Mung bean mixed fermentation product rich in micromolecule polypeptide and preparation method and application of mung bean mixed fermentation product

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to a mung bean mixed fermentation product rich in micromolecule polypeptide and a preparation method and application thereof. The method adopts a strain synergistic fermentation technology, and specifically comprises the following steps: fermenting mung beans by using two probiotics including saccharomyces cerevisiae and lactobacillus plantarum as fermentation strains, and carrying out homogenization wall breaking, centrifugation, ultrafiltration, ethanol precipitation and macroporous adsorption resin purification on the fermentation liquor to obtain a fermentation stock solution rich in micromolecule polypeptides. The obtained fermentation stock solution shows certain free radical scavenging and anti-allergic effects. The fermentation liquor is clear and transparent, has good room-temperature stability, special fragrance and rich small-molecular-weight polypeptides, and has good application value in the fields of skin care and dietary supplements.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a mixed-culture fermentation product of mung beans rich in small molecule polypeptides, its preparation method, and its application. Background Technology

[0002] Mung beans, also known as green beans, belong to the genus *Vigna* and are one of the most important legumes in Asia. They have a long history of cultivation in my country and are widely grown, mainly concentrated in the Yellow River, lower Yangtze River, and North China regions, with Inner Mongolia and Henan having the largest cultivation areas. Mung beans contain 22%–26% protein, which can supplement protein intake. They also contain various bioactive components, such as resistant starch, polysaccharides, flavonoids, and antifungal proteins, which have effects such as lowering blood lipids, antioxidation, antitumor, antibacterial properties, and improving immunity. They also have health benefits such as strengthening the spleen and promoting diuresis, clearing heat and detoxifying, and promoting blood circulation and removing pus. Currently, there are many mung bean products on the market, such as mung bean protein milk drinks, mung bean cakes, and mung bean starch sheets. In addition, mung beans can also be fermented to make soy sauce.

[0003] Mung bean protein is rich in essential amino acids, such as leucine, lysine, phenylalanine, and valine, and is considered an important source of dietary protein with high nutritional value. Mung bean protein has various physiological activities, such as anti-cancer, anti-proliferative, and ACE-inhibiting activities. Although mung bean protein can be used to supplement protein, some protein fragments are allergenic (Tan Chao et al. Preparation of polyclonal antibodies against mung bean allergens and establishment of indirect competitive enzyme-linked immunosorbent assay method. Chinese Journal of Food Science, 2013, 13(7):170-173.). In addition, mung bean protein also exhibits excellent functional properties. The emulsifying ability of mung bean protein (0.58) is higher than that of albumin (0.45), and the water-holding capacity (2.75 mL / g) is higher than that of gourd shredded protein concentrate (1.56 mL / g). Mung bean protein may become a good ingredient in various processed food formulations. It has been reported that Brishti et al. used mung bean protein as an additive for vegetarian meat. Mung bean protein treated with heat and ultrasound has good solubility and stability and can be used as a raw material for the production of high-protein beverages.

[0004] Mung bean polypeptides are a mixture of oligopeptides derived from the hydrolysis of mung bean protein. They have a small relative molecular mass, contain a large number of essential amino acids, and are highly water-soluble, low in viscosity, and highly stable. Mung bean polypeptides have a wider range of applications in the food and pharmaceutical fields. They promote the growth of beneficial bacteria in the intestines and can be made into intestinal nutrients or supplementary foods to help people with weak digestive abilities to quickly restore their absorption function.

[0005] In traditional technology, bioactive peptides can be obtained through two methods: protease hydrolysis and microbial fermentation, or a combination of both. Enzymatic hydrolysis uses mung bean flour as a raw material, removing starch to produce mung bean protein, which is then hydrolyzed with neutral or alkaline proteases to obtain mung bean polypeptides. This method suffers from drawbacks such as limited material availability and high cost. Currently, the preparation of hydrolyzed mung bean polypeptides mostly employs enzymatic hydrolysis, using mung bean flour as a raw material, removing starch to produce mung bean protein, and then hydrolyzing it with neutral or alkaline proteases to obtain mung bean polypeptides. This method also suffers from drawbacks such as limited material availability and high cost, and loses various mung bean active substances such as flavonoids during the process. Furthermore, enzymatic hydrolysis involves small batch sizes and large enzyme usage, resulting in a high unit price for hydrolyzed mung bean protein, limiting its commercial use.

[0006] Due to differences in amino acid sequences and molecular sizes, peptide fragments exhibit vastly different activities. For example, enzymatic hydrolysis has been reported to obtain mung bean peptides, yielding peptides with antibacterial or hypotensive activities. The activities of peptide products obtained by different preparation methods vary (CN201310256458.9; Wu Jinhong. Isolation, purification and characterization of antimicrobial peptides in mung beans and their crystal culture [D]. Fuzhou University, 2004.).

[0007] Therefore, there is still considerable room for development in the field of mung bean polypeptide products, both in terms of manufacturing methods and product applications. Summary of the Invention

[0008] To address a series of problems existing in the fermentation and extraction technology of small-molecule soybean peptides, this invention has two objectives. First, it provides a mixed-culture fermentation product rich in mung bean hydrolyzed peptides, achieved by fermenting the abundant protein in mung beans with two types of probiotics to hydrolyze it into peptides with smaller molecular weights. Second, it provides a method for preparing the fermentation broth of this mixed-culture fermentation product rich in mung bean hydrolyzed peptides. This preparation method and the resulting mixed-culture fermentation product rich in mung bean peptides exhibit good anti-allergic and antioxidant effects, and are expected to be used in the care of sensitive skin.

[0009] To achieve the above objectives, the main solutions adopted are as follows:

[0010] Two probiotic strains, Bacillus subtilis and Saccharomyces cerevisiae, were used as fermentation starters. Fermentation process parameters were controlled, and mung bean syrup was added to the fermentation system. At the end of the fermentation, the bacterial residue was removed, and the fermentation liquid was filtered to obtain a clear solution rich in small molecule peptides.

[0011] Based on this approach, the present invention first provides a mung bean mixed-culture fermentation product rich in small molecule polypeptides:

[0012] This mung bean mixed-culture fermented product is prepared by crushing and pulping mung beans, sterilizing them, inoculating them with two kinds of probiotics for synergistic fermentation, and then purifying them through homogenization, centrifugation, ultrafiltration, ethanol precipitation, and macroporous adsorption resin. The probiotics are a mixture of Saccharomyces cerevisiae and Lactobacillus plantarum; the molecular weight cutoff for the ultrafiltration operation is 1000 Da. The term "mung bean" refers to the mature seed of Vigna radiata (L.) R. Wilczek, an annual erect herbaceous plant belonging to the genus Vigna in the legume family.

[0013] Preferably, the weight ratio of Saccharomyces cerevisiae to Lactobacillus plantarum in the mixed bacteria is 1:2.

[0014] Preferably, the inoculum size of the mixed bacteria is 6%, the fermentation temperature is 30℃±5℃, the initial pH is 5.0±1.0℃, and the fermentation speed is 150 rpm.

[0015] Based on this solution, the present invention further provides a method for preparing the above-mentioned mung bean mixed-culture fermentation product, comprising the following steps:

[0016] S1 mung bean pretreatment: Weigh mung beans, add water and grind into a paste, stir and transfer to a fermentation tank, sterilize to obtain sterilized mung bean paste;

[0017] S2 Inoculation and Fermentation: A mixture of brewer's yeast and Lactobacillus plantarum is inoculated into the sterilized mung bean slurry from step S1 for fermentation to obtain crude fermentation liquid;

[0018] S3 Post-treatment of crude fermentation broth: Homogenize and break the cell walls of the crude fermentation broth obtained in step S2, centrifuge the obtained suspension to remove the residue, obtain fermentation broth, adjust the pH, add an ultrafiltration membrane for ultrafiltration, and the obtained external liquid is the filtrate.

[0019] S4 Filtrate Purification: Add ethanol to the filtrate obtained in step S3, allow it to stand and precipitate, and filter to remove the precipitate; use macroporous adsorption resin to purify the mung bean mixed bacteria fermentation product; the molecular weight cutoff of the ultrafiltration operation is 1000 Da.

[0020] Preferably, the weight ratio of the brewing yeast and the lactobacillus plantarum is 1:2.

[0021] Preferably, the inoculum amount of the mixed bacteria is 6%, the fermentation temperature is 30℃±5℃, the initial pH is 5.0±1.0℃, and the fermentation speed is 150 rpm.

[0022] Preferably, the conditions for high-pressure homogenization and cell wall disruption are: disruption pressure 70 MPa, number of cycles ≥ 5, and circulation temperature ≤ 20°C. The purpose of high-pressure homogenization and cell wall disruption is to destroy the cell wall of the bacterial strain, which is a conventional technique in the art. Obviously, those skilled in the art will find it sufficient within this limited range to obtain various parameters or combinations of parameters to achieve cell wall disruption. More preferably, the number of cycles is 6–11, and the circulation temperature is 15–20°C.

[0023] Preferably, the ultrafiltration operation involves a feed solution temperature of 30±5℃ and a pressure of 0.2 MPa, with circulation treatment until the internal liquid volume is less than or equal to 5L. The purpose of ultrafiltration is to separate large molecules from small molecules using pressure as the driving force and physical barriers. Obviously, those skilled in the art will find that within this limited range, a technical solution is sufficient to separate molecules of the desired size. More preferably, the ultrafiltration operation involves circulation treatment until the internal liquid volume is 3-5L.

[0024] Preferably, the macroporous adsorption resin is HP20SS type macroporous resin, and the mobile phase for purification by the macroporous adsorption resin is water and ethanol.

[0025] Studies on this mung bean mixed-culture fermented product have revealed its antioxidant and anti-allergic properties, making it suitable for sensitive skin care. Therefore, this invention further provides the application of this mung bean mixed-culture fermented product in the preparation of antioxidants and anti-allergic agents. Since the human gastrointestinal tract and skin have a high absorption rate for peptides with a molecular weight below 1000 Da, this product is suitable as a dietary supplement or skincare agent.

[0026] The present invention has the following beneficial effects:

[0027] This invention separates proteins and other substances contained in mung beans by selecting strains of bacteria and optimizing the fermentation process, and reasonably controls the degree of degradation. The resulting mung bean mixed-culture fermented product has dual effects of anti-oxidation and anti-allergy.

[0028] The mung bean mixed-culture fermentation of the present invention, through mixed-culture fermentation and purification, yields polypeptide molecules with small molecular weight and easy absorption, suitable for sensitive skin care or as a dietary supplement. Detailed Implementation

[0029] This invention relates to a fermentation broth rich in small molecule polypeptides and its preparation method. Lactobacillus plantarum and Saccharomyces cerevisiae are used to co-ferment mung bean slurry. The resulting fermentation broth is then subjected to membrane treatment and macroporous resin extraction and enrichment to obtain the fermentation stock solution.

[0030] The preparation method is described in detail below through preferred embodiments. It should be particularly noted that those skilled in the art can refer to the main technical route and embodiments of this invention and make appropriate modifications and alterations to the preparation method without departing from the scope of this invention to achieve equivalent or similar technical effects. All similar substitutions and modifications are obvious to those skilled in the art and should be considered as included within the scope of protection of this invention. Unless otherwise specified, the parameters used in the following experiments follow common sense and conventional understanding in the art; for example, the total inoculation amount is 6%, and the volume is expressed as a percentage of volume.

[0031] Example 1: Pretreatment of mung beans

[0032] Weigh 30 kg of mung beans, add a small amount of water, and grind them into a paste using an industrial fruit and vegetable crusher. Add water to a total weight of 120 kg, stir thoroughly, and transfer to a 200 L fermentation tank. Heat with steam to boiling and maintain for 1 hour. Seal the fermentation tank, switch to sterilization mode, set the sterilization temperature to 121℃, the sterilization pressure to 0.13 MPa, and sterilize for 30 minutes.

[0033] Example 2: Pretreatment of bacterial strains

[0034] Prepare MRS culture medium according to the following formula: 10 g / L peptone, 5 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L diammonium hydrogen citrate, 0.5 g / L magnesium sulfate heptahydrate, 0.005 g / L manganese sulfate, and 20 g / L agar. Adjust the pH to 6.5, sterilize at 121℃ for 20 minutes, and set aside for use.

[0035] Prepare PDA culture medium according to the following formula: 200g / L potato, 20g / L glucose, 20g / L agar, sterilize at 115℃ for 20min and set aside.

[0036] In a sterile operating table, Lactobacillus plantarum (strain number: DSM 9843) was inoculated into MRS medium and incubated on a shaker at 35°C and 140 rpm for 24 h to activate it.

[0037] In a sterile operating table, the brewing yeast (strain number: AH109) was inoculated into PDA medium and incubated on a shaker at 32°C and 140 rpm for 24 h to activate it.

[0038] Example 3 Inoculation and Fermentation

[0039] The activated *Lactobacillus plantarum* and *Saccharomyces cerevisiae* from Example 2 were inoculated into the fermenter of Example 1, and fermentation was carried out according to the following parameters:

[0040] Inoculation ratio: Saccharomyces cerevisiae: Lactobacillus plantarum = 1:2 (by weight);

[0041] Total vaccination volume: 6%;

[0042] Culture temperature: Control the temperature at 30℃±5℃

[0043] Initial pH: 5.0 ± 1.0℃;

[0044] Speed: 150 rpm;

[0045] Molasses addition amount: 10%–20%;

[0046] Defoamer: Triton-100, addition amount 1%

[0047] Example 4 Post-treatment of fermentation broth

[0048] After the bacterial culture in Example 3 stopped increasing at the OD value (absorbance value), the resulting fermentation broth was processed using a high-pressure homogenizer with the following process parameters:

[0049] Cell wall breaking pressure: 70 MPa; Number of cycles: 6; Cycle temperature: 18℃.

[0050] The resulting dark yellow-green slurry was centrifuged with a disc centrifuge to remove the residue, yielding a light yellow-brown fermentation broth. The pH was adjusted to 6.0±0.5, and the broth was added to a 1000Da ultrafiltration membrane treatment system. The temperature of the broth was controlled at 30±5℃ and the pressure at 0.2Mpa. The broth was circulated until the internal liquid volume reached 4L. The resulting external liquid was the filtrate.

[0051] Example 5 Stepwise precipitation

[0052] Add 20% ethanol to the filtrate obtained in Example 4 (the volume ratio of filtrate to ethanol is 4:1), let it stand for 12 hours to allow sufficient precipitation, filter it with a plate and frame filter press to remove the precipitate, and then filter it with a 1-micron filter cartridge to obtain a clear liquid.

[0053] Add ethanol to the obtained filtered liquid again until the total volume of ethanol is 40%, let it stand for 12 hours to fully precipitate, and then filter it with a 1-micron filter to obtain the ethanol precipitate.

[0054] Example 6 Macroporous Resin Treatment

[0055] HP20SS macroporous resin, soaked in ethanol, was loaded onto a chromatographic column with a diameter of 50 mm and a packing height of 60 cm. The column was repeatedly rinsed with deionized water to remove the ethanol, and then rinsed with 8 column volumes (BV) each of 5% sodium hydroxide solution, deionized water, 5% hydrochloric acid solution, and deionized water in sequence.

[0056] Example 7: Filtrate Treatment

[0057] The ethanol precipitate from Example 5 was adjusted to pH 5.0, diluted with water to a volume percentage of 5%, and loaded onto the macroporous adsorption resin column of Example 6 at a flow rate of 2 BV / h. Elution was performed according to the following procedure:

[0058] Table 1. Column Elution Procedure

[0059] Elution solution Flow rate Elution volume Deionized water 6BV / h 6BV 5% ethanol 4BV / h 6BV 40% ethanol 4BV / h 2BV Deionized water 6BV / h 12BV

[0060] Collect the 40% ethanol eluent, concentrate under reduced pressure to remove the ethanol, add 12L to 15L of water, and adjust the pH to 6.5 ± 0.5 until the solution is no longer turbid. This yields a fermentation stock solution rich in small molecule peptides and various cellular secretory nutrients. A batch of fermentation broth can yield approximately 50L of stock solution.

[0061] Example 8: Determination of Total Peptide Content

[0062] The yield of peptides was determined using the biuret method: 1.0 mL of the peptide extract from Example 7 was accurately measured and placed in a 10 mL volumetric flask. Then, 1.0 mL of biuret reagent A and 0.2 mL of biuret reagent B were added, and the mixture was brought to a final volume with distilled water. The mixture was shaken well and allowed to stand for 10 min. The absorbance was measured at 540 nm. The measured data were substituted into the regression equation of the standard curve to obtain the mass concentration of the sample diluted 10 times. A standard curve was plotted using casein phosphopeptide as the standard, and the absorbance was used to correspond to the peptide mass concentration of the standard curve.

[0063] The standard curve regression equation is: ρ1 = 0.0535A - 0.0011.

[0064] Where A is the absorbance at a wavelength of 540 nm; ρ1 is the mass concentration of casein phosphopeptide (mg / mL).

[0065] Formula for calculating peptide yield:

[0066] Peptide yield = ρ² × V × 10m × 100%

[0067] In the formula: ρ2 is the mass concentration of mung bean polypeptide (mg / mL); V is the total volume of the sample (mL); and m is the mass of mung bean protein (g).

[0068] The obtained fermentation broth contains 60 mg / mL of polypeptides. Based on the protein content of 25% in mung beans, the yield of small molecule polypeptides obtained by this invention is not less than 40%.

[0069] Example 9: Determination of antioxidant activity of fermentation broth

[0070] DPPH free radical scavenging is a common method for assessing antioxidant activity. Its alcoholic solution is purple, with maximum absorption at 517 nm. DPPH has a single electron and can therefore accept one electron or hydrogen ion. When a free radical scavenger is added, the unpaired electron of DPPH is paired by the scavenger, causing its color to lighten. This process results in a decrease in absorbance at the maximum absorption wavelength of 517 nm. The decrease in absorbance level is positively correlated with the antioxidant's scavenging ability; that is, the lower the absorbance, the stronger the antioxidant activity. By measuring the change in absorbance, the antioxidant's scavenging ability can be quantitatively analyzed. The inhibition rate reflects the antioxidant's efficiency in scavenging DPPH free radicals; the higher the inhibition rate, the stronger the antioxidant activity.

[0071] The antioxidant capacity of this product was studied using a DPPH free radical scavenging assay kit, following the kit's instructions and employing a standard DPPH free radical scavenging test. Based on a peptide mass concentration of 60 mg / mL, the fermentation stock broth prepared in Example 7 was added to pH 7.0 phosphate buffer to prepare three concentration solutions: 1 mg / mL, 5 mg / mL, and 8 mg / mL. One mL of each sample solution was mixed with 2 mL of 0.1 mmol / L DPPH solution (stored protected from light) in a colorimetric tube, incubated at room temperature in the dark for 30 min, and then centrifuged (7320 r / min, 5 min). The supernatant was collected. Vitamin C was used as a positive control, and the absorbance was measured at 517 nm. The DPPH free radical scavenging rate (%) was calculated according to the kit's instructions. The sample solutions exhibited significant and clear DPPH free radical scavenging capacity. The DPPH free radical scavenging rates at 1 mg / mL, 5 mg / mL, and 8 mg / mL were 62.7 ± 2.9%, 75.4 ± 2.1%, and 87.2 ± 3.5%, respectively, showing a dose-dependent relationship. The DPPH radical scavenging rate of the 5 mg / mL sample was not significantly different from that of the 8 μg / mL Vc positive control solution; the DPPH radical scavenging rate of the 8 mg / mL sample was not significantly different from that of the 10 μg / mL Vc positive control solution.

[0072] Example 10: Determination of the anti-allergic activity of the fermentation broth

[0073] Hyaluronidase is involved in type I hypersensitivity reactions. Hyaluronidase is strongly associated with inflammation and allergies. It has been reported that histamine-like substances released by mast cells can regulate hyaluronidase activity, while anti-allergy drugs can inhibit hyaluronidase activity. Therefore, measuring hyaluronidase activity is a reliable in vitro method for measuring anti-allergic activity.

[0074] Referring to the literature (Sun Peidong et al. Study on the inhibitory effect of irisin on hyaluronidase [J]. Chemical Bulletin, 2013), its anti-allergic ability was determined by the hyaluronidase inhibition method. The fermentation stock solution prepared in Example 7 was added to pH 7.0 phosphate buffer to prepare three concentration solutions of 1 mg / mL, 5 mg / mL, and 8 mg / mL, based on a mass concentration of 60 mg / mL. Hyaluronidase (concentration not less than 500 U / mL) was freshly prepared using an acetate-sodium acetate buffer system, with the acetate-sodium acetate buffer system as a blank control. The analyte and hyaluronidase were co-incubated at 37°C, and the absorbance value was measured at 530 nm to calculate the hyaluronidase inhibition rate. Each concentration was measured in triplicate. All three concentrations of the sample showed significant and clear hyaluronidase inhibition ability. The inhibition rates of hyaluronidase at concentrations of 1 mg / mL, 5 mg / mL and 8 mg / mL were 16.9±3.7%, 43.8±1.6% and 51.6±2.3%, respectively, and were dose-dependent.

Claims

1. A mung bean mixed-culture fermentation product rich in small molecule polypeptides, characterized in that, The mung bean mixed-culture fermentation product is prepared by crushing and pulping mung beans, sterilizing them, inoculating them with two kinds of probiotics for synergistic fermentation, and then homogenizing, centrifuging, ultrafiltration, ethanol precipitation, and purification with macroporous adsorption resin. The probiotics are a mixture of Saccharomyces cerevisiae and Lactobacillus plantarum. The molecular weight cutoff of the ultrafiltration operation is 1000 Da.

2. The mung bean mixed-culture fermentation product according to claim 1, characterized in that, The weight ratio of Saccharomyces cerevisiae and Lactobacillus plantarum in the mixed bacteria is 1:

2.

3. The mung bean mixed-culture fermentation product according to claim 1, characterized in that, The inoculum size of the mixed bacteria was 6%, the fermentation temperature was 30℃±5℃, the initial pH was 5.0±1.0℃, and the fermentation speed was 150 rpm.

4. The method for preparing the mung bean mixed-culture fermentation product according to claim 1, the method comprising the following steps: S1 mung bean pretreatment: Weigh mung beans, add water and grind into a paste, stir and transfer to a fermentation tank, sterilize to obtain sterilized mung bean paste; S2 Inoculation and Fermentation: A mixture of brewer's yeast and Lactobacillus plantarum is inoculated into the sterilized mung bean slurry from step S1 for fermentation to obtain crude fermentation liquid; S3 Post-treatment of crude fermentation broth: Homogenize and break the cell walls of the crude fermentation broth obtained in step S2, centrifuge the obtained suspension to remove the residue, obtain fermentation broth, adjust the pH, add an ultrafiltration membrane for ultrafiltration, and the obtained external liquid is the filtrate. S4 Filtrate Purification: Add ethanol to the filtrate obtained in step S3, let it stand to precipitate, filter to remove the precipitate; purify with macroporous adsorption resin to obtain mung bean mixed bacteria fermentation product. The ultrafiltration operation has a molecular weight cutoff of 1000 Da.

5. The manufacturing method according to claim 4, characterized in that, The weight ratio of the brewing yeast and Lactobacillus plantarum is 1:

2.

6. The manufacturing method according to claim 4, characterized in that, The inoculum size of the mixed bacteria was 6%, the fermentation temperature was 30℃±5℃, the initial pH was 5.0±1.0℃, and the fermentation speed was 150 rpm.

7. The manufacturing method according to claim 4, characterized in that, The conditions for high-pressure homogenization cell disruption are a cell disruption pressure of 70 MPa and a cycle number of 5 or more.

8. The manufacturing method according to claim 4, characterized in that, The ultrafiltration operation involves a feed temperature of 30±5℃ and a pressure of 0.2Mpa, with the liquid being circulated until the internal liquid volume is less than or equal to 5L.

9. The use of the mung bean mixed-culture fermentation product according to any one of claims 1 to 3 in the preparation of antioxidants.

10. The use of the mung bean mixed-culture fermentation product according to any one of claims 1 to 3 in the preparation of an anti-allergy agent.