Method for extracting highland barley polyphenol based on probiotic fermentation

Abstract

The invention belongs to the field of polyphenol extraction, and particularly relates to a method for extracting highland barley polyphenol based on probiotic fermentation. According to the preparation method disclosed by the invention, firstly, the highland barley powder is fermented by utilizing lactobacillus acidophilus, so that the extraction rate of highland barley polyphenol is increased, and the antioxidant activity and alpha-glucosidase inhibitory activity of highland barley polyphenol are changed; in the extraction process, the fermentation residues are pretreated, extraction of the highland barley polyphenol is further improved, meanwhile, the highland barley polyphenol extracted by fermenting highland barley has the potential of assisting in treating type II diabetes, and a foundation is laid for further development and utilization of highland barley.

Description

Technical Field

[0001] This invention belongs to the field of polyphenol extraction, specifically relating to a method for extracting barley polyphenols based on probiotic fermentation. Background Technology

[0002] highland barley ( Hordeum vulgare Linn var. nudum Hook.f. Barley (Horse barley), a plant belonging to the genus *Horse* of the Poaceae family, is also known as naked barley, rye, or naan barley because its grains are exposed. It is a staple food for Tibetans in China and a major raw material for brewing highland barley wine. It is distributed in high-altitude and cold regions such as Qinghai, Tibet, and Ganzi and Aba prefectures in Sichuan. Polyphenols are abundant secondary metabolites in barley, and current research shows that the content of phenolic substances in barley is much higher than in other grains.

[0003] Most polyphenols naturally occurring in edible plants exist in free or bound forms, and the appropriate extraction methods vary among different plants due to differences in their chemical composition. Xu Fei et al. optimized the extraction process of bound polyphenols from barley, but their method mainly used barley bran as raw material, and the extraction rate still needs improvement. Currently, there is limited research on the extraction and activity of polyphenols from barley. Summary of the Invention

[0004] To address the problems existing in the prior art, the present invention provides a method for extracting barley polyphenols based on probiotic fermentation.

[0005] The technical solution adopted by the present invention to achieve the above objectives is as follows: This invention provides a method for extracting barley polyphenols based on probiotic fermentation, comprising the following steps: (1) After sterilizing and drying the barley flour, cool it to room temperature, then inoculate it with seed liquid of probiotic strains, and carry out shaker culture and fermentation. After fermentation, sterilize it. (2) Extraction of free phenols from barley using ultrasonic-assisted methanol: Add methanol to fermented barley powder, extract by ultrasonication, centrifuge, and take the supernatant; (3) Add NaOH solution and sorbitol to the residue remaining after the extraction of free phenols, mix thoroughly and shake to digest, adjust pH, extract the bound phenols in the reaction system with ethyl acetate; collect and combine the above organic solution layers, concentrate by vacuum rotation to obtain barley bound phenolic substances.

[0006] Preferably, in step (1), the ratio of barley flour to seed liquid is 5g:1-1.5mL; the concentration of the seed liquid is 10. 8 CFU / mL.

[0007] Preferably, in step (1), the fermentation is carried out at 37°C, with a shaking speed of 90 r / min and an initial pH of 5.5 for 36 h.

[0008] Preferably, in step (2), the ratio of fermented barley flour to methanol is 1:10; the ultrasonic conditions are: extraction for 30-40 min at a temperature of 30-35℃ and a power of 200-250 W; and centrifugation for 30 min at 4000 rpm.

[0009] Preferably, in step (3), the concentration of the NaOH solution is 2 mol / L; the sorbitol accounts for 1-3% of the mass of highland barley flour; and the pH of the reaction solution is adjusted to 2.0 using concentrated hydrochloric acid.

[0010] Preferably, in step (3), the time for the oscillation digestion is 4 hours; and the temperature for the vacuum rotary concentration is 45°C.

[0011] The beneficial effects of this invention are as follows: (1) In this invention, Lactobacillus acidophilus was first used to ferment barley flour, which improved the extraction rate of barley polyphenols and changed its antioxidant activity and α-glucosidase inhibitory activity. (2) In the extraction process of this invention, the extraction of barley polyphenols is further improved by pre-treating the fermentation residue. At the same time, the barley polyphenols obtained by this invention through fermentation of barley have the potential to assist in the treatment of type 2 diabetes, laying the foundation for the further development and utilization of barley. Attached Figure Description

[0012] Figure 1 Content of bound phenols and free phenols before and after fermentation of different strains; Figure 2 HPLC was used to determine the composition and content of bound phenols and free phenols before and after fermentation. Figure 3 The ability of free and bound phenols to scavenge DPPH free radicals before and after fermentation; Figure 4 To inhibit the α-glucosidase activity of fermented barley polyphenols. Detailed Implementation

[0013] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0014] The Lactobacillus acidophilus used in this invention was purchased from Shandong Zhongke Jiayi Bioengineering Co., Ltd.

[0015] Example 1 1. Materials and Instruments The experimental material was highland barley (Tibetan barley 3000). The dried highland barley grains were ground, passed through a 60-mesh sieve, and stored in a sealed bag at 4℃ for later use. The reagents included methanol, NaOH, concentrated hydrochloric acid, and ethyl acetate.

[0016] Electronic balance, vertical pressure steam sterilizer, shaker, Jinghong electric thermostatic drying oven, Thermo Fisher fully automatic cell counter, microscope, clean bench, drying oven, ultrasonic cleaner, centrifuge, EYELA rotary evaporator, fume hood.

[0017] 2. Methods 2.1 Preparation of probiotic-fermented highland barley polyphenols 2.1.1 Probiotic culture Five probiotics—LPL (Lactobacillus plantarum), LRH (Lactobacillus rhamnosus), LBU (Lactobacillus bulgaricus), LAC (Lactobacillus acidophilus), and LCA (Lactobacillus casei)—were inoculated into sterilized MRS medium and cultured for 1 day to obtain seed culture.

[0018] 2.1.2 Probiotic-fermented highland barley Add 5 g of highland barley flour (passed through a 60-mesh sieve) and 90 mL of distilled water to six 250 mL Erlenmeyer flasks respectively, and sterilize in an autoclave at 121 °C (separately placed in each Erlenmeyer flask with the highland barley flour) for 25 min. Then dry in an oven for 1 h and cool to room temperature. Inoculate each highland barley sample with 1.2 mL of different inoculum seed solution (concentration 10) at a 6% (w / v) inoculation rate. 8 The barley flour was fermented at 37°C (CFU / mL) for 36 h under shaking conditions at 90 r / min and initial pH 5.5. After fermentation, the fermentation flask was inactivated at 80°C for 10 min.

[0019] 2.1.3 Preparation of free phenols from barley Ultrasonic-assisted extraction of free phenols from highland barley using methanol: anhydrous methanol was used, the solid-liquid ratio was 1:10, the ultrasonic temperature was 30 ℃, the ultrasonic power was 200 W, and the extraction time was 30 min. The mixture was then centrifuged (4000 rpm, 30 min), and the supernatant was collected and diluted to 100 mL in a volumetric flask. The phenol content was then determined. After the analysis, the phenols were evaporated to dryness at 45 ℃ and stored at -20 ℃.

[0020] 2.1.4 Barley-based phenol extraction 20 mL of 2 mol / L NaOH solution and 0.1 g of sorbitol were added to the residue remaining after the extraction of free phenols, and the mixture was thoroughly mixed and shaken for digestion for 4 h. The pH of the reaction solution was then adjusted to 2.0 with concentrated hydrochloric acid. The bound phenols in the reaction system were extracted three times with ethyl acetate. The organic solution layers were collected and combined, concentrated under vacuum (45 °C), and then diluted to 10 mL with anhydrous methanol to obtain the barley bound phenolic extract, which was stored at -20 °C for subsequent experiments.

[0021] 2.1.5 Establishment of the standard curve for highland barley polyphenols Dissolve 6 mg of gallic acid standard in anhydrous methanol and dilute to volume in a 10 mL volumetric flask. Perform serial dilutions of 0, 20, 60, 100, 150, 200, 300, 400, 500, and 600 μg / mL gallic acid standard solution in 5 mL centrifuge tubes. Add 100 μL of Folin-Ciocalteu reagent to each concentration gradient tube, react for 6 min, add 1 mL of 7% Na₂CO₃ and 0.8 mL of water, mix well, react for 90 min, and measure the absorbance at 760 nm using a microplate reader. Plot gallic acid concentration (mg / mL) on the x-axis and absorbance on the y-axis to obtain the standard curve: the formula is y = 0.002x + 0.0521(R² / 2π)² / 2π. 2 = 0.9951).

[0022] 2.1.6 Determination of Polyphenol Content in Fermented Highland Barley Detection of polyphenol content in fermented highland barley: The bound phenols and free phenols in the methanol extract after final volume determination were measured according to the gallic acid standard detection method to obtain their absorbance values. The corresponding concentrations were calculated using the regression equation of the gallic acid standard curve and the OD760 value.

[0023] 2.1.7 Determination of the composition and content of polyphenols in fermented highland barley Polyphenol composition analysis: High-performance liquid chromatography (HPLC) was used to analyze the composition of different polyphenol extracts using a digital ultraviolet detector (DAD). Appropriately diluted polyphenol extracts were filtered through a 0.22 μm organic filter before HPLC analysis. A Sunfire C18 reversed-phase column (250 × 4.6 mm, 5 μm) was used at 30℃; the sample injection volume was 10 µL; the mobile phase consisted of phase A (phosphate buffer, pH=2.5) and phase B (acetonitrile); the elution flow rate was 0.8 mL / min, using gradient elution (0–35 min, 15% B; 35–36 min, 50% B; 36–40 min, 80% B; 40–45 min, 20% B; 45–55 min, 85% B); the elution time was 55 min. Polyphenol spectra were recorded, and the extraction chromatogram wavelength was 280 nm.

[0024] 2.2 In vitro activity detection ① DPPH free radical scavenging activity assay Mix 0.1 mL of sample solution with 0.1 mL of DPPH reagent, react at room temperature in the dark for 30 min, and then measure the absorbance at 517 nm. The sample solvent serves as a blank control. Calculate the DPPH free radical inhibition rate: Inhibition rate (%) = ×100 In the formula, A0 is the absorbance of the blank control; A1 is the absorbance of the sample.

[0025] ② Determination of α-glucosidase inhibitory activity Solution preparation (1) Preparation of α-glucosidase: Prepare 0.2 U / mL, i.e., 1 mg of enzyme dissolved in 50 mL PBS buffer and stored at 4℃.

[0026] (2) Preparation of P-NPG substrate: Prepare to 2 mmol / L, that is, accurately weigh 60.25 mg and dissolve it in 100 mL PBS buffer and store at low temperature.

[0027] (3) Sample solution preparation: Weigh 1 mg of polyphenol extract and dissolve it in 1 mL of PBS buffer and store at low temperature.

[0028] (4) Preparation of positive control solution: Weigh 1 mg of acarbose and dissolve it in 1 mL of PBS buffer and store at low temperature.

[0029] (5) Preparation of 0.2 mol / L Na2CO3 solution: Weigh 2.12 g Na2CO3 into a beaker, add an appropriate amount of distilled water solution, and make up to 100 mL. Store at 4℃.

[0030] 2.3 Determination of the inhibitory activity of barley polyphenol extract against α-glucosidase: α-glucosidase inhibitory activity was determined using the p-NPG method. The activity assay was performed on 96-well plates. The experimental group was treated with 25 μL of sample solution and α-glucosidase (0.2 U / mL, 25 μL), the background group with only sample solution, and the blank group with only PBS buffer. After addition, the plates were incubated at 37°C for 15 min. Then, 50 μL of p-NPG substrate was added to each well, and the plates were incubated at 37°C for 20 min. The reaction was terminated by adding Na₂CO₃ to each well, and a yellow color was observed. The OD value was measured at 405 nm using a microplate reader.

[0031] Inhibition rate (%) = ×100% 3. Results 3.1 Content of polyphenols in fermented highland barley The absorbance of the samples at 760 nm was measured using an ELISA reader. The results were then substituted into the regression equation of the gallic acid standard curve to determine the content of free and bound phenols in barley fermented by different bacterial strains. (Specific details are as follows...) Figure 1 As shown in the figure. Calculations showed that Lactobacillus acidophilus had the highest total phenol content; therefore, Lactobacillus acidophilus-fermented highland barley was selected for the following activity study.

[0032] 3.2 Composition and content of polyphenols in fermented highland barley Preliminary HPLC analysis revealed significant differences in the composition and content of polyphenols in the barley polyphenol extracts before and after Lactobacillus acidophilus fermentation. Figure 2 As shown.

[0033] 3.3 Antioxidant activity of fermented barley polyphenols The changes in the antioxidant activity of polyphenols before and after fermentation with Lactobacillus acidophilus were determined. Specific results are as follows: Figure 3 As shown. Free phenols after fermentation ( Figure 3 A) The DPPH free radical scavenging ability was enhanced compared to the unfermented group. Within the range of 0.2-1 mg / mL, the dependence increased with increasing concentration. At 1 mg / mL, the DPPH free radical scavenging rates of fermented and unfermented free phenols reached 52.06% and 29.72%, respectively; after fermentation, the bound phenols ( Figure 3 B) The DPPH free radical scavenging ability was enhanced compared with the unfermented group. In the range of 0.02-0.1 mg / mL, the dependence increased with the increase of concentration. At 0.1 mg / mL, the DPPH free radical scavenging rate of fermented and unfermented bound phenols reached 46.66% and 40.15%, respectively. Overall, fermentation improved the antioxidant activity of polyphenols.

[0034] 3.4 α-Glucosidase Inhibitory Activity of Fermented Barley Polyphenols The α-glucosidase inhibitory activity was determined using 1 mg / mL of free phenol and conjugated phenol extracts from highland barley. The results showed that... Figure 4 As shown, the α-glucosidase inhibitory activity of both free and bound polyphenol extracts was significantly enhanced after fermentation. (F—Free polyphenolic; B—Bound polyphenolic) 4. Summary Fermentation of highland barley flour using Lactobacillus acidophilus improved the extraction rate of highland barley polyphenols and altered their antioxidant and α-glucosidase inhibitory activities. Fermented highland barley shows potential as an adjunct treatment for type 2 diabetes, laying the foundation for further development and utilization of highland barley.

Claims

1. A method for extracting barley polyphenols based on probiotic fermentation, characterized in that, The following steps are involved: (1) After sterilizing and drying the barley flour, cool it to room temperature, then inoculate it with seed liquid of probiotic strains, and carry out shaker culture and fermentation. After fermentation, sterilize it. (2) Extraction of free phenols from barley using ultrasonic-assisted methanol: Add methanol to fermented barley powder, extract by ultrasonication, centrifuge, and take the supernatant; (3) Add NaOH solution and sorbitol to the residue remaining after the extraction of free phenols, mix thoroughly and shake to digest, adjust the pH, and extract the bound phenols in the reaction system with ethyl acetate; The organic solution layers were collected and combined, and then concentrated under vacuum to obtain barley-bound phenolic substances.

2. The method according to claim 1, characterized in that, In step (1), the ratio of barley flour to seed liquid is 5g:1-1.5mL; the concentration of the seed liquid is 10. 8 CFU / mL.

3. The method according to claim 2, characterized in that, In step (1), the fermentation is carried out at 37°C, with a shaking speed of 90 r / min and an initial pH of 5.5 for 36 h.

4. The method according to claim 1, characterized in that, In step (2), the ratio of fermented barley flour to methanol is 1:10; the ultrasonic conditions are: extraction for 30-40 min at 30-35℃ and 200-250 W power; and centrifugation for 30 min at 4000 rpm.

5. The method according to any one of claims 1-4, characterized in that, In step (3), the concentration of the NaOH solution is 2 mol / L; the sorbitol accounts for 1-3% of the mass of highland barley flour; and the pH of the reaction solution is adjusted to 2.0 using concentrated hydrochloric acid.

6. The method according to claim 1 or 5, characterized in that, In step (3), the time for the oscillation digestion is 4 hours; the temperature for the vacuum rotation concentration is 45°C.

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