucosidase inhibitors, such as acarbose, often cause adverse reactions and liver damage with long-term use. Traditional Chinese medicines with high flavonoid content have the potential to be formulated into alpha-glucosidase inhibitors, but they suffer from drawbacks such as metabolic instability, significant differences in in vitro and in vivo activity, multiple targets, and a tendency to produce side effects.

[0003] Currently, papaya is rich in active precursors such as polysaccharides, polyphenols, and flavonoids, which can be converted into highly active inhibitory components through microbial fermentation. The product of papaya fermentation by *Lactobacillus plantarum* exhibits higher α-glucosidase inhibitory activity compared to papaya fermentation products from other lactobacilli. Furthermore, the preparation process of α-glucosidase inhibitors offers advantages over traditional separation, purification, and extraction methods, including greater operability, higher reproducibility, and simpler operation.

[0004] Existing technologies use a single lactic acid bacteria to ferment papaya to prepare functional products. However, single-strain fermentation does not fully utilize the papaya substrate, resulting in poor inhibitory activity of the papaya fermentation product, namely the α-glucosidase inhibitor, against α-glucosidase. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides an α-glucosidase inhibitor, its preparation method, and its application, which can solve the technical problem that existing α-glucosidase inhibitors have poor inhibitory activity against α-glucosidase.

[0006] This invention is achieved by adopting the following technical solution: An α-glucosidase inhibitor, wherein the α-glucosidase inhibitor is prepared by mixing Saccharomyces cerevisiae, Lactobacillus plantarum, and papaya substrate at a ratio of 0.5 × 10⁻⁶. 6 CFU: (1×10) 6 CFU~5×10 6 CFU): 1 mL of the mixture was obtained by anaerobic fermentation. Furthermore, an α-glucosidase inhibitor is prepared by combining *Saccharomyces cerevisiae*, *Lactobacillus plantarum*, and papaya substrate at a concentration of 0.5 × 10⁻⁶. 6 CFU: 1×10 6 CFU: obtained by fermentation at a ratio of 1 mL.

[0007] Furthermore, an α-glucosidase inhibitor is prepared by combining *Saccharomyces cerevisiae*, *Lactobacillus plantarum*, and papaya substrate at a concentration of 0.5 × 10⁻⁶. 6 CFU: 5×10 6 CFU: obtained by fermentation at a ratio of 1 mL.

[0008] The method for preparing the α-glucosidase inhibitor includes the following steps: Mix peeled red papaya with water and homogenize to make papaya puree; Mix papaya homogenate, cellulase and pectinase, heat, filter, and obtain filtrate; The filtrate, white sugar, and skim milk were mixed, and the pH was adjusted to 6.2-6.8 with baking soda. The mixture was then sterilized to obtain the papaya substrate. Saccharomyces cerevisiae, Lactobacillus plantarum, and papaya substrate were mixed in a certain proportion and fermented anaerobically at 35℃~39℃ for 47h~49h to obtain an α-glucosidase inhibitor.

[0009] Furthermore, edible alkali is used to adjust the pH to 6.2.

[0010] Furthermore, edible alkali is used to adjust the pH to 6.8.

[0011] Further, anaerobic fermentation was carried out at 35°C for 49 hours.

[0012] Furthermore, anaerobic fermentation was carried out at 39°C for 47 hours.

[0013] Furthermore, the peeled red-fleshed papaya is 2.5cm in size. 3 ~3cm 3 .

[0014] Furthermore, the peeled red-fleshed papaya is 2.5cm in size. 3 .

[0015] Furthermore, the peeled red-fleshed papaya is 3cm in size. 3 .

[0016] Furthermore, the mass ratio of the peeled red papaya to water is 1:(0.8~1.2).

[0017] Furthermore, the mass ratio of the peeled red papaya to water is 1:0.8.

[0018] Furthermore, the mass ratio of the peeled red papaya to water is 1:1.2.

[0019] Furthermore, the papaya homogenate, cellulase, and pectinase are mixed in a mass ratio of 100:0.05:(0.04~0.06).

[0020] Furthermore, the papaya homogenate, cellulase, and pectinase are mixed in a mass ratio of 100:0.05:0.04.

[0021] Furthermore, the papaya homogenate, cellulase, and pectinase are mixed in a mass ratio of 100:0.05:0.06.

[0022] Furthermore, the heating temperature is 45℃~55℃; the heating time is 1 hour.

[0023] Furthermore, the heating temperature is 45°C; the heating time is 1 hour.

[0024] Furthermore, the heating temperature is 55°C; the heating time is 1 hour.

[0025] Furthermore, the filtrate, white sugar, and skim milk are mixed in a ratio of 100mL: 5g: (4.5mL~5.5mL); the skim milk is prepared by mixing 10g of skim milk powder with 100mL of water.

[0026] Furthermore, the filtrate, white sugar, and skim milk are mixed in a ratio of 100mL:5g:4.5mL.

[0027] Furthermore, the filtrate, white sugar, and skim milk are mixed in a ratio of 100mL:5g:5.5mL.

[0028] Furthermore, the sterilization is pasteurization.

[0029] The application of the aforementioned papaya fermentation broth in the preparation of health products that help maintain healthy blood sugar levels.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention provides an α-glucosidase inhibitor, wherein the α-glucosidase inhibitor is composed of *Saccharomyces cerevisiae*, *Lactobacillus plantarum*, and papaya substrate at a concentration of 0.5 × 10⁻⁶. 6 CFU: (1×10) 6 CFU~5×10 6 CFU): The α-glucosidase inhibitor is obtained by anaerobic fermentation after mixing at a ratio of 1 mL. Compared with α-glucosidase inhibitors obtained by fermentation with a single Lactobacillus plantarum or other single lactic acid bacteria, the α-glucosidase inhibitor has a higher α-glucosidase activity inhibition effect. It can be used to prepare health products that help maintain healthy blood sugar levels and adds fermented flavor.

[0031] 2. The method for preparing the α-glucosidase inhibitor provided by this invention uses the optimal lactic acid bacteria strain to ferment papaya to prepare the α-glucosidase inhibitor, and the co-fermentation system of Saccharomyces cerevisiae and Lactobacillus plantarum solves the problem of insufficient fermentation activity of single strains.

[0032] 3. The preparation method of the α-glucosidase inhibitor provided by this invention has a clearly defined strain selection. *Lactobacillus plantarum* is the optimal single lactic acid bacterium for preparing inhibitors from fermented papaya, exhibiting superior activity compared to other common lactic acid bacteria. Combined fermentation enhances the effect; *Saccharomyces cerevisiae* secretes extracellular enzymes such as pectinase during fermentation, partially degrading the pectin structure in plant cell walls, thereby promoting the release of cell contents and improving the availability of polyphenols and other bioactive substances, providing a substrate basis for subsequent biotransformation by lactic acid bacteria. *Lactobacillus plantarum* utilizes the active precursor released by yeast cell wall disruption for targeted biotransformation, generating highly active α-glucosidase inhibitory components, significantly enhancing inhibitory activity. The inhibitory activity is significantly enhanced; the α-glucosidase inhibitory activity of the combined fermentation product is significantly higher than that of *Lactobacillus plantarum* fermentation alone. It is natural and safe, using pure fruit and vegetable fermentation with food-grade microorganisms, without chemical additives, suitable for food and health product applications. The process is mild, conditions are easily controlled, and it is suitable for industrial production. The activity enhancement is clear and the data is reproducible. The product is natural, safe, and palatable. Attached Figure Description

[0033] Figure 1 This study analyzed the α-glucose inhibitory activity of papaya pulp fermented with different lactic acid bacteria. Different letters, such as a and b, a and c, b and d, represent significant differences between the two groups; however, the same letter, such as a and a, represents no significant difference between the two groups.

[0034] Figure 2 Analysis of α-glucosidase inhibitory activity in Lactobacillus plantarum during single and combined fermentation.

[0035] Figure 3 The colony counts in papaya fermentation broth samples were calculated using different fermentation ratios of Lactobacillus plantarum and Saccharomyces cerevisiae. Among them, (A) represents the Lactobacillus plantarum colony count, and (B) represents the Saccharomyces cerevisiae colony count.

[0036] Figure 4 The α-glucosidase inhibitory activity of papaya fermentation broth samples and in vitro simulated digestion samples under different fermentation ratios of Lactobacillus plantarum and Saccharomyces cerevisiae are shown. The upper pink part represents the α-glucosidase inhibitory activity of the papaya fermentation broth samples, and the lower green part represents the α-glucosidase inhibitory activity of the in vitro simulated digestion samples. Detailed Implementation

[0037] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods. Specific explanations are needed as follows:

[0038] Lactobacillus plantarum ( Lactobacillus plantarum (From "Jin Xiaofan. Study on mixed fermentation of mango pulp by lactic acid bacteria and yeast [D]. Hainan University, 2019. DOI:10.27073 / d.cnki.ghadu.2019.000962"); Saccharomyces cerevisiae ( Saccharomyces cerevisiae Yeast Life O™ is from Raman Company.

[0039] Lactobacillus acidophilus has the accession number CICC 20248; Lactobacillus thermophilus has the accession number CICC 20375; and Lactobacillus delbrueckii subsp. bulgaricus has the accession number CICC 20353; all of them are from the China Industrial Microbial Culture Collection Center.

[0040] The cellulase unit is 100,000 U / g, and the pectinase unit is 50,000 U / g.

[0041] Example 1: Preparation of an α-glucosidase inhibitor, the specific steps are as follows: S1. Fully ripe, clean-skinned, undamaged, and unspoiled red papaya; peel and cut into 3cm pieces. 3 Small pieces, to get papaya chunks.

[0042] S2. Mix papaya chunks and water in a 1:1 mass ratio to make papaya homogenate.

[0043] S3. For every 100g of papaya homogenate, add 0.05g of cellulase and 0.05g of pectinase, incubate in a 50℃ water bath for 1 hour, cool to 25℃, filter, and retain the filtrate; for every 100mL of filtrate, add 5g of white sugar and 5mL of skim milk; the skim milk is prepared by mixing 10g of skim milk powder and 100mL of water. Finally, edible alkali was added to adjust the pH of the papaya filtrate to 6.5, and the solution was pasteurized at 75°C for 30 minutes to obtain the papaya substrate.

[0044] S4. Mix brewer's yeast, lactobacillus plantarum, and papaya substrate at a ratio of 0.5 × 10⁻⁶. 6 CFU: 4×10 6 CFU: 1 mL was mixed and fermented anaerobically at 37℃ for 48 h to obtain papaya fermentation broth, which is an α-glucosidase inhibitor; the ratio was set at 1:8.

[0045] Comparative Example 1: Preparation of a papain α-glucosidase inhibitor Based on Example 1, brewer's yeast, lactobacillus plantarum, and papaya substrate were mixed at a ratio of 0.5 × 10⁻⁶. 6 CFU: 4×10 6 CFU: Mix at a ratio of 1 mL, and replace with brewer's yeast and papaya substrate at a ratio of 0.5 × 10⁻⁶.6 CFU: Mix at a ratio of 1 mL; set to 1:0.

[0046] Comparative Example 2 Based on Example 1, brewer's yeast, lactobacillus plantarum, and papaya substrate were mixed at a ratio of 0.5 × 10⁻⁶. 6 CFU: 4×10 6 CFU: 1 mL was mixed, and the substrate was changed to brewer's yeast, lactobacillus plantarum and papaya at a ratio of 0.5 × 10⁻⁶. 6 CFU: 1×10 6 CFU: Mix at a ratio of 1 mL; set the ratio as 1:2.

[0047] Comparative Example 3 Based on Example 1, brewer's yeast, lactobacillus plantarum, and papaya substrate were mixed at a ratio of 0.5 × 10⁻⁶. 6 CFU: 4×10 6 CFU: 1 mL was mixed, and the substrate was changed to brewer's yeast, lactobacillus plantarum and papaya at a ratio of 0.5 × 10⁻⁶. 6 CFU: 2×10 6 CFU: Mix at a ratio of 1 mL; set the ratio as 1:4.

[0048] Comparative Example 4 Based on Example 1, brewer's yeast, lactobacillus plantarum, and papaya substrate were mixed at a ratio of 0.5 × 10⁻⁶. 6 CFU: 4×10 6 CFU: 1 mL was mixed, and the substrate was changed to brewer's yeast, lactobacillus plantarum and papaya at a ratio of 0.5 × 10⁻⁶. 6 CFU: 3×10 6 CFU: Mix at a ratio of 1 mL; set the ratio as 1:6.

[0049] Comparative Example 5 Based on Example 1, brewer's yeast, lactobacillus plantarum, and papaya substrate were mixed at a ratio of 0.5 × 10⁻⁶. 6 CFU: 4×10 6 CFU: 1 mL was mixed, and the substrate was changed to brewer's yeast, lactobacillus plantarum and papaya at a ratio of 0.5 × 10⁻⁶. 6 CFU: 5×10 6 CFU: Mix at a ratio of 1 mL; set to 1:10.

[0050] Comparative Example 6 Based on Example 1, brewer's yeast, lactobacillus plantarum, and papaya substrate were mixed at a ratio of 0.5 × 10⁻⁶. 6 CFU: 4×10 6 CFU: 1 mL was mixed, and the ratio of Lactobacillus plantarum and papaya substrate was changed to 4 × 10⁻⁶. 6CFU: Mix at a ratio of 1 mL.

[0051] Comparative Example 7 Based on Example 1, brewer's yeast, lactobacillus plantarum, and papaya substrate were mixed at a ratio of 0.5 × 10⁻⁶. 6 CFU: 4×10 6 CFU: 1 mL was mixed, and the ratio of Lactobacillus acidophilus and papaya substrate was changed to 4 × 10⁻⁶. 6 CFU: Mix at a ratio of 1 mL.

[0052] Comparative Example 8 Based on Example 1, brewer's yeast, lactobacillus plantarum, and papaya substrate were mixed at a ratio of 0.5 × 10⁻⁶. 6 CFU: 4×10 6 CFU: 1 mL was mixed, and the ratio of thermophilic lactobacillus and papaya substrate was changed to 4 × 10⁻⁶. 6 CFU: Mix at a ratio of 1 mL.

[0053] Comparative Example 9 Based on Example 1, brewer's yeast, lactobacillus plantarum, and papaya substrate were mixed at a ratio of 0.5 × 10⁻⁶. 6 CFU: 4×10 6 CFU: 1 mL was mixed, and the mixture was changed to Lactobacillus delbrueckii subsp. bulgaricus and papaya substrate were mixed at a ratio of 4 × 10⁻⁶. 6 CFU: Mix at a ratio of 1 mL.

[0054] Experiment 1: α-glucose inhibitory activity of papaya pulp fermented by different lactic acid bacteria An assay was performed to detect the α-glucosidase inhibitory activity of papaya fermentation broth using p-nitrophenyl-α-D-glucopyranoside (PNPG) as a substrate.

[0055] The papaya fermentation broth obtained from papaya substrate without any added bacteria was used as a control.

[0056] The inhibitory activity of papaya fermentation broths from control and comparative examples 6 to 9 against α-glucosidase was detected.

[0057] The results are as follows Figure 1 As shown, compared with Lactobacillus acidophilus, Lactobacillus thermophilus, and Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus plantarum has higher α-glucosidase inhibitory activity.

[0058] Experiment 2: α-glucose inhibitory activity of co-fermented papaya pulp The papaya fermentation broth obtained from papaya substrate without any added bacteria was used as the blank group.

[0059] The inhibitory activity of papaya fermentation broth from the blank group, comparative example 6, and example 1 on α-glucosidase was detected.

[0060] The results are as follows Figure 2 As shown, co-fermentation with *Saccharomyces cerevisiae* and *Lactobacillus plantarum* exhibits higher enzyme inhibitory activity than fermentation with *Lactobacillus plantarum* alone. Repeated tests showed that the α-glucosidase inhibitory activity in the control group was 20.57%, 19.46%, 19.79%, and 19.94%, respectively; the α-glucosidase inhibitory activity in Comparative Example 6 was 95.25%, 94.61%, and 95.48% in three tests, respectively; and the α-glucosidase inhibitory activity in Example 1 was 98.25%, 98.49%, and 99.01% in three tests, respectively.

[0061] Experiment 3 Colony Counting in Papaya Fermentation Broth Samples with Different Fermentation Ratios of Lactobacillus plantarum and Saccharomyces cerevisiae Experiment 4: The papaya substrate fermented without any bacteria was tested and represented as 0:0; the α-glucosidase inhibitory activity of Comparative Examples 1-4, Example 1, and Comparative Example 5 was tested.

[0062] The results of Experiments 3 and 4 are as follows: like Figure 3 and Figure 4 As shown in the plate count results, the 1:8 ratio of *Saccharomyces cerevisiae* to *Lactobacillus plantarum* significantly increased the colony count of *Lactobacillus plantarum* during co-fermentation. Simultaneously, the α-glucosidase inhibition experiment showed that the 1:8 ratio exhibited higher α-glucosidase inhibitory activity, especially showing the best α-glucosidase inhibitory activity after in vitro simulated digestion. This demonstrates the significant potential for the preparation of α-glucosidase inhibitors from co-fermented papaya beverages.

[0063] Single-strain substrate utilization is limited; relying solely on the lactic acid bacteria's own enzyme system is insufficient to fully break down large molecules such as papaya cellulose and pectin, resulting in inadequate release of active precursors and ultimately low inhibitory activity. Poor strain compatibility is also a concern; most lactic acid bacteria show low compatibility with papaya substrates and have simple metabolic pathways, making it difficult to generate highly active inhibitory components. Combined fermentation enhances the effect by employing a yeast + lactic acid bacteria synergistic fermentation approach, enhancing activity through complementary microbial metabolism.

[0064] Co-fermentation of papaya results in more thorough substrate degradation, higher conversion efficiency of active substances, and sufficient α-glucosidase inhibitory activity. The enzyme inhibitory activities of the co-fermentation products show little difference. The combined microbial fermentation system is specifically matched to papaya substrates, achieving a significant synergistic effect in inhibitory activity.

[0065] Using papaya pulp as raw material, a method of co-fermentation of Saccharomyces cerevisiae and Lactobacillus plantarum was adopted. By exploring the optimal fermentation ratio of the two microorganisms, the best α-glucosidase inhibitory activity was achieved, and an α-glucosidase inhibitor with higher fermentation activity than Lactobacillus plantarum alone was prepared, which also enhanced the fermentation flavor.

[0066] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. An α-glucosidase inhibitor, characterized in that, The α-glucosidase inhibitor was prepared from Saccharomyces cerevisiae, Lactobacillus plantarum, and papaya substrate at a ratio of 0.5 × 10⁻⁶. 6 CFU: (1×10) 6 CFU~5×10 6 CFU: Obtained by anaerobic fermentation after mixing at a ratio of 1 mL.

2. The method for preparing the α-glucosidase inhibitor according to claim 1, characterized in that, Includes the following steps: Mix peeled red papaya with water and homogenize to make papaya puree; Mix papaya homogenate, cellulase and pectinase, heat, filter, and obtain filtrate; The filtrate, white sugar, and skim milk were mixed, the pH was adjusted to 6.2-6.8, and the mixture was sterilized to obtain the papaya substrate. Saccharomyces cerevisiae, Lactobacillus plantarum, and papaya substrate were mixed in a certain proportion and fermented anaerobically at 35℃~39℃ for 47h~49h to obtain an α-glucosidase inhibitor.

3. The preparation method according to claim 2, characterized in that, The peeled red papaya measures 2.5cm in size. 3 ~3cm 3 .

4. The preparation method according to claim 2, characterized in that, The mass ratio of the peeled red papaya to water is 1:(0.8~1.2).

5. The preparation method according to claim 2, characterized in that, The papaya homogenate, cellulase, and pectinase were mixed in a mass ratio of 100:0.05:(0.04~0.06).

6. The preparation method according to claim 2, characterized in that, The heating temperature is 45℃~55℃; the heating time is 1 hour.

7. The preparation method according to claim 2, characterized in that, The filtrate, white sugar, and skim milk are mixed in a ratio of 100mL: 5g: (4.5mL~5.5mL); the skim milk is prepared by mixing 10g of skim milk powder with 100mL of water.

8. The preparation method according to claim 2, characterized in that, The sterilization method is pasteurization.

9. The use of the α-glucosidase inhibitor according to claim 1 in the preparation of health products that help maintain healthy blood glucose levels.

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