Production method of green coffee bean extract capable of improving alpha-glucosidase inhibitory activity

Abstract

The invention discloses a production method of a green coffee bean extract capable of improving alpha-glucosidase inhibitory activity. Cellulase and pectinase are used for improving the yield of chlorogenic acid and a derivative (CGAs) of chlorogenic acid, tannase and esterase are used for conducting structural modification on polyphenol and CGAs in green coffee bean extract clear liquid, and then lactobacillus rhamnosus, lactobacillus paracasei and bacillus subtillis form a composite strain for fermentation. According to the method, the proportion of CGAs components and the proportion of caffeic acid in the green coffee bean extract are improved by combining an enzymic method with the probiotics to ferment the green coffee bean extract, and the alpha-glucosidase inhibitory activity of CGAs and metabolites thereof is improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of food processing, and particularly relates to a method for improving the alpha-glucosidase inhibitory activity of green coffee bean extract by enzyme method and microbial fermentation. BACKGROUND

[0002] In recent years, the importance of primary prevention of type 2 diabetes mellitus has been increasingly recognized, which has stimulated interest in the role of dietary factors in the etiology of type 2 diabetes mellitus. Coffee is one of the most popular beverages in the world, and epidemiological studies have shown that regular coffee consumption can prevent type 2 diabetes mellitus. Experimental studies using animal models of diabetes mellitus have further demonstrated that chlorogenic acids in coffee have good hypoglycemic effects. Chlorogenic acid derivatives (CGAs) are one of the main coffee polyphenols, and their content in green coffee beans is 8-12%. CGAs are caffeoylquinic acids, which are esters of caffeic acid and quinic acid. CGAs include mono-caffeoylquinic acids and di-caffeoylquinic acids. According to the different esterification positions, mono-caffeoylquinic acids mainly include 3-O-caffeoylquinic acid (3-CQA, chlorogenic acid), 4-CQA (cryptochlorogenic acid) and 5-CQA (neochlorogenic acid) and three other isomers; di-caffeoylquinic acids mainly include 3,5-diCQA (isochlorogenic acid A), 3,4-diCQA (isochlorogenic acid B) and 4,5-diCQA (isochlorogenic acid C) and three other isomers. CGAs mainly exist in the form of mono-caffeoylquinic acids in plants, and the contents of 3-CQA in coffee, 4-CQA and 5-CQA in sweet potato peel are relatively rich.

[0003] CGAs can achieve the purpose of regulating glucose metabolism by reducing insulin resistance index and inhibiting the activity of α-amylase and α-glucosidase. As Zhang Yanli's research shows, the inhibition rate of sunflower disc 3-CQA on α-amylase and α-glucosidase is as high as 65% and 61%; Oboh et al. reported that the IC50 value of 3-CQA for inhibiting α-amylase is 9.10 μg / mL, and the IC50 value for inhibiting α-glucosidase is 9.24 μg / mL; however, in the research of Nyambe Silavwe, the crude extract of chlorogenic acid (CGAs) has little effect on α-amylase, and when the concentration of CGAs is 5 mmol / L, the inhibition rate on α-amylase is <20%. In addition, the inhibition mechanism of CGAs on α-amylase is complex, and different concentrations have different effects on α-amylase, that is, it may act as an inhibitor or an activator. Zhang et al. reported that iso-chlorogenic acid A isolated from sweet potato leaves, i.e. 3,5-diCQA, has a strong inhibitory effect on α-glucosidase, and the more caffeoyl groups in quinic acid, the stronger the inhibitory effect on α-glucosidase, however, acetylated chlorogenic acid derivatives have weak inhibitory effect on α-glucosidase. These studies show that pure chlorogenic acid and its derivatives, different CGAs composition in crude chlorogenic acid extract, and whether different proportions of CQAs will affect the inhibitory activity on α-amylase and α-glucosidase have not been reported. SUMMARY

[0004] The present application aims to improve the composition of CGAs and the proportion of CQAs and caffeic acid in green coffee bean extract by enzyme combined with probiotic fermentation of green coffee bean extract supernatant, and to improve the α-glucosidase inhibitory activity of green coffee bean extract.

[0005] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: The present application provides a method for producing green coffee bean extract with improved α-glucosidase inhibitory activity.

[0006] The method for improving the α-glucosidase inhibitory activity of green coffee bean extract provided by the present application comprises the following steps: (1) Preparation of green coffee bean extract: green coffee beans are slurried at a material-to-liquid ratio of 1:8-10, and 80 U / mL-160 U / mL cellulase, 30 U / mL-40 U / mL pectinase, and 2-4 U / mg substrate of tannase are added to the slurrying liquid for enzymolysis, followed by centrifugation to obtain green coffee bean extract supernatant; The temperature for enzymolysis is 45℃-50℃, and the time is 0.5 h-1.0 h; The cellulase is a product of Beijing Solarbio Technology Co., Ltd., with an enzyme activity of 500000 U / g; Pectinase is a product of Shandong Longket Enzyme Preparation Co., Ltd., with an enzyme activity of 30,000 U / g; The tanninase is a product of Cangzhou Xiasheng Biotechnology Co., Ltd., with an enzyme activity of 500 U / mL.

[0007] (2) Restrictive enzymatic hydrolysis of CGAs: Add 0.2-0.5 U / mg of the substrate porcine liver esterase to the clear liquid of green coffee bean extract, based on the total amount of CGAs, and perform enzymatic hydrolysis. After the enzymatic hydrolysis is completed, put it into a container to be fermented and keep it at 100℃ for 5 min-8 min to inactivate the enzyme and complete pasteurization. Then, quickly cool it to room temperature to obtain the restricted hydrolysate of green coffee bean CGAs. The enzymatic hydrolysis was carried out at a temperature of 35℃-40℃ for 0.5 h-1.0 h. Porcine liver esterase is a product of Shanghai Aladdin Biochemical Technology Co., Ltd., with an enzyme activity of 15000 U / g; (3) Culture of fermentation strains: The freeze-dried Lactobacillus rhamnosus and Lactobacillus paracasei powders were inoculated into MRS liquid medium and cultured anaerobicly at 37℃ for 24-36 h to obtain seed culture. The concentration of the seed culture was adjusted to OD. 600nm Value 0.36-0.37; Bacillus subtilis powder was inoculated into PCA liquid medium and cultured at 37°C with shaking for 20–24 h to obtain seed culture. The concentration (OD) of the seed culture was then adjusted. 600nm Value 0.36-0.37; Among them, Lactobacillus rhamnosus (JYLR005) and Lactobacillus paracasei (LPC14) are products of Shaanxi Qianwei Biotechnology Co., Ltd., with an active lactic acid bacteria content of ≥10 billion CFU / g; Bacillus subtilis (CGMCC) is a product of Guanying Biotechnology Co., Ltd., with a live bacteria count ≥100 billion CFU / g; (4) Preparation of fermentation system for green coffee bean extract: 1-1.5% (v / v) seed liquid combination was added to the restricted hydrolysate of green coffee beans CGAs, wherein Lactobacillus rhamnosus: Lactobacillus paracasei: Bacillus subtilis = 1-1.5: 2-2.5: 2-3 (v / v), and fermented at 35-37℃ for 8-12 hours. After centrifugation, fermented coffee liquid was obtained.

[0008] The fermented coffee liquid obtained by the above method is also within the scope of protection of this invention.

[0009] The fermented coffee liquid has a caffeic acid content ≤175mg / L, a total CGAs content ≥3400mg / L, and a chlorogenic acid derivative with a 3-CQA:4-CQA:5-CQA:caffeic acid (CA) mass ratio of 16.0-17.8:2.1-2.2:1.5-1.7:1. The fermented coffee liquid diluted 10 times showed an α-glucosidase inhibition rate of ≥70%.

[0010] This invention hydrolyzes chlorogenic acid derivatives in green coffee bean extract using tanninase and esterase, and then uses probiotic fermentation to obtain mixtures of CQAs and caffeic acid in different proportions, which significantly improves the α-glucosidase inhibition rate.

[0011] This invention utilizes enzyme-linked microbial fermentation of green coffee bean extract to modify the structure of CGAs, obtaining a mixture of CQAs and caffeic acid in a certain proportion, which greatly enhances the α-glucosidase inhibitory activity of green coffee bean extract. Attached Figure Description

[0012] Figure 1 HPLC chromatograms of different chlorogenic acid derivatives and caffeic acid standards; Figure 2 This is the HPLC chromatogram of the fermented coffee liquid obtained in Example 1 of the present invention; Figure 3 The HPLC chromatogram of the fermented coffee liquid obtained in Example 2 of this invention; Figure 4 The HPLC chromatogram of the green coffee bean extract prepared in Comparative Example 1 is shown below. Figure 5 The HPLC chromatogram of the enzymatically hydrolyzed coffee extract prepared in Comparative Example 2 is shown below. Figure 6 The HPLC chromatogram of the fermented coffee extract containing the complex intestinal probiotics prepared in Comparative Example 3 is shown below. Figure 7 The HPLC chromatogram of the fermented coffee liquid prepared in Comparative Example 4 is shown below. Figure 8 The HPLC chromatogram of the fermented coffee liquid prepared in Comparative Example 5 is shown below. Figure 9a Standard curve for chlorogenic acid derivative 3-CQA standard; Figure 9b Standard curve for chlorogenic acid derivative 4-CQA standard; Figure 9c Standard curve for chlorogenic acid derivative 5-CQA standard; Figure 9d Standard curves for chlorogenic acid derivatives 3,4-diCQA and 3,5-diCQA; Figure 9e Standard curve for chlorogenic acid derivative 4,5-diCQA standard; Figure 9f This is the standard curve for caffeic acid standards. Detailed Implementation

[0013] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0014] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0015] In the examples below, the cellulase was a product of Beijing Solarbio Technology Co., Ltd. (Solarbio), with an enzyme activity of 500,000 U / g; Pectinase is a product of Shandong Longket Enzyme Preparation Co., Ltd., with an enzyme activity of 30,000 U / g; Tanninase is a product of Cangzhou Xiasheng Biotechnology Co., Ltd., with an enzyme activity of 500 U / mL; Porcine liver esterase is a product of Shanghai Aladdin Biochemical Technology Co., Ltd., with an enzyme activity of 15000 U / g; Lactobacillus rhamnosus (JYLR005) and Lactobacillus paracasei (LPC14) are products of Shaanxi Qianwei Biotechnology Co., Ltd., with an active lactic acid bacteria content of ≥10 billion CFU / g; Bacillus subtilis (CGMCC) is a product of Guanying Biotechnology Co., Ltd., with a live bacteria count ≥100 billion CFU / g.

[0016] The relevant detection methods in the following embodiments are as follows: Determination of α-glucosidase inhibition rate: Reagents were added to the 96-well plates according to Table 1. First, 60 μL of 0.01 mol / L phosphate buffer solution (pH 6.8, PBS) was placed in each well of the 96-well plate. Then, 20 μL of the samples prepared in the examples and comparative examples were added to the sample group and the sample control group. The plates were equilibrated at 37°C for 5 min. The control group and the blank control group were replaced with an equal volume of PBS. Then, 20 μL of 0.2 U / mL α-glucosidase was added to the control group and the sample group. The blank control group and the sample control group were replaced with an equal volume of PBS. The plates were kept at 37°C for 30 min. Then, 20 μL of p-nitrobenzene-α-D-glucopyranoside (pNPG) was added to initiate the reaction. The reaction was carried out at 37°C for 30 min. 30 μL of Na2CO3 was added to terminate the reaction. The absorbance was measured at 405 nm.

[0017] Table 1. Reaction system for α-glucosidase activity inhibition rate

[0018]

[0019] Determination of chlorogenic acid composition and content of chlorogenic acid derivatives and caffeic acid: Preparation of standard solutions: Accurately weigh 20 mg of reference standards for 3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, 4,5-diCQA, and caffeic acid, respectively. Dissolve each in methanol and dilute to 10 mL in volumetric flasks, shake well, and obtain single reference standard solutions. Then, measure a certain amount of the above reference standard solutions into 10 mL volumetric flasks and dilute to volume with methanol. Dilute stepwise with 0.1% phosphoric acid-methanol solution to prepare standard working solutions with mass concentrations of 0.5 mg / L, 5 mg / L, 20 mg / L, 50 mg / L, and 100 mg / L. Filter the solutions through a 0.45 μm aqueous phase membrane for later use.

[0020] Sample preparation: Take 0.5 mL of the patent-prepared sample solution into a 200 mL beaker, add 80 mL of 0.1% phosphoric acid solution, incubate in a boiling water bath for 30 min, shaking occasionally. Remove and cool to room temperature, transfer to a 100 mL volumetric flask, dilute to the mark with 0.1% phosphoric acid solution, shake well, let stand, and filter through a 0.45 μm aqueous filter membrane for high performance liquid chromatography (HPLC) determination.

[0021] Liquid chromatography conditions: Shiseido CAPCELL PAK MGⅡC18 column (4.6 mm × 250 mm, 5.0 μm), mobile phase: methanol-0.10% phosphoric acid (12:88), flow rate: 1.00 mL / min, detection wavelength: 327 nm, column temperature: 35.0℃, injection volume: 10.0 μL.

[0022] Example 1: Enzymatic fermentation of compound intestinal probiotics enhances the α-glucosidase inhibitory activity of green coffee bean extract. (1) Preparation of green coffee bean extract: After washing off the dust, green coffee beans were pulped at a material-liquid ratio of 1:10. 80 U / mL cellulase, 30 U / mL pectinase, and 2 U / mg substrate tanninase were added to the pulp. The mixture was enzymatically hydrolyzed at 50℃ for 1.0 h and centrifuged at 3500 r / min to obtain a clear liquid of green coffee bean extract. (2) Restrictive enzymatic hydrolysis of CGAs: 0.2 U / mg of porcine liver esterase was added to the clear liquid of green coffee bean extract based on the total chlorogenic acid content. The enzymatic hydrolysis temperature was 37℃ for 1.0 h. After the enzymatic hydrolysis was completed, the liquid was placed in a beverage bottle to be fermented and kept at 100℃ for 5 min to inactivate the enzyme and simultaneously complete pasteurization. The liquid was then rapidly cooled to room temperature to obtain the restrictive hydrolysate of green coffee bean CGAs.

[0023] (3) Culture of fermentation strains: The freeze-dried Lactobacillus rhamnosus and Lactobacillus paracasei powders were inoculated into MRS liquid medium and cultured anaerobicly at 37℃ for 24 h to obtain seed culture. The concentration of the seed culture was adjusted to OD. 600nm Value: 0.36-0.37.

[0024] Bacillus subtilis (food grade) powder was inoculated into PCA liquid medium and cultured at 37℃ with shaking for 20 h to obtain seed culture. The concentration OD of the seed culture was then adjusted. 600nm Value: 0.36-0.37.

[0025] (4) Preparation of fermentation system for green coffee bean extract: 1% (v / v) seed liquid combination was added to the restricted hydrolysate of green coffee beans CGAs, wherein Lactobacillus rhamnosus: Lactobacillus paracasei: Bacillus subtilis (food grade) = 1:2:2 (v / v), and fermented at 35℃ for 8 hours. The mixture was then centrifuged at 3500 r / min to obtain fermented coffee liquid.

[0026] The fermented coffee liquid diluted 10 times showed an α-glucosidase inhibition rate of 71.50%, a caffeic acid content of 175.0 mg / L, and a CGAs content of 3445.0 mg / L.

[0027] Example 2: Enzymatic compound intestinal probiotics enhance the α-glucosidase inhibitory activity of green coffee bean extract. (1) Preparation of green coffee bean extract: After washing off the dust, green coffee beans were pulped at a material-to-liquid ratio of 1:8. 160 U / mL cellulase, 40 U / mL pectinase, and 4 U / mg substrate tanninase were added to the pulp. The mixture was enzymatically hydrolyzed at 45℃ for 1.0 h and centrifuged at 3500 r / min to obtain a clear liquid of green coffee bean extract. (2) Restrictive enzymatic hydrolysis of chlorogenic acid derivatives: 0.5 U / mg of porcine liver esterase based on the chlorogenic acid content was added to the clear liquid of green coffee bean extract, and enzymatic hydrolysis was carried out at 35℃ for 0.5 h. After enzymatic hydrolysis, the liquid was placed in a beverage bottle to be fermented and kept at 100℃ for 5 min to inactivate the enzyme and simultaneously complete pasteurization. The liquid was then rapidly cooled to room temperature to obtain the restricted hydrolysate of green coffee bean CGAs.

[0028] (3) Culture of fermentation strains: The freeze-dried Lactobacillus rhamnosus and Lactobacillus paracasei powders were inoculated into MRS liquid medium and cultured anaerobicly at 37℃ for 24 h to obtain seed culture. The concentration of the seed culture was adjusted to OD. 600nm Value: 0.36-0.37.

[0029] Bacillus subtilis (food grade) powder was inoculated into PCA liquid medium and cultured at 37℃ with shaking for 20 h to obtain seed culture. The concentration OD of the seed culture was adjusted. 600nm Value: 0.36-0.37.

[0030] (4) Preparation of fermentation system for green coffee bean extract: 1.5% (v / v) seed liquid combination was added to the restricted hydrolysate of green coffee beans CGAs, wherein Lactobacillus rhamnosus: Lactobacillus paracasei: Bacillus subtilis (food grade) = 1.5: 2.5: 3 (v / v), and fermented at 35℃ for 8 hours. The fermented coffee liquid was obtained by centrifugation at 3500 r / min.

[0031] The fermented coffee liquid diluted 10 times showed an α-glucosidase inhibition rate of 81.45%, a caffeic acid content of 159.1 mg / L, and a CGAs content of 3431.7 mg / L.

[0032] Comparative Example 1: Green Coffee Bean Extract The preparation of green coffee bean extract is the same as step (1) in Example 1; The 10-fold dilution showed an α-glucosidase inhibition rate of -21.51%, a caffeic acid content of 192.70 mg / L, and a CGAs content of 2257.0 mg / L.

[0033] Comparative Example 2: Enzymatic hydrolysis of coffee extract (without probiotic fermentation) (1) The preparation of green coffee bean extract is the same as step (1) in Example 1; (2) The restricted enzymatic hydrolysis of chlorogenic acid derivatives is the same as step (2) in Example 1; The α-glucosidase inhibition rate of the 10-fold diluted enzymatic hydrolysate was 5.20%, the caffeic acid content was 186.7 mg / L, and the CGAs content was 2984.6 mg / L.

[0034] Comparative Example 3: Fermented Coffee Extract with Complex Intestinal Probiotics (Unenzymatically Hydrolyzed) (1) The preparation of green coffee bean extract is the same as step (1) in Example 1; (2) The fermentation strain was cultured in the same way as in step (3) of Example 1; (3) The preparation of the green coffee bean extract fermentation system is the same as step (4) in Example 1; The α-glucosidase inhibition rate of the fermentation broth diluted 10 times was 12.96%, the caffeic acid content was 186.7 mg / L, and the CGAs content was 3114.1 mg / L.

[0035] Comparative Example 4: Enzymatic Fermentation of Coffee Extract with Two Types of Probiotic Intestinal Bacteria (1) The preparation of green coffee bean extract is the same as step (1) in Example 1; (2) The restricted enzymatic hydrolysis of chlorogenic acid derivatives is the same as step (2) in Example 1; (3) Culture of fermentation strains: The freeze-dried Lactobacillus rhamnosus and Lactobacillus paracasei powders were inoculated into MRS liquid medium and cultured anaerobicly at 37℃ for 24 h to obtain seed culture. The concentration of the seed culture was adjusted to OD. 600nm Value: 0.36-0.37.

[0036] (4) Preparation of fermentation system for green coffee bean extract: 1% (v / v) combined bacterial culture was added to the restricted hydrolysate of green coffee beans CGAs, wherein Lactobacillus rhamnosus: Lactobacillus paracasei = 1:2 (v / v), and the mixture was placed at 37℃ and allowed to ferment for 8 hours. The mixture was then centrifuged at 3500 r / min to obtain fermented coffee liquid.

[0037] The α-glucosidase inhibition rate of 10-fold fermented coffee liquid was 31.79%, the caffeic acid content was 194.3 mg / L, and the CGAs content was 3164.5 mg / L.

[0038] Comparative Example 5: Enzymatic Fermentation of Coffee Extract with Other Probiotic Groups (1) The preparation of green coffee bean extract is the same as step (1) in Example 1; (2) The restricted enzymatic hydrolysis of chlorogenic acid derivatives is the same as step (2) in Example 1; (3) Cultivation of fermentation strains and dilution of freeze-dried bacteria: The freeze-dried bacteria powders of Lactobacillus rhamnosus and Lactobacillus paracasei were inoculated into MRS liquid medium and cultured anaerobicly at 37℃ for 24 h to obtain seed culture. The concentration of the seed culture was adjusted to OD. 600nm Value: 0.36-0.37.

[0039] Lactobacillus curvature was inoculated into MRS liquid medium and anaerobic cultured at 37°C for 24 h to obtain a seed culture. The concentration of the seed culture was adjusted to OD. 600nm Value 0.36-0.37; Bacillus subtilis (food grade) powder was inoculated into PCA liquid medium and cultured at 37℃ with shaking for 20 h to obtain seed culture. The concentration OD of the seed culture was then adjusted. 600nm Value: 0.36-0.37.

[0040] Dilute the freeze-dried Bifidobacterium with sterile water to OD0.05 600nm Value: 0.36-0.37.

[0041] Among them: Lactobacillus curvaturei (PGLCJ22) and Bifidobacterium are from Shaanxi Qianwei Biotechnology Co., Ltd., with an active lactic acid bacteria content of ≥10 billion CFU / g; (4) Preparation of fermentation system for green coffee bean extract: 1% (v / v) seed liquid combination was added to the restricted hydrolysate of green coffee beans CGAs, wherein Lactobacillus rhamnosus: Lactobacillus paracasei: Bacillus subtilis (food grade) = 1:2:2, and then 0.25% (v / v) Bifidobacterium and 0.25% (v / v) Lactobacillus curvature were added respectively. The mixture was placed at 37℃ and allowed to ferment for 8 h. After centrifugation at 3500 r / min, fermented coffee liquid was obtained.

[0042] The α-glucosidase inhibition rate of the fermented coffee liquid diluted 10 times was 22.09%, the caffeic acid content was 128.4 mg / L, and the CGAs content was 1206.9 mg / L.

[0043] Comparative Example 6: Pure chlorogenic acid (3-CQA) A solution with a concentration of 3.4 mg / mL was prepared using pure 3-CQA. After a 10-fold dilution, the α-glucosidase inhibition rate was -3.12%, the caffeic acid content was 0 mg / L, and the chlorogenic acid content was 3400 mg / L.

[0044] Comparative Example 7: Pure Chlorogenic Acid Derivative Compound A chlorogenic acid derivative with a ratio of 3-CQA:4-CQA:5-CQA:caffeic acid = 17.5:2.1:1.6:1 and a CGAs content of 3.4 mg / mL was used. The 10-fold dilution showed an α-glucosidase inhibition rate of 63.8%, a caffeic acid content of 160 mg / L, and a CGAs content of 3400 mg / L.

[0045] Test results of embodiments and comparative examples of the present invention The results of α-glucosidase inhibition rate are shown in Table 2.

[0046] Table 2. α-glucosidase inhibition rate of the products obtained from the embodiments and comparative examples of the present invention.

[0047] As shown in the table, the green coffee bean extract (Comparative Example 1) had no inhibitory effect on α-glucosidase, but rather exhibited a certain activating effect. The fermented coffee liquid prepared using the methods described in this patent example showed an α-glucosidase inhibition rate of 71.5%-81.45%, significantly enhancing its α-glucosidase inhibitory activity. Enzymatic hydrolysis of the green coffee bean extract (Comparative Example 2) did not improve its α-glucosidase inhibitory activity, only showing a slight inhibitory effect. Direct fermentation using compound microorganisms (Comparative Example 3) did not significantly improve the inhibition rate. However, using enzyme modification and fermentation with two types of microorganisms (Comparative Example 4) increased the α-glucosidase inhibition rate from 5.2% to 31.79%, indicating that the treatment simulating small intestinal enzymatic hydrolysis followed by further fermentation by large intestinal microorganisms significantly improved the α-glucosidase inhibition of the green coffee bean extract. However, using more types of microorganisms for synergistic fermentation based on this patent embodiment did not improve the α-glucosidase inhibitory activity, possibly because excessive microorganisms in this patent-like fermentation time were over-metabolized into other substances by CGAs. The dynamic hydrolysis of CGAs by other gut microbes and its relationship with α-glucosidase inhibitory activity remain unclear.

[0048] It is worth noting that although the total CGA content of all 3-CQA (Comparative Example 6) which is similar to the total CGA content in the embodiments of this patent is significantly higher than that of Comparative Example 1, it does not show α-glucosidase inhibitory activity. This indicates that although 3-CQA accounts for the majority of CGAs in this patent, the α-glucosidase inhibitory activity of 3-CQA alone is relatively low. On the other hand, the combination of CGAs with a content and composition ratio of 3-CQA, 4-CQA, and 5-CQA similar to that in this patent, combined with a certain amount of caffeic acid, shows higher α-glucosidase inhibitory activity (Comparative Example 7). This indicates that the proportion of CGAs that inhibit α-glucosidase needs to be determined. At the same time, in the embodiments of this patent, other phenols and other active substances generated by the fermentation of the compound bacteria also play a certain auxiliary role in the α-glucosidase inhibitory activity.

[0049] The composition of chlorogenic acid, and the contents of chlorogenic acid and caffeic acid in the coffee extract are shown in Table 3. As can be seen from the table, the total CGAs content of the green coffee bean extract (Comparative Example 1) at a 1:10 dilution was only 2257 mg / L, while the 3-CQA content in Example 1 of this patent was 61.9% higher than in Comparative Example 1, and the total CGAs content was more than 52% higher. Although the comparative example and the actual example used the same supernatant, the enzymatic hydrolysis combined with lactic acid bacteria fermentation further enzymatically hydrolyzed the polyphenols in the coffee extract, releasing more 3-CQA, 4-CQA, 5-CQA, and dual CQAs. The dynamic hydrolysis of polyphenols to generate CQAs, and the dynamic hydrolysis of CQAs to caffeic acid, resulted in an increase in the overall CGAs content, which is one of the main reasons for the improved α-glucosidase inhibitory activity in this patent.

[0050] Table 3. Composition of chlorogenic acid derivatives in the examples and comparative examples of this patent (unit: mg / L)

[0051] After enzymatic extraction with cellulase and pectinase, and enzymatic modification with tanninase and esterase (Comparative Example 2), the CGAs content in green coffee bean extract increased to some extent, but the increase was not significant. However, after fermentation with the probiotic combination of this patent following enzymatic hydrolysis, the CGAs yield of green coffee bean extract (Comparative Example 3) was significantly increased, although caffeic acid showed some decomposition. Probiotic fermentation after enzymatic extraction and modification significantly affected the chlorogenic acid composition, CGAs, and caffeic acid content of the coffee extract. Specifically, the enzymatic combined with two-strain combination (Comparative Example 4), while weaker than the probiotic combination of this patent, was significantly higher than that of enzymatic modification alone or fermentation with only microbial combinations. The addition of *Lactobacillus flavus* and *Bifidobacterium* (Comparative Example 5) to the bacterial combination of this patent caused a significant decrease in CGAs content. This may be because excessive microorganisms during the fermentation time of other examples in this patent increased the further degradation of generated CGAs, resulting in a lower caffeic acid content in the fermentation broth. The higher total CGAs and lower caffeic acid content are somewhat correlated with α-glucosidase inhibitory activity.

[0052] Dividing the different CQAs contents in Table 3 by the caffeic acid content, and dividing Comparative Example 6 (which did not contain caffeic acid) by 160 (according to the contents of the examples), yields Table 4. Table 4 shows that by analyzing the ratios of CQAs and caffeic acid in the examples and comparative examples of this patent, it can be found that the ratio of 3-CQA: 4-CQA: 5-CQA: caffeic acid is 16.0-17.8: 2.1-2.2: 1.5-1.7: 1, with caffeic acid content ≤175 mg / L, the highest CQAs content (≥ 3400 mg / mL), and the highest α-glucosidase inhibition rate.

[0053] Table 4. Composition ratio of each chlorogenic acid derivative in the examples and comparative examples of this patent (with caffeic acid content as 1 part).

[0054] This invention utilizes a specific enzymatic method combined with specific probiotics to ferment the clear liquid of green coffee bean extract under defined conditions, thereby bio-hydrolyzing the polyphenols in the liquid. This significantly increases the content of CGAs in the fermentation broth, adjusts the ratio of CQAs to caffeic acid in the green coffee bean extract, and enhances the α-glucosidase inhibitory activity of the green coffee bean extract.

[0055] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for improving the inhibitory activity of green coffee extract on α-glucosidase, comprising the following steps: (1) Preparation of green coffee extract: green coffee beans are slurried at a material-to-liquid ratio of 1:8-10, and the slurry is added with cellulase at 80 U / mL-160 U / mL, pectinase at 30 U / mL-40 U / mL, and tannase at 2-4 U / mg of substrate, and then subjected to enzymolysis, centrifugation, and thus green coffee extract supernatant is obtained; wherein the enzymolysis is performed at a temperature of 45℃-50℃ for 0.5 h-1.0 h; the cellulase is a product of Solarbio, Beijing, with an enzyme activity of 500000 U / g; the pectinase is a product of Shandong Longkete Enzyme Preparation Co., Ltd., with an enzyme activity of 30000 U / g; the tannase is a product of Cangzhou Xiasheng Biotechnology Co., Ltd., with an enzyme activity of 500 U / mL; (2) Restrictive enzymolysis of chlorogenic acid derivatives: porcine liver esterase is added to the green coffee extract supernatant at a dosage of 0.2-0.5 U / mg of substrate, and then subjected to enzymolysis, and after the enzymolysis is completed, the solution is loaded into a container to be fermented, subjected to heat preservation at 100℃ for 5 min-8 min to inactivate the enzyme and complete pasteurization, and then rapidly cooled to room temperature to obtain green coffee CGAs restrictive hydrolysate; wherein the enzymolysis is performed at a temperature of 35℃-40℃ for 0.5 h-1.0 h; the porcine liver esterase is a product of Shanghai Aladdin Biochem Technology Co., Ltd., with an enzyme activity of 15000 U / g; (3) Culture of fermentation strains: Lactobacillus rhamnosus and Paracasei freeze-dried bacteria powder were inoculated into MRS liquid medium, and seed liquid was obtained by anaerobic culture at 37°C for 24-36 h. The concentration of the seed liquid was adjusted to OD 600nm 0.36-0.

37. Bacillus subtilis powder was inoculated into PCA liquid medium, and the seed liquid was obtained by shaking culture at 37°C for 20-24 h. The concentration of the seed liquid was adjusted to OD 600nm 0.36-0.37; wherein: Lactobacillus rhamnosus and Lactobacillus paracasei are products of Shaanxi Qianwei Biotechnology Co., Ltd., with a content of active lactic acid bacteria ≥100 billion CFU / g; Bacillus subtilis is a product of Guanying Biotechnology Co., Ltd., with a content of active bacteria ≥1000 billion CFU / g; (4) Preparation of green coffee extract fermentation system: 1-1.5% (v / v) seed liquid combination is inoculated into the green coffee CGAs restrictive hydrolysate, wherein the ratio of Lactobacillus rhamnosus: Lactobacillus paracasei: Bacillus subtilis is 1-1.5: 2-2.5: 2-3 (v / v), and the mixture is subjected to static fermentation at 35-37℃ for 8-12 h, and then centrifuged to obtain fermented coffee liquid.

2. Fermented coffee liquid prepared by the method of claim 1.

3. The fermented coffee liquor according to claim 2, characterized in that, The fermented coffee liquid has a caffeic acid content ≤175 mg / L, a total CGAs content ≥3400 mg / L, and a mass ratio of 3-CQA:4-CQA:5-CQA:caffeic acid in chlorogenic acid derivatives of 16.0-17.8:2.1-2.2:1.5-1.7:

1.

4. The fermented coffee liquor according to claim 2 or 3, characterized in that, The α-glucosidase inhibition rate of the fermented coffee liquid diluted 10 times is ≥70%.

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