Blueberry and rhizoma polygonati compound as well as preparation method and application thereof

Abstract

The invention discloses a blueberry and rhizoma polygonati compound as well as a preparation method and application thereof, and relates to the technical field of deep processing of blueberries. The blueberry and rhizoma polygonati compound is prepared by fermenting a water bath sterilization mixture of blueberries and rhizoma polygonati through lactobacillus plantarum P9. In the blueberry and rhizoma polygonati compound, the mass ratio of rhizoma polygonati to blueberries is 1: (2-8). By utilizing the method provided by the invention, the content of functional components (or effective components) in the blueberry fermentation product can be greatly increased, and the number of viable bacteria in the fermentation process is increased. The preparation method has the beneficial effects that the additional value of the blueberries is increased through compound fermentation of the blueberries and the rhizoma polygonati, and the obtained fermented product has the function of protecting intestinal tracts.

Description

Technical Field

[0001] The invention relates to the technical field of blueberry deep processing, and in particular to a blueberry-polygonatum complex, and a preparation method and application thereof. Background Art

[0002] Blueberry, commonly known as bilberry, belongs to the genus Vaccinium of the Ericaceae family and is a perennial shrub. Li Shizhen recorded in Compendium of Materia Medica that Cangshan bilberry has the effects of "soothing qi, relieving fullness, benefiting the kidney and consolidating essence, strengthening tendons and improving eyesight". Blueberry has the functions of preventing brain nerve aging, anti-cancer, anti-inflammation, protecting the liver, improving the body's immunity, and protecting eyesight; Polygonatum sibiricum is a perennial herb of the genus Polygonatum of the Asparagaceae family. It is a medicinal and edible raw material. Traditional Chinese medicine believes that Polygonatum sibiricum has the functions of tonifying qi and nourishing yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys. It is used for spleen and stomach qi deficiency, fatigue, insufficient stomach yin, lung deficiency and dry cough and other symptoms. It contains polysaccharides, volatile oils, steroidal saponins, cardiac glycosides, anthrone compounds, lignans, vitamins, trace elements, amino acids and other ingredients, which have the effects of tonifying qi and nourishing yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys. It is often used to treat symptoms such as spleen and stomach qi deficiency, fatigue, insufficient stomach yin, dry mouth and poor appetite, lung deficiency and dry cough, fatigue cough and hemoptysis, insufficient essence and blood, soreness of the waist and knees, premature graying of hair, internal heat and thirst.

[0003] Although blueberries have good medicinal and edible effects, they have a short fruiting period and are difficult to store. The fruits are easily damaged by mechanical damage and microbial decay, which inevitably causes economic losses. Therefore, processing them into products is an effective way to improve the situation. However, the processing rate of blueberries is low. In 2020, the global processing rate of blueberries was only 30.7%, and the technical content of processed blueberry products was low. They were mainly low-tech products such as juice drinks, dried fruits, and jams, lacking high-value-added products oriented to functions. Therefore, it is particularly important to develop high-value-added blueberry deep-processing products.

[0004] At present, there are few studies on the combination of blueberries by using fermentation technology, and there is still a blank in the combination of blueberries and polygonatum. Direct fermentation after blueberry pulping is not conducive to the growth of strains and fermentation due to the low initial pH.

[0005] Research on blueberry fermentation mainly focuses on compound strain fermentation. Using a single strain fermentation instead of a compound strain fermentation can also increase the anthocyanin content. At present, most blueberry fermented products are deeply processed based on blueberry enzymatic hydrolysate, and Polygonatum sibiricum is deeply processed based on Polygonatum sibiricum extract. In this process, there is a problem of loss of effective ingredients in blueberries and Polygonatum sibiricum. In addition, the anthocyanins in blueberries are easily lost due to the need for high temperature and high pressure sterilization during the fermentation process, and anthocyanins are extremely sensitive to heat.

[0006] Therefore, it is particularly important to improve the fermentation efficiency of blueberries by compounding blueberry and Polygonatum and using a single strain, and to retain the effective ingredients in blueberries and improve their functional activity by compounding blueberry and Polygonatum and optimizing the sterilization method in the fermentation process.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The purpose of the present invention is to provide a blueberry-polygonatum complex, and a preparation method and application thereof to solve the problems of low blueberry processing rate, poor stability of bioactive compounds, low content of effective ingredients after fermentation, and insufficient fermentation.

[0009] The present invention provides a method for preparing a blueberry-polygonatum complex, which is a method for deep development and comprehensive utilization of blueberries. The method provided by the present invention can greatly increase the functional components (or effective ingredients) in blueberries, increase the number of live bacteria in the fermentation process, facilitate fermentation, and provide a solution for increasing the added value of blueberries.

[0010] The present invention is achieved in that:

[0011] In a first aspect, the present invention provides a blueberry-polygonatum complex, which is prepared by fermenting a water-bath sterilized mixture of blueberry and polygonatum with Lactobacillus plantarum P9; in the blueberry-polygonatum complex, the mass ratio of the raw materials polygonatum to blueberry is 1:(2-8).

[0012] The inventor found that the reason for the low number of live bacteria after blueberry fermentation is that the initial pH of the blueberry raw material itself is low (usually the initial pH is about 3.5), and it is difficult to find a fermentation bacterium adapted to blueberry fermentation under this condition. The present invention, through screening, found that plant lactobacillus P9 is a probiotic lactic acid bacteria with excellent gastrointestinal fluid tolerance, which can achieve the effect of composite strain fermentation by single strain fermentation, and a large amount of blueberry effective ingredients (such as anthocyanins and polyphenols) are retained in the fermentation product. In order to adjust the initial pH of blueberry, the present invention adjusts the initial pH of blueberry by compounding Polygonatum sibiricum, so that the initial pH reaches about 3.9, which is more conducive to the growth of the strain, and the large-scale growth of the strain increases the content of short-chain unsaturated fatty acids in the fermentation product, which helps to improve the taste after fermentation.

[0013] Lactobacillus plantarum P9 is a probiotic isolated from naturally fermented sour porridge in Bayannaoer, Inner Mongolia. It has probiotic functions such as regulating intestinal flora, reducing inflammation and allergic reactions in the body. Lactobacillus plantarum P9 is deposited in the General Microbiological Culture Collection Center of the China Microbiological Culture Collection Administration (the Patent Microbiological Culture Collection Center designated by the State Patent Office), address: Beijing, China. The deposit number is CGMCC No.16662, and the deposit time is June 22, 2018. For the specific deposit information of the strain, please refer to patent CN202010351922.2.

[0014] There is still no research on the combined fermentation of blueberry and polygonatum. Polygonatum and blueberry are fermented in a mass ratio of 1:(2-8), which complements each other, can optimize the taste, and is more suitable for fermentation. The mass ratio of polygonatum to blueberry is, for example, selected from 1:2, 1:3, 1:4, 1:5, 1:6, 1:7 or 1:8.

[0015] The sterilization process before fermentation is related to the content of anthocyanins. High temperature can easily destroy anthocyanin components. Water bath sterilization before fermentation can significantly retain the content of anthocyanins in the fermentation sample.

[0016] In a preferred embodiment of the present invention, the water bath sterilization condition is 85-95°C for 10-20 minutes. Under the above water bath sterilization conditions, it is helpful to retain more effective ingredients. The water bath sterilization conditions include but are not limited to 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C or 95°C for 10-20 minutes.

[0017] In a preferred embodiment of the present invention, the fermentation temperature is 25-41°C and the fermentation time is 16-48h. The fermentation temperature is, for example, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C or 41°C. The fermentation time is, for example, 16h, 18h, 20h, 22h, 24h, 28h, 30h, 32h or 48h.

[0018] In a preferred embodiment of the present invention, the inoculation amount of Lactobacillus plantarum P9 is 0.02%-0.06%, for example, the inoculation amount is selected from 0.02%, 0.03%, 0.04%, 0.05% or 0.06%.

[0019] In a preferred embodiment of the present invention, after the fermentation is completed, post-processing of the fermentation product is also included; the post-processing of the fermentation product includes centrifuging the fermentation product, filtering the centrifuge liquid in sequence, sterilizing, and adding a sweetener.

[0020] In a preferred embodiment of the present invention, the sterilization in the post-treatment of the fermentation product is water bath sterilization or high temperature instantaneous sterilization.

[0021] In a preferred embodiment of the present invention, the centrifugal separation conditions are 5000-10000 rpm, 0-20°C, and 10-20 min.

[0022] In a second aspect, the present invention provides a method for preparing a blueberry-polygonatum complex, comprising pulping a mixture of blueberries and polygonatum, and then sterilizing the pulped product in a water bath; and then fermenting the sterilized material using Lactobacillus plantarum P9; the mass ratio of polygonatum to blueberries in the mixture of blueberries and polygonatum is 1:(2-8).

[0023] The initial pH of blueberries is adjusted by compounding Polygonatum sibiricum to about 3.9, which is more conducive to the growth of the strain. The large-scale growth of the strain increases the content of short-chain unsaturated fatty acids in the fermentation product, which helps to improve the taste after fermentation.

[0024] Polygonatum sibiricum and blueberry are fermented in a mass ratio of 1:(2-8), the two complement each other, can optimize the taste, and are more suitable for fermentation. The mass ratio of Polygonatum sibiricum to blueberry is, for example, selected from 1:2, 1:3, 1:4, 1:5, 1:6, 1:7 or 1:8.

[0025] The sterilization process before fermentation is related to the effective content of anthocyanins. High temperature can easily destroy the activity of anthocyanin components. Water bath sterilization before fermentation can significantly retain the content of anthocyanins in the fermentation sample.

[0026] In a preferred embodiment of the present invention, the fermentation temperature is 25-41°C and the fermentation time is 16-48h. The fermentation temperature is, for example, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C or 41°C. The fermentation time is, for example, 16h, 18h, 20h, 22h, 24h, 28h, 30h, 32h or 48h.

[0027] In a preferred embodiment of the present invention, the inoculation amount of Lactobacillus plantarum P9 is 0.02%-0.06%, for example, the inoculation amount is selected from 0.02%, 0.03%, 0.04%, 0.05% or 0.06%.

[0028] The present invention utilizes Lactobacillus plantarum P9 for fermentation, and by controlling parameters such as the amount of fermentation bacteria added, fermentation temperature, fermentation time, material-liquid ratio and mass ratio, it is proved that the present invention can significantly increase the content of functional components in blueberry polygonatum, especially the content of anthocyanins and polyphenols.

[0029] When the fermentation temperature is within the above temperature range, the taste of the samples is better, especially after fermentation at 37°C, the taste is better.

[0030] In a preferred embodiment of the present invention, the fermentation is carried out at a rotation speed of 100-150 rpm.

[0031] The conditions for water bath sterilization are 85-95°C for 10-20 minutes. Under the above water bath sterilization conditions, it is helpful to retain more active ingredients. Water bath sterilization conditions include but are not limited to 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C or 95°C for 10-20 minutes.

[0032] In a preferred embodiment of the present invention, after the fermentation is completed, post-processing of the fermentation product is also included; the post-processing of the fermentation product includes centrifuging the fermentation product, filtering the centrifuge liquid in sequence, sterilizing, and adding a sweetener.

[0033] The sterilization in the post-treatment of the fermentation product is water bath sterilization or high temperature instantaneous sterilization, for example, sterilization at 85-95°C water bath conditions for 10-20 minutes; high temperature instantaneous sterilization is ultra-high temperature instantaneous sterilization (UHT); the conditions are 135-150°C, 2-8s.

[0034] In a preferred embodiment of the present invention, the centrifugal separation is carried out at 5000-10000 rpm and 18° C. for 10-20 min. The centrifugal temperature includes but is not limited to 0° C.-20° C.

[0035] In a preferred embodiment of the present invention, the sweetener is selected from at least one of xylitol, compound sweeteners, sucralose, maltose syrup, xylo-oligosaccharides, galacto-oligosaccharides, fructo-oligosaccharides, protein sugars, aspartame, sorbitol, mannitol, maltitol, isomalt and glucose syrup.

[0036] The invention does not contain any additives except sweeteners, such as white sugar, preservatives, flavors, thickeners, etc.

[0037] In a preferred embodiment of the present invention, the sweetener is selected from a compound sweetener, which is a mixture of erythritol and steviol glycoside.

[0038] In a preferred embodiment of the present invention, the composite sweetener is a mixture of 99.6%-99.8% by mass of erythritol and 0.4%-0.2% by mass of steviol glycoside.

[0039] The addition amount of the compound sweetener accounts for 3%-9% of the total material mass, for example 7%. After adding 7% of the compound sweetener, the fermentation product has higher odor, color, sourness, sweetness, overall flavor and aftertaste.

[0040] In a preferred embodiment of the present invention, a carbon source is added during the pulping process;

[0041] In a preferred embodiment of the present invention, the carbon source is anhydrous glucose, and the amount of anhydrous glucose added accounts for 2%-5% of the total pulping material mass.

[0042] In a preferred embodiment of the present invention, the beating is to mix the mixture of blueberry and polygonatum with 4-6 times the volume of water, and the beating is performed; the beating time is 2-5 minutes;

[0043] Preferably, the beating speed is 1000-50000 rpm, so as to achieve sufficient mixing.

[0044] In a preferred embodiment of the present invention, the mass ratio of polygonatum to blueberry in the mixture of blueberry and polygonatum is 1:6. Under the above ratio, a better fermentation effect is achieved. The two complement each other and can optimize the taste.

[0045] In a preferred embodiment of the present invention, the Huangjin extract is selected from at least one of the root, stem, leaf, flower and fruit of the plant of the polygonatum cyrtonema, preferably the root of the polygonatum cyrtonema.

[0046] In a third aspect, the present invention further provides a product, which includes the above-mentioned blueberry-polygonatum complex or the blueberry-polygonatum complex prepared by the above-mentioned preparation method of the blueberry-polygonatum complex.

[0047] The above products are food or medicine.

[0048] Food includes but is not limited to dairy products, puddings, cream, cream cheese, whipped cream, curd, milkshakes, custards, nutrition bars, energy bars, candy, baked goods, cereal chips, baby food, spreads, sugar-free cookies and chocolates, calcium chews, meat products, condiments, gravy, soups, shortening, beverages, and alcohol;

[0049] In a preferred embodiment of the present invention, beverages include but are not limited to blueberry polygonatum juice. Wines include but are not limited to brewed wines such as liquor, beer, and red wine.

[0050] In a fourth aspect, the present invention also provides the use of the blueberry-polygonatum complex in the preparation of functional foods for resisting inflammatory bowel disease.

[0051] The fermented beverage provided by the present invention has a good protective effect on the intestinal epithelium and is beneficial to maintaining intestinal health.

[0052] Inflammatory bowel disease includes, but is not limited to, any of acute colitis, ulcerative colitis, Crohn's disease, microscopic colitis, diversion colitis, Behcet's disease immuno-oncology colitis, chemotherapy or radiation colitis, graft-versus-host disease colitis, collagenous colitis, lymphocytic colitis, and pouchitis.

[0053] In a preferred embodiment of the present invention, the fermented beverage has at least one of the following uses:

[0054] (1) Maintain the integrity of the intestinal mucosa;

[0055] (2) Inhibit the levels of intestinal inflammatory factors;

[0056] (3) Treatment of acute ulcerative colitis.

[0057] The present invention has the following beneficial effects:

[0058] The present invention, through screening, finds that plant lactobacillus P9 is a probiotic lactic acid bacteria with excellent gastrointestinal fluid tolerance, and can achieve the effect of composite strain fermentation by single strain fermentation, and a large amount of effective ingredients (such as anthocyanins and polyphenols) of blueberry are retained in the fermentation product, and the number of viable bacteria in the fermentation process is high, and the fermentation is sufficient. In order to adjust the initial pH of blueberry, the present invention adjusts the initial pH of blueberry by compounding polygonatum, so that the initial pH reaches about 3.9, which is more conducive to the growth of strains. The large-scale growth of strains increases the content of short-chain unsaturated fatty acids in the fermentation product, which contributes to the improvement of taste after fermentation.

[0059] The blueberry-polygonatum composite provided by the invention can be prepared into beverages, such as drinks and wine.

[0060] The blueberry-polygonatum complex provided by the present invention has a good protective effect on intestinal epithelium, is beneficial to maintaining intestinal health, has a good therapeutic effect on inflammatory bowel disease, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0062] Figure 1 Schematic diagram of the process flow of the preparation method of Example 1 of the present invention;

[0063] Figure 2 This is a statistical result of anthocyanin content in blueberry polygonatum fermented liquid after sterilization by different sterilization methods before fermentation;

[0064] Figure 3 This is the statistical result of anthocyanin content before and after fermentation;

[0065] Figure 4 This is a statistical result diagram of anthocyanin content in fermentation of Lactobacillus plantarum P9 and composite strains;

[0066] Figure 5 This is the statistical result diagram of total phenol content before and after fermentation;

[0067] Figure 6This is a statistical result diagram of total phenol content in fermentation of Lactobacillus plantarum P9 and composite strains;

[0068] Figure 7 This is a statistical diagram of the comprehensive score of the electronic tongue at different fermentation temperatures;

[0069] Figure 8 The results of different compounds detected by the electronic nose at different fermentation temperatures are shown in the figure;

[0070] Fig. 9 This is a statistical chart of electronic tongue sensory test of fermentation products under different sweeteners;

[0071] Fig.10 It is a statistical result diagram of the mRNA expression levels of ZO-1, Occludin-1 and Claudin4 in the blank group, model group, non-fermented liquid intervention group (using the blueberry polygonatum liquid prepared in Comparative Example 3), and fermented liquid intervention group (using the blueberry polygonatum liquid prepared in Example 1);

[0072] Fig.11 It is a graph showing the TEER statistical results of the blank group, the model group, the non-fermented liquid group (using the blueberry polygonatum liquid prepared in Comparative Example 3), and the fermented liquid group (using the blueberry polygonatum liquid prepared in Example 1);

[0073] Fig.12 serum levels of TNF-α, IL-10, MCP-1, and D-mannitol;

[0074] Fig.13 Representative images of H&E staining (100X) of colon tissue pathological structure (the fermentation liquid group is the blueberry polygonatum liquid prepared in Example 1). DETAILED DESCRIPTION

[0075] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0076] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0077] Materials and Reagents

[0078] Blueberry, Polygonatum sibiricum, and Lactobacillus plantarum P9 freeze-dried powder.

[0079] Lactobacillus plantarum P9 is deposited in the General Microbial Culture Collection Center of China Microbiological Culture Collection Administration (the Patent Microbiological Culture Collection Center designated by the State Patent Office), Beijing, China. The deposit number is CGMCC No.16662, and the deposit date is June 22, 2018;

[0080] The plant lactobacillus P9 is a probiotic strain isolated from naturally fermented sour porridge in Bayannaoer, Inner Mongolia, and has probiotic functions such as regulating intestinal flora, reducing inflammation and allergic reactions in the body, etc.

[0081] Cyanidin-3-O-glucoside standard, gallic acid standard, Shanghai Yuanye Biotechnology Co., Ltd., and methanol are all chromatographic grade; Folin, sodium carbonate, anhydrous sodium acetate, potassium chloride, hydrochloric acid, and formic acid are all analytical grade; anhydrous glucose standard, China Food and Drug Inspection Institute.

[0082] Example 1

[0083] This embodiment provides a method for preparing a blueberry polygonatum complex. The preparation process is as follows Figure 1 shown.

[0084] (1) Remove rotten and moldy blueberries, rinse the surface with tap water, blanch for 5 minutes, remove impurities, add Polygonatum sibiricum (Polygonatum sibiricum root) soaked in pure water for 1 hour at a mass ratio of 6:1, and beat at a material-liquid ratio of 1:4. Adjust the speed to 4-6 and continue beating for 2-3 minutes at a speed of 5000 rpm. During beating, add 2% anhydrous glucose of the total material and continue beating for 1-2 minutes.

[0085] (2) The beaten material was sterilized in a 95°C water bath for 15 min, immediately cooled to room temperature, inoculated with plant lactobacillus P9 freeze-dried powder to make the number of viable bacteria in each bottle 20 billion (the inoculation amount of plant lactobacillus P9 was 0.05%), and fermented at 37°C for 32 h. After the fermentation was completed, centrifuged at 10,000 rpm and 18°C ​​for 20 min in a centrifuge, and the supernatant was centrifuged. Filtered, the filtrate was sterilized in a water bath, and the sterilization conditions were 95°C for 10 min.

[0086] (3) adding a composite sweetener accounting for 7% of the total mass of the total material, wherein the composite sweetener is a mixture of 99.8% by mass of erythritol and 0.2% by mass of stevioside, and performing aseptic filling to obtain a finished product.

[0087] Example 2

[0088] Compared with Example 1, the only difference is that the fermentation conditions in step (2) are different. In this example, the fermentation conditions are 25° C. and fermentation for 32 hours.

[0089] Example 3

[0090] Compared with Example 1, the only difference is that the fermentation conditions in step (2) are different. In this example, the fermentation conditions are 29° C. for 32 hours.

[0091] Example 4

[0092] Compared with Example 1, the only difference is that the fermentation conditions in step (2) are different. In this example, the fermentation conditions are 33° C. for 32 hours.

[0093] Example 5

[0094] Compared with Example 1, the only difference is that the fermentation conditions in step (2) are different. In this example, the fermentation conditions are 41° C. and fermentation for 32 hours.

[0095] Example 6

[0096] Compared with Example 1, the only difference is that the sweetener in step (3) is different. The sweetener added in this example is 8% xylitol.

[0097] Example 7

[0098] Compared with Example 1, the only difference is that the sweetener in step (3) is different. The sweetener added in this example is 0.025% sucralose.

[0099] Example 8

[0100] Compared with Example 1, the only difference is that the mass ratio of polygonatum to blueberry in step (1) is different. In this embodiment, the ratio is 1:2.

[0101] Example 9

[0102] Compared with Example 1, the only difference is that the mass ratio of polygonatum to blueberry in step (1) is different. In this embodiment, the ratio is 1:4.

[0103] Example 10

[0104] Compared with Example 1, the only difference is that the mass ratio of polygonatum to blueberry in step (1) is different. In this embodiment, the ratio is 1:8.

[0105] Comparative Example 1

[0106] Compared with Example 1, the only difference is that the sterilization conditions before fermentation in step (2) are different. The sterilization conditions before fermentation in this comparative example are sterilization in an autoclave for 15 minutes.

[0107] Comparative Example 2

[0108] Compared with Example 1, the only difference is that the sterilization conditions before fermentation in step (2) are different. The sterilization conditions before fermentation in this comparative example are sterilization in an electric ceramic stove for 15 minutes.

[0109] Comparative Example 3

[0110] Compared with Example 1, the only difference is that in step (2), the freeze-dried powder of Lactobacillus plantarum P9 is not inoculated, the mixture is placed at 37° C. for 32 hours, and the supernatant is obtained by centrifugation.

[0111] Comparative Example 4

[0112] Compared with Example 1, the only difference is that Polygonatum sibiricum is not added in step (1).

[0113] Comparative Example 5

[0114] Compared with Example 1, the only difference is that the strain inoculated in step (2) is different, and the composite strain of Lactobacillus plantarum P9 and Lactobacillus casei is added in this comparative example.

[0115] Experimental Example 1

[0116] The content of anthocyanin after fermentation of Example 1, Comparative Example 1 and Comparative Example 2 was measured respectively, and the content of anthocyanin after fermentation of Example 1 and Comparative Example 3 was also measured.

[0117] (1) Drawing of standard curve for determination of anthocyanin content:

[0118] Dilute the cyanidin-3-O-glucoside standard to 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.03125 mg / mL with 1% formic acid in 80% MeOH. Transfer 250 μL of pH 1 and pH 4.5 buffers to the 96-well parallel wells in duplicate and add 25 μL of the standard solution to the appropriate wells. One set of standards should be added to pH 1 buffer in duplicate and the other set of standards should be added to pH 4.5 buffer in duplicate. Use 80% MeOH with 1% formic acid as a blank and zero the standard curve. Read at 535 nm and 700 nm and plot the net absorbance of anthocyanidins against concentration.

[0119] (2) Sample processing:

[0120] The sample was centrifuged to obtain the supernatant, the debris was filtered out, and the anthocyanin content was determined after the fermentation broth was diluted.

[0121] (3) Determination of anthocyanin content in samples

[0122] Dilute the sample to 10 times, transfer 250 μL of pH 1 and pH 4.5 buffer in duplicate to 96-well parallel wells, and add 25 μL of sample solution to the appropriate wells. One set of sample solutions should be added to pH 1 buffer in duplicate, and the other set of sample solutions should be added to pH 4.5 buffer in duplicate. 80% MeOH containing 1% formic acid was used as a blank for correction. The plate was shaken in a plate reader for 30 seconds and then read at 535nm and 700nm. The anthocyanin concentration of the sample was calculated according to the linear equation.

[0123] The calculation formula is as follows:

[0124] Absorbance (net value) = (A535 nm pH 1.0 - A700 nm pH 1.0) - (A535 nm pH 4.5 - A700 nm pH 4.5).

[0125] The samples prepared in Example 1 and Comparative Examples 1 and 2 were respectively taken for anthocyanin determination. The anthocyanin determination results in different heat treatment methods are shown in Table 1. Figure 2 The results showed that boiling water bath sterilization could significantly retain the content of anthocyanins in the fermentation samples.

[0126] The anthocyanin content of blueberry yellow sperm after fermentation without adding Lactobacillus plantarum P9 (Comparative Example 3) and adding Lactobacillus plantarum P9 (Example 1) is shown in Table 1. Figure 3 As shown, the results showed that the anthocyanin content of blueberry Huangjing liquid fermented by Lactobacillus plantarum P9 was significantly higher than that of blueberry Huangjing liquid not fermented by P9.

[0127] The anthocyanin content of the blueberry yellow sperm liquid fermented by the composite strain (Comparative Example 5) and the blueberry yellow sperm liquid fermented by adding plant lactobacillus P9 (Example 3) is shown in Table 1. Figure 4 As shown, the results showed that the anthocyanin content of blueberry yellow sperm fermented by single strain P9 was higher than that of blueberry yellow sperm fermented by composite strains.

[0128] Experimental Example 2

[0129] The polyphenol contents after fermentation of Example 1 and Comparative Example 3, and Example 1 and Comparative Example 5 were measured respectively.

[0130] (1) Drawing of standard curve:

[0131] Using 0.5mg / mL gallic acid solution as the standard, dilute the gallic acid standard to 0.25mg / mL, 0.125mg / mL, 0.0625mg / mL, 0.03125mg / mL, 0.0156mg / mL, and 0.0078mg / mL. Transfer 25uL of the standard to a 96-well plate in triplicate, use 25uL of aqueous solution as a blank control, then add 125uL of FCR, shake gently and let stand for 10min, finally add 125uL of 7.5% Na2CO3 solution, seal the plate, let stand for 30min, and measure the absorbance at 765nm in the 96-well plate. Use concentration-absorbance value to make a standard curve.

[0132] (2) Sample processing

[0133] The sample was centrifuged to obtain the supernatant, the debris was filtered out, and the fermentation broth was diluted 10 times for determination of polyphenol content.

[0134] (3) Sample processing

[0135] The sample was diluted 10 times, and 25uL of the sample was transferred to a 96-well plate in triplicate. Then, 125uL of FCR was added, and the plate was gently shaken and allowed to stand for 10 minutes. Finally, 125uL of 7.5% Na2CO3 solution was added, the plate was sealed, and allowed to stand for 30 minutes. The absorbance was measured at 765nm in the 96-well plate.

[0136] Figure 5 The experimental results show that the fermentation of blueberry polygonatum with Lactobacillus plantarum P9 in Example 1 has the following beneficial effects: it can significantly increase the content of polyphenols in blueberry polygonatum, and provide a reference approach for the efficient utilization of blueberries.

[0137] Figure 6 The experimental results show that the Lactobacillus plantarum P9 fermentation of blueberry and polygonatum in Example 1 is due to the use of a composite strain to ferment blueberry and polygonatum.

[0138] Experimental Example 3

[0139] The number of viable bacteria after fermentation with and without adding Polygonatum sibiricum (Comparative Example 4) was detected respectively.

[0140] Refer to "GB 4789.35-2016 National Food Safety Standard Food Microbiology Inspection Lactic Acid Bacteria Inspection".

[0141] (1) Sample processing:

[0142] Accurately weigh 25g of uniform sample solution, add sterile water to quantify to 250g, and tap at 12.0 / S for 2min;

[0143] (2) Dilute the sample:

[0144] Use a 1mL sterile pipette or micropipette to draw 1mL of the 1:10 sample solution, and slowly inject it into a sterile test tube containing 9mL of physiological saline along the tube wall. Shake the test tube to mix it evenly to make a 1:100 sample solution. Take another 1mL sterile pipette or micropipette tip and make 10-fold increase of sample solution according to the above operation sequence until 10 -7 , 10 -8 Dilution;

[0145] (3) Making a plate:

[0146] From 10 -7 , 10 -8 Pipette 1 mL of sample homogenate from the diluent into a sterile plate, and prepare two plates for each dilution. After the diluent is transferred into the plate, pour about 15 mL of MRS agar medium cooled to 48°C into the plate, and rotate the plate to mix evenly.

[0147] (4) Cultivation: Anaerobic cultivation at 36℃±1℃ for 72h±2h.

[0148] The statistical results of the number of live bacteria are shown in the following table. The results show that adding Polygonatum sibiricum increases the initial pH of the blueberry fermentation liquid, is beneficial to the growth of the strain, and the number of live bacteria increases significantly after fermentation.

[0149]

[0150] Experimental Example 4

[0151] This experimental example uses super bionic technology to optimize the taste.

[0152] (1) The sensory analysis of fermented blueberry Polygonatum cyrtonema samples was performed using the Heracles II ultrafast gas phase electronic nose. The analysis parameters are as follows.

[0153] Electronic nose detection parameters:

[0154]

[0155]

[0156] (2) The taste of fermented blueberry Polygonatum odoratum samples was detected by using the SA402B electronic tongue taste analysis. The instrument consists of a host, a sensor, and an operating computer. The sensor consists of a taste sensor, a ceramic reference electrode, and a temperature sensor. The potential of the taste sensor film is detected based on the phase change with the reference electrode. Taste sensors are mainly divided into three types: positively charged membrane, mixed membrane, and negatively charged membrane. The sourness, bitterness, astringency, umami, and saltiness of the sample were detected.

[0157] Electronic tongue is based on the comprehensive score results Figure 7 As shown by Figure 7It can be seen that among the five fermentation temperatures in Examples 1-5, 37°C is the best fermentation temperature and has the best score.

[0158] The electronic nose is based on the four main peak areas (select the main peaks with relevant parameters above 85 points in the integral report). The sum of the relative peak areas of the four main peaks at 37°C is the largest. The statistical results of the relative peak areas at different fermentation temperatures refer to Figure 8 .

[0159] (3) Sensory evaluation

[0160] 0.0125% sucralose (Example 7), 7% compound sweetener (Example 1), and 8% xylitol (Example 6) were respectively mixed, and the optimal taste ratio was judged with six characteristic descriptions as indicators:

[0161]

[0162]

[0163] The results were tested on 20 volunteers who participated in the sensory test. Fig. 9 As shown, it indicates that:

[0164] 1. Among the different sugars added in the sensory evaluation, the sweetness and sourness are quite different. The overall flavor, aftertaste, and color are quite different from those of competing products.

[0165] The total score of 2.7% compound sweetener was 19.48 points, slightly higher than xylitol's 19.455 points.

[0166] 3. All three samples scored higher than competing products.

[0167] Experimental Example 5

[0168] A pharmacodynamic study on Caco-2 cell inflammation and barrier damage was conducted. The improvement effect of blueberry and polygonatum fermented liquid on lipopolysaccharide-induced Caco-2 cell inflammation was evaluated by inflammatory factors, and the protective effect of blueberry and polygonatum fermented liquid on Caco-2 cell barrier damage was evaluated by detecting the expression level of tight junction protein (JT).

[0169] In this experiment, Caco-2 cells were collected and diluted to 1.5×10 5 / mL, and then transferred to a 6-well plate for growth and attachment. It was divided into a blank group, a model group, a non-fermented liquid intervention group (using the liquid prepared in Comparative Example 3), and a fermented liquid intervention group (using the blueberry polygonatum liquid prepared in Example 1).

[0170] (1) Determination of JT protein expression level. The unfermented liquid intervention group (using the liquid prepared in comparative example 3) and the fermented liquid intervention group (using the blueberry polygonatum liquid prepared in example 1) were given 10 μg / ml of unfermented liquid and fermented liquid respectively in advance. After 24 hours, 5% DSS was added and incubated for 24 hours. The supernatant was discarded, and 150 μl of lysis solution was added to each well. The cells were completely lysed on ice, transferred to a centrifuge tube, centrifuged at 12000 rpm and 4°C for 15 minutes, and the supernatant was stored at -80°C for later use. After quantitative protein analysis by the BCA kit, gel preparation → electrophoresis → membrane transfer → blocking → incubation with primary antibody → incubation with secondary antibody → exposure to determine the expressed protein content.

[0171] (2) Effect on transmembrane resistance of Caco-2 cells. Add 500 μL of cell suspension to the chamber of a 12-well plate, add 1 mL of complete culture medium to the lower chamber, change the medium every 2 days in the first two weeks, change the medium every day in the last week to measure the resistance value, measure the resistance value on the 21st day, and the next experiment can be carried out if TEER (Ω*cm2)>250. The model group was added with 5% DSS, and the unfermented liquid intervention group (using the liquid prepared in Comparative Example 3) and the fermented liquid intervention group (using the blueberry polygonatum liquid prepared in Example 1) were each given 20 uL of unfermented liquid and fermented liquid in advance, and 5% DSS was added for induction after 24 hours. The resistance values ​​were measured at 2, 6, 18, and 24 hours after treatment.

[0172] The experimental results are as follows Fig.10 As shown, the addition of DSS in the Caco-2 monolayer model can induce protein damage. Tight junctions are widely present at the top of the junctions between gastrointestinal mucosal epithelial or endothelial cells, and are composed of proteins such as occludin, claudin, and ZO protein. TJ proteins form a barrier in the intestine. Fig.10 In the fermentation intervention group, the expression levels of ZO-1 and Occludin-1 were increased, and the level of Claudin4 was decreased, indicating that the fermentation liquid (the blueberry polygonatum liquid prepared in Example 1) can maintain the integrity of the intestinal mucosa by adjusting the expression level of tight junction proteins.

[0173] The experimental results are as follows Fig.11 As shown in the figure, the addition of DSS to the Caco-2 monolayer model can induce an increase in intestinal cell membrane permeability. The transmembrane resistance value can be used to evaluate the permeability of the intestinal cell membrane. Fig.11 The resistance value of the model group was significantly lower than that of the blank group, and a significant correction was observed at 24 hours after the intervention with the fermentation liquid (the blueberry and polygonatum liquid prepared in Example 1), indicating that the fermentation liquid pretreatment can enhance the protective function of the intestinal barrier.

[0174] Experimental Example 6

[0175] Male C57 mice (6-8 weeks old) were randomly divided into four groups: a blank group (mice had normal diet and drinking water); a model group (mice received basal food, and drinking water contained 3% DSS); a fermentation liquid group (using the blueberry Polygonatum liquid prepared in Example 1) (mice received basal food, drinking water contained 3% DSS, and mice were given 0.2 ml of fermentation liquid by gavage daily). Mice in the blank group received normal drinking water for 8 days. Mice in the model group and the fermentation liquid group were given drinking water containing 3% DSS for 5 days, and then given normal drinking water on the 5th to 8th day. At the end of the experiment, blood samples were collected, and then the serum was stored at -80°C after centrifugation at 3000rpm for 10min. Colon tissue was taken, and the proximal colon was stored in 4% paraformaldehyde for histopathological examination.

[0176] (1) Enzyme-linked immunosorbent assay (ELISA) kits were used to detect the levels of TNF-α, IL-10, MCP-1 and D-mannitol in mouse serum according to the manufacturer's instructions.

[0177] (2) Liver tissue was stained with hematoxylin and eosin (H&E). First, paraffin sections were dewaxed, and the sections were placed in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% alcohol for 5 min, and then washed with tap water. Then the sections were stained with hematoxylin stain for 3-5 min, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with blue regeneration solution, and rinsed with running water. Then the sections were dehydrated in 85% and 95% gradient alcohol for 5 min each, and stained in eosin stain for 5 min. Finally, the sections were placed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, and sealed with neutral gum. Microscope examination, image acquisition and analysis.

[0178] The experimental results are as follows Fig.12 As shown, the level of pro-inflammatory factor TNF-α in the serum of the fermentation broth intervention group was significantly reduced, and the level of anti-inflammatory factor IL-10 was increased. Overexpression of MCP-1 can cause inflammation, and the fermentation broth can reduce serum MCP-1 levels (*P<0.01, *P<0.05). D-mannitol was used to detect intestinal leakage, and the level of D-mannitol in the serum of the model group mice was significantly higher than that of the blank group mice. However, the fermentation broth inhibited the increase of D-mannitol concentration in the serum (**P<0.01). This suggests that the fermentation broth can improve inflammation in mice with ulcerative colitis and repair the intestinal barrier.

[0179] The experimental results are as follows Fig.13 As shown, colon H&E staining showed that the crypts in the model group were atrophied, the number was reduced, and the goblet cells in the crypts were reduced compared with the fermentation liquid group. This suggests that the fermentation liquid can improve intestinal damage in ulcerative colitis mice and treat acute ulcerative colitis.

[0180] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A blueberry-polygonatum complex, characterized in that: The method is prepared by fermenting a water-bath sterilized mixture of blueberry and polygonatum with plant lactobacillus P9; in the blueberry-polygonatum complex, the mass ratio of the raw materials polygonatum to blueberry is 1:(2-8).

2. The blueberry-polygonatum complex according to claim 1, characterized in that The water bath sterilization conditions are 85-95°C, 10-20min; Preferably, the fermentation temperature is 25-41°C and the fermentation time is 16-48h; Preferably, the inoculation amount of the Lactobacillus plantarum P9 is 0.02%-0.06%.

3. The blueberry-polygonatum complex according to claim 1, characterized in that After the fermentation is completed, the fermentation product is further treated; the fermentation product is treated by centrifugation, and the centrifuge is filtered, sterilized, and sweetened in turn; Preferably, the sterilization in the post-treatment of the fermentation product is water bath sterilization or high temperature instantaneous sterilization; Preferably, the centrifugal separation is performed at 5000-10000 rpm and 0-20° C. for 10-20 min.

4. A method for preparing a blueberry-polygonatum complex, characterized in that: The mixture of blueberry and polygonatum is pulped, and then the pulped product is sterilized in a water bath; then the sterilized material is fermented by using plant lactobacillus P9; the mass ratio of polygonatum to blueberry in the mixture of blueberry and polygonatum is 1:(2-8).

5. The method for preparing the blueberry-polygonatum complex according to claim 4, characterized in that: The fermentation temperature is 25-41°C and the fermentation time is 16-48h; Preferably, the inoculation amount of the Lactobacillus plantarum P9 is 0.02%-0.06%; the fermentation temperature is 25-37° C.; the fermentation time is 16-32 hours; Preferably, the fermentation is carried out at a rotation speed of 100-150 rpm; Preferably, the water bath sterilization conditions are 85-95°C, 10-20 min.

6. The method for preparing the blueberry-polygonatum complex according to claim 5, characterized in that: After the fermentation is completed, the fermentation product is further treated; the fermentation product is treated by centrifugation, and the centrifuge is filtered, sterilized, and sweetened in turn; Preferably, the sterilization in the post-treatment of the fermentation product is water bath sterilization or high temperature instantaneous sterilization; Preferably, the centrifugal separation conditions are 5000-10000 rpm, 0-20°C, 10-20 min; Preferably, the sweetener is selected from at least one of xylitol, compound sweeteners, sucralose, maltose syrup, xylooligosaccharides, galacto-oligosaccharides, fructo-oligosaccharides, protein sugars, aspartame, sorbitol, mannitol, maltitol, isomalt and glucose syrup; Preferably, the added amount of the compound sweetener accounts for 3%-9% of the total material mass.

7. The method for preparing the blueberry-polygonatum complex according to claim 1, characterized in that: adding a carbon source during the pulping process; Preferably, the carbon source is anhydrous glucose, and the amount of anhydrous glucose added accounts for 2%-5% of the total pulping material mass; Preferably, the beating is performed by mixing the mixture of blueberry and polygonatum with 4-6 times the volume of water; Preferably, the beating time is 2-5 min; Preferably, the beating speed is 1000-50000 rpm; Preferably, the mass ratio of polygonatum to blueberry in the mixture of blueberry and polygonatum is 1:6; Preferably, the Huangjian is selected from at least one of the roots, stems, leaves, flowers and fruits of the Polygonatum cyrtonema plant.

8. A product, characterized in that The invention comprises the blueberry-polygonatum complex described in claims 1-3 or the blueberry-polygonatum complex prepared by the preparation method of the blueberry-polygonatum complex described in any one of claims 4-7.

9. The product according to claim 8, characterized in that The product is a food or a drug; Preferably, the food is a dairy product, pudding, cream, cream cheese, fresh cream, curd, milkshake, custard, nutrition bar, energy bar, candy, baked food, cereal fragments, baby food, spread, sugar-free biscuits and chocolate, calcium chewable tablets, meat product, condiment, gravy, soup, shortening, beverage or wine.

10. Use of the blueberry-polygonatum complex according to any one of claims 1 to 3 or the blueberry-polygonatum complex prepared by the method for preparing the blueberry-polygonatum complex according to any one of claims 4 to 7 in preparing functional foods for resisting inflammatory bowel disease; Preferably, the functional food has at least one of the following uses: (1) Maintain the integrity of the intestinal mucosa; (2) Regulate the levels of intestinal inflammatory factors; (3) Treatment of acute ulcerative colitis.

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