Method for preparing hawthorn pulp fermentation product by improving polyphenol content and antioxidant activity of hawthorn pulp through lactobacillus fermentation

Abstract

The invention discloses a method for preparing a hawthorn pulp fermentation product by improving the polyphenol content and antioxidant activity of hawthorn pulp through lactobacillus fermentation. The method comprises the following steps: (1) removing stems and kernels of haws, adding water, crushing and pulping to obtain haw pulp; (2) adjusting the pH value of the hawthorn pulp, adding a carbon source and a nitrogen source, and sterilizing to obtain a hawthorn pulp mixture; and (3) inoculating lactic acid bacteria into the hawthorn pulp blend for fermentation, and obtaining the hawthorn pulp fermentation product with high polyphenol content and high antioxidant activity after fermentation. According to the preparation method, the content of free phenol and bound phenol in the hawthorn pulp can be effectively increased through fermentation, conversion of phenolic compounds is caused, and the antioxidant activity is enhanced. The method not only effectively maintains the nutritional quality of the hawthorn pulp, but also widens the application field of the hawthorn, provides reference for developing a hawthorn lactobacillus fermentation product, provides a new idea for popularization of the hawthorn, and provides new possibility for development of a large healthy industry.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for preparing a hawthorn pulp fermentation product by increasing the polyphenol content and antioxidant activity of hawthorn pulp through lactic acid bacteria fermentation. Background Art

[0002] Hawthorn (Crataegus pinnatifida Bunge), also known as "fairy fruit" and "mountain red", belongs to the Rosaceae family, Crataegus genus, and has been included in the first list of medicine and food by the Ministry of Health. Hawthorn is rich in a variety of bioactive ingredients, such as polysaccharides, dietary fiber, organic acids, polyphenolic compounds, triterpenoid compounds, etc., especially anthocyanins, phenolic acids, flavonoids and other polyphenolic compounds are rich in content and the most types. In addition, hawthorn has the functions of improving cardiovascular function, anti-inflammation, anti-oxidation, lowering blood sugar, lowering blood lipids, and promoting digestion. However, hawthorn is not suitable for fresh consumption due to its sour taste. It is usually processed into products. If moutan bark, hawthorn slices, hawthorn juice, hawthorn canned food, etc., a large amount of sugar will be added or over-cooked during the processing process, which will cause serious problems of nutrient loss. Therefore, in order to maintain the nutrition and quality of hawthorn, a new hawthorn processing technology is urgently needed.

[0003] At present, lactic acid bacteria fermentation technology has been widely used in the field of fruit and vegetable product processing, which can not only give the product good sensory flavor, but also protect and improve the nutritional quality and functional activity. Polyphenols are important plant secondary metabolites, usually present in plants in two forms (free polyphenols and bound polyphenols). Among them, free polyphenols are easily digested and absorbed by the human body, but bound polyphenols usually interact with cellulose, protein or polysaccharides in plant cell walls to form covalent compounds, which are difficult to extract and have low bioavailability. The conversion of phenolic compounds can be promoted by lactic acid bacteria fermentation, which effectively maintains the nutritional quality of fruits and vegetables and can be applied to hawthorn processing. However, hawthorn has a high acidity and is not suitable for direct lactic acid bacteria fermentation. The main ways to solve this technical problem include domesticating lactic acid bacteria to improve its acid resistance, mixing hawthorn with other sugary fruits for fermentation, screening acid-resistant strains, etc. These technical methods are relatively complicated, and there are problems such as low lactic acid bacteria activity and difficulty in improving biological activity. Therefore, how to carry out lactic acid bacteria fermentation of hawthorn pulp to increase the number of viable bacteria, total phenol content and antioxidant activity in the fermented hawthorn pulp, especially to promote the release of free polyphenols and the extraction of bound polyphenols and the bioavailability of phenolic compounds, is still a technical problem that needs to be solved urgently. Summary of the invention

[0004] The purpose of the present invention is to provide a method for improving the polyphenol content and antioxidant activity of hawthorn pulp by lactic acid bacteria fermentation. The method uses hawthorn as a raw material and Lactobacillus rhamnosus as a fermentation strain, can effectively increase the content of free phenol and bound phenol, and transform hawthorn pulp polyphenols, thereby improving the antioxidant activity and the bioavailability of phenolic compounds, and maintaining the nutritional quality of hawthorn.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a method for preparing a hawthorn pulp fermentation product by increasing the polyphenol content and antioxidant activity of hawthorn pulp by lactic acid bacteria fermentation, comprising the following steps:

[0007] (1) removing the stems and cores of hawthorn, adding water to crush and pulp, and obtaining hawthorn pulp;

[0008] (2) adjusting the pH value of the hawthorn pulp, adding a carbon source and a nitrogen source, and sterilizing the mixture to obtain a hawthorn pulp formulation;

[0009] (3) Inoculating lactic acid bacteria into the hawthorn pulp formulation for fermentation, and obtaining the hawthorn pulp fermentation product with high polyphenol content and high antioxidant activity after fermentation.

[0010] In the above method, further, in the crushing and pulping step, the ratio of hawthorn to water is 1: (2-6). As an example, the hawthorn is fresh hawthorn. The method further includes blanching the hawthorn with boiling water before crushing and pulping in step (1). Blanching with boiling water can achieve the effect of inactivating enzymes.

[0011] In the above method, further, step (1) also includes the step of enzymatically hydrolyzing the slurry obtained by crushing and beating; preferably, the enzymatic hydrolysis step includes: adding 0.5g / kg pectinase to the slurry and enzymatically hydrolyzing it at 50°C for 2h.

[0012] In the above method, further, in the step of adjusting the pH value of the hawthorn pulp, the pH value is adjusted to 5.0-7.0; and / or,

[0013] In the step of adjusting the pH value of hawthorn pulp, food grade sodium carbonate, sodium bicarbonate or sodium hydroxide is used for adjustment.

[0014] In the above method, the carbon source can be glucose, sucrose and other sugar substances, and the nitrogen source can be soy protein isolate, skim milk powder and the like.

[0015] Furthermore, the carbon source is glucose, and the amount of glucose added is 2% to 10% based on the mass of the hawthorn pulp after adjusting the pH value; and / or,

[0016] The nitrogen source is soy protein isolate, and the added amount of the soy protein isolate is 0.1% to 0.5% based on the mass of the hawthorn pulp after adjusting the pH value.

[0017] In the above method, as an example, the sterilization is performed by high temperature and high pressure sterilization at 90°C for 15 minutes, followed by natural cooling to room temperature (25°C).

[0018] In the above method, further, the lactic acid bacteria is lactobacillus, preferably Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus paracasei or Lactobacillus casei; more preferably, the lactic acid bacteria is Lactobacillus rhamnosus, and the inoculation amount of Lactobacillus rhamnosus in the initial viable count of the hawthorn pulp formulation is 5×10 5 CFU / mL~6×10 6 CFU / mL; As an example, the Lactobacillus rhamnosus was added in the form of a bacterial suspension, and its effective viable count was 1×10 8 CFU / mL, based on the volume of the hawthorn pulp formulation, the inoculation amount of the bacterial suspension is 0.5% to 6%. More preferably, the bacterial suspension is obtained by the following steps: inoculating Lactobacillus rhamnosus into MRS broth medium for activation, taking the activated bacterial solution and centrifuging it, pouring out the supernatant, adding sterile physiological saline and resuspending and mixing to the required effective viable bacterial count. As an example, the absorbance of the bacterial suspension is measured at 600nm, and OD is adjusted to about 0.500. At this time, the effective viable bacterial count is about 1×10 8 CFU / mL. As an example, the inoculation amount of Lactobacillus rhamnosus in the MRS broth medium is 1%, cultured at 37°C for 24h, and activated repeatedly twice. The speed of the centrifugation is 6000rpm, the time is 10min, and the temperature is 4°C. The concentration of the physiological saline is 0.85%. As an example, the plant lactobacillus is plant lactobacillus H87; the casei lactobacillus is casei lactobacillus SB27; the rhamnosus lactobacillus is rhamnosus lactobacillus FN518; the paracasei lactobacillus is paracasei lactobacillus ML446.

[0019] In the above method, further, in the fermentation step, the fermentation temperature is 27°C to 47°C; and / or,

[0020] In the fermentation step, the fermentation time is 18h to 66h;

[0021] The fermentation is carried out in a closed environment.

[0022] In a preferred embodiment of the present invention, in the crushing and beating step, the ratio of pitted hawthorn to water is 1:3; in the step of adjusting the initial pH value of hawthorn pulp, the pH value is adjusted to 6.5; the amount of glucose added is 6% of the hawthorn pulp; the amount of soy protein isolate added is 0.3% of the hawthorn pulp; based on the initial viable count of the hawthorn pulp formulation, the inoculation amount of Lactobacillus rhamnosus is 4×10 6 CFU / mL (i.e., the Lactobacillus rhamnosus was added in the form of bacterial suspension, and its effective viable count was 1×10 8 CFU / mL, based on the volume of the hawthorn pulp formulation, the inoculation amount of the rhamnosus lactobacillus is 4%); in the fermentation step, the fermentation temperature is 37°C; in the fermentation step, the fermentation time is 48h. Under this condition, the number of viable bacteria, total phenol content and DPPH clearance rate in the fermented hawthorn pulp are the highest.

[0023] In the above method, further, the effective viable count in the hawthorn pulp fermentation product is higher than 8×10 8 CFU / mL, with prebiotic functional characteristics; the improvement of the polyphenol content of hawthorn pulp includes increasing the free polyphenol content, bound polyphenol content and total phenol content of hawthorn pulp; the present invention can not only achieve the improvement of the free polyphenol content, but also increase the bound polyphenol content, which may be because the ether bond or ester bond connecting the bound phenol and the cell wall is broken under the action of lactic acid bacteria, making the bound phenol easier to extract; the improvement of the polyphenol content of hawthorn pulp is achieved by mediating the flavonoid biosynthesis pathway through the action of lactic acid bacteria, and at the same time promoting the conversion of macromolecular phenolic compounds to small molecular phenolic compounds; in the present invention, Lactobacillus rhamnosus fermentation can effectively increase the content of phenolic compounds in hawthorn pulp and promote the conversion of phenolic compounds; the improvement of antioxidant activity is reflected in increasing at least one of DPPH scavenging rate, hydroxyl radical scavenging rate and FRAP; in the present invention, Lactobacillus rhamnosus fermentation improves the antioxidant activity of hawthorn pulp, and may be related to the production of phenolic metabolites with antioxidant activity under the action of Lactobacillus rhamnosus.

[0024] In a second aspect, the present invention provides a hawthorn pulp fermentation product prepared by any of the methods described above.

[0025] The present invention has the following beneficial effects: the present invention adopts lactic acid bacteria fermentation technology, which can effectively increase the free phenol and bound phenol content of hawthorn pulp, cause the conversion of phenolic compounds, enhance antioxidant activity, and give hawthorn pulp a probiotic effect. It not only effectively maintains the nutritional quality of hawthorn pulp, but also broadens the application field of hawthorn, provides a reference for the development of hawthorn lactic acid bacteria fermentation products, provides new ideas for the promotion of hawthorn, and provides new possibilities for the development of the big health industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1The effects of the fermentation of four lactic acid bacteria on the viable bacterial count (a), pH (b), total phenol content (c) and DPPH clearance rate (d) in hawthorn in the present invention;

[0027] Figure 2 The effects of different fermentation times on the viable bacterial count, total phenol content and DPPH clearance rate of fermented hawthorn pulp in the present invention;

[0028] Figure 3 The effects of different fermentation temperatures on the viable bacterial count, total phenol content and DPPH clearance rate of fermented hawthorn pulp in the present invention;

[0029] Figure 4 The effects of different inoculation amounts on the number of viable bacteria, total phenol content, and DPPH clearance rate of fermented hawthorn pulp in the present invention;

[0030] Figure 5 The effects of different material-liquid ratios on the viable bacterial count, total phenol content and DPPH clearance rate of fermented hawthorn pulp in the present invention;

[0031] Figure 6 The effects of different initial pH values ​​on the viable bacterial count, total phenol content, and DPPH clearance rate of fermented hawthorn pulp in the present invention;

[0032] Figure 7 The effects of different glucose addition amounts on the viable bacterial count, total phenol content and DPPH clearance rate of fermented hawthorn pulp in the present invention;

[0033] Figure 8 The effects of different amounts of soy protein isolate added on the viable bacterial count, total phenol content and DPPH clearance rate of fermented hawthorn pulp in the present invention;

[0034] Fig. 9 The change of the number of live bacteria in the hawthorn pulp during the lactic acid bacteria fermentation process of the present invention;

[0035] Fig.10 The change of DPPH clearance rate of hawthorn pulp during lactic acid bacteria fermentation in the present invention;

[0036] Fig.11 The change of scavenging rate of hydroxyl radicals of hawthorn pulp during lactic acid bacteria fermentation in the present invention;

[0037] Fig.12 The change of FRAP of hawthorn pulp during lactic acid bacteria fermentation in the present invention;

[0038] Fig.13 In the present invention, metabolomics was used to explore the biotransformation pathways of phenolic compounds in hawthorn pulp before and after fermentation: PCA diagram (a), differential metabolite volcano diagram (b) and key differential metabolite heat map (c) before and after fermentation of hawthorn pulp. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0040] The methods used in the following examples, unless otherwise specified, are all conventional methods, carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0041] The sources of the raw materials in the following examples are as follows:

[0042] Lactobacillus plantarum H87 is provided by the Functional Dairy and Probiotic Engineering Laboratory of the School of Food Science and Engineering, Ocean University of China, and is recorded in the document "Liang C, Zhou XH, Gong PM, et al. Lactiplantibacillus plantarum H-87prevents high-fat diet-induced obesity by regulating bile acidmetabolismin C57BL / 6J mice[J]. Food&function, 2021, 12(10): 4315-24." The public can obtain it from the applicant. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0043] Lactobacillus casei SB27 is provided by the Functional Dairy and Probiotics Engineering Laboratory of the School of Food Science and Engineering, Ocean University of China, and is recorded in the document "Wang Shumei, Di Wei, Yu Jiarui, et al. Study on the effect of Lactobacillus casei SB27 on reducing the toxicity of N-nitrosamines on IEC-6 cells [J]. China Brewing, 2022, 41(04): 54-58." The public can obtain it from the applicant. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0044] Lactobacillus rhamnosus FN518 was provided by the Functional Dairy and Probiotic Engineering Laboratory of the School of Food Science and Engineering, Ocean University of China, and is recorded in the document "Yang Wenjun, Liu Tongjie, Liang Xi, et al. Effects of 2'-fucosyllactose on the colonization and anti-inflammatory ability of probiotics [J]. Food Industry Science and Technology, 2021, 42(20): 355-364." The public can obtain it from the applicant. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0045] Lactobacillus paracasei ML446 was provided by the Functional Dairy and Probiotic Engineering Laboratory of the School of Food Science and Engineering, Ocean University of China, and is recorded in the document "Yang Wenjun, Liu Tongjie, Liang Xi, et al. Effects of 2'-fucosyllactose on the colonization and anti-inflammatory ability of probiotics [J]. Food Industry Science and Technology, 2021, 42(20): 355-364." The public can obtain it from the applicant. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0046] Pectinase was purchased from Henan Wanbang Chemical Technology Co., Ltd. with an enzyme activity of 30,000 U / g.

[0047] Soy protein isolate was purchased from Beijing Solebow Technology Co., Ltd. with product number S9510.

[0048] The compositions of the culture media in the following examples are as follows: MRS broth medium (g / L): peptone 10.0; beef powder 10.0; yeast powder 5.0; glucose 20.0; magnesium sulfate 0.1; sodium acetate 5.0; ammonium citrate 2.0; potassium hydrogen phosphate 2.0; manganese sulfate 0.05; Tween 80 1.0.

[0049] Example 1: Selection of fermentation process

[0050] Follow the steps below to carry out lactic acid fermentation of hawthorn pulp:

[0051] (1) Pretreatment: The hawthorns are screened to select those that are insect-free, complete, and not dark in color, wipe off dust, rinse with clean water, remove stems and cores, and weigh.

[0052] (2) Blanching: Take a certain amount of ultrapure water, heat it to boil, add the processed hawthorn, and blanch it for 30 seconds to inactivate the enzyme.

[0053] (3) Pulping: add distilled water at a solid-liquid ratio of 1:2-1:6, and use a wall breaker to crush and pulp.

[0054] (4) Enzymatic hydrolysis: add 0.5 g / kg pectinase and perform enzymatic hydrolysis at 50°C for 2 h to obtain hawthorn pulp, which is then placed in a refrigerator at 4°C for later use.

[0055] (5) Preparation of hawthorn pulp: In order to make the bacteria grow more appropriately in hawthorn pulp, it is necessary to prepare it. Use food-grade sodium carbonate to adjust the pH to 5.0-7.0, add 2%-10% glucose as a carbon source, and add 0.1%-0.5% soy protein isolate as a nitrogen source.

[0056] (6) Sterilize the prepared hawthorn pulp under high temperature and high pressure at 90°C for 15 min, and then cool it naturally to room temperature (25°C).

[0057] (7) The frozen Lactobacillus rhamnosus strains were thawed to room temperature, and the strains were inoculated into a conical flask containing 20 mL of MRS broth medium at a ratio of 1% under sterile conditions, and cultured at 37° C. for 24 h. After repeated activation twice, strains with good vitality were obtained.

[0058] (8) The activated bacterial suspension was centrifuged (6000 rpm, 10 min, 4°C), the supernatant was discarded, and sterile 0.85% saline was added to resuspend and mix to obtain a bacterial suspension for fermentation. The live bacterial content was about 1×10 8 CFU / mL, 5×10 5 CFU / mL (0.5%)-6×10 6 An inoculum of 6% CFU / mL was inoculated into 100 mL of hawthorn pulp, sealed with a sealing film, and cultured at 27°C-47°C for 18h-66h.

[0059] The specific experimental method is to determine the number of viable bacteria using the standard plate count method. The pH value is determined using a pH meter. The total phenol content is determined using the Folin phenol colorimetric method. Take 1 mL of the diluted fermented hawthorn pulp supernatant in a 15 mL test tube, add 0.5 mL of Folin phenol reagent, react for 8 minutes, add 1.5 mL of 20% sodium carbonate solution and 7 mL of distilled water, shake well and react in the dark for 2 hours, then measure the absorbance at 765 nm. The DPPH clearance rate is determined by mixing 1 mL of the diluted sample with 2 mL of 0.2 mM DPPH solution, reacting in the dark for 20 minutes, and measuring the absorbance at 517 nm. Three parallel samples are set up for each group of experiments, and the independent parallel operation is repeated three times.

[0060] 1. Effect of unadjusted pH on fermentation

[0061] The solid-liquid ratio was adjusted to 1:3, and Lactobacillus plantarum (PL), Lactobacillus rhamnosus (LR), Lactobacillus paracasei (LP), and Lactobacillus casei (LC) were inoculated into hawthorn pulp as fermentation strains, with an inoculation volume of 4 × 10 6 CFU / mL (4%), fermentation temperature was 37°C, fermentation time was 48h, pH was not adjusted, no carbon source and nitrogen source were added, and the initial pH of hawthorn pulp was 2.83. The effect of unadjusted pH on the viable bacterial count and pH change of fermented hawthorn pulp was explored. The specific measurement experiments and result data are shown in Table 1.

[0062] As shown in Table 1, after 48 h of fermentation, the number of effective live bacteria of each strain did not reach 1×10 6 CFU / mL, and the pH value did not change significantly, indicating that lactic acid bacteria could not adapt to the acidic environment of the original hawthorn pulp and could not grow well, so it was necessary to adjust the hawthorn pulp.

[0063] Table 1 Growth of four lactic acid bacteria in original hawthorn pulp after 48h of fermentation

[0064]

[0065] 2. Effect of not adding carbon or nitrogen source on fermentation

[0066] The solid-liquid ratio was adjusted to 1:3, and Lactobacillus rhamnosus was inoculated into the hawthorn pulp as the fermentation strain. The inoculation amount of the strain was 4×10 6 CFU / mL (4%), fermentation temperature was 37°C, fermentation time was 48h, pH was adjusted to 6.5, no carbon source (glucose) was added, 0.3% soy protein isolate was added, no nitrogen source (soy protein isolate) was added, and 6% glucose was added. The effect of not adding carbon source or nitrogen source on the number of viable bacteria in fermented hawthorn pulp was explored. The specific determination experiment and result data are shown in Table 2.

[0067] As shown in Table 2, after 48 hours of fermentation, the number of live bacteria in the hawthorn pulp without adding a carbon source was 6.97±0.12Log CFU / mL, and the number of live bacteria in the hawthorn pulp without adding a nitrogen source was 6.62±0.15Log CFU / mL. The growth of Lactobacillus rhamnosus was poor, so it was necessary to add carbon and nitrogen sources.

[0068] Table 2 Effect of not adding carbon source or nitrogen source on the number of viable bacteria in fermented hawthorn pulp

[0069]

[0070] 3. Selection of strains

[0071] The material-liquid ratio was adjusted to 1:3, the initial pH of hawthorn pulp was 6.5, the amount of glucose added was 4%, the amount of soy protein isolate added was 0.3%, and Lactobacillus plantarum (PL), Lactobacillus rhamnosus (LR), Lactobacillus paracasei (LP), and Lactobacillus casei (LC) were inoculated into hawthorn pulp as fermentation strains, and the inoculation amount of the strains was 4×10 6 CFU / mL (4%), fermentation temperature was 37°C, and fermentation time was 72h. To explore the effects of different lactic acid bacteria on the viable count, pH, total phenol content, and DPPH clearance rate of fermented hawthorn pulp, see the specific determination experiment and result data. Figure 1 .

[0072] Depend on Figure 1 It can be seen that after the fermentation, the pH of hawthorn pulp fermented by Lactobacillus rhamnosus was the lowest, the total phenol content and DPPH clearance rate increased the most, and the number of viable bacteria reached 8.00±0.07log CFU / mL, indicating that Lactobacillus rhamnosus has stronger growth adaptability and polyphenol conversion ability in hawthorn pulp. Lactobacillus rhamnosus was used as the fermentation strain in the future.

[0073] 4. Fermentation time

[0074] The material-liquid ratio was adjusted to 1:3, the initial pH of hawthorn pulp was 6.0, the amount of glucose added was 4%, the amount of soy protein isolate added was 0.3%, and the inoculum amount of Lactobacillus rhamnosus was 4×10 6 CFU / mL (4%), the fermentation temperature was 37°C, and the effects of different fermentation times (18h, 24h, 30h, 36h, 42h, 48h, 54h, 60h, 66h) on the number of viable bacteria, total phenol content, and DPPH clearance rate of fermented hawthorn pulp were investigated. For specific determination experiments and result data, see Figure 2 .

[0075] Depend on Figure 2 It can be seen that with the change of fermentation time, the number of live bacteria in the fermented hawthorn pulp reached the maximum value at 48h, which was 8.28±0.04Log CFU / mL, and the number of live bacteria decreased significantly from 48h to 66h (p<0.05). The total phenol content increased first and then decreased, and the total phenol content was the highest at 48h of fermentation. The DPPH clearance rate increased significantly (p<0.05), and the change was gentle after 48h.

[0076] 5. Fermentation temperature

[0077] The material-liquid ratio was adjusted to 1:3, the initial pH of hawthorn pulp was 6.0, the amount of glucose added was 4%, the amount of soy protein isolate added was 0.3%, and the inoculation amount of Lactobacillus rhamnosus was 6×10 6 CFU / mL (4%), fermentation time was 48h, and the effects of different fermentation temperatures (27℃, 32℃, 37℃, 42℃, 47℃) on the number of viable bacteria, total phenol content, and DPPH clearance rate of fermented hawthorn pulp were investigated. For specific determination experiments and result data, see Figure 3 .

[0078] Depend on Figure 3 It can be seen that with the increase of fermentation temperature, the number of viable bacteria first increased and then decreased significantly. The total phenol content and DPPH clearance rate were the highest at 37°C.

[0079] 6. Inoculation volume

[0080] The material-liquid ratio was adjusted to 1:3, the initial pH of hawthorn pulp was 6.0, the amount of glucose added was 4%, the amount of soy protein isolate added was 0.3%, the fermentation temperature was 37 °C, and the fermentation time was 48 h. The present invention explored the effects of different inoculum sizes (5 × 10 5 CFU / mL (0.5%), 1×10 6 CFU / mL (1%), 2×10 6 CFU / mL (2%), 3×10 6 CFU / mL (3%), 4×10 6 CFU / mL (4%), 5×106 CFU / mL (5%), 6×10 6 CFU / mL (6%)) on the number of viable bacteria, total phenol content, and DPPH clearance rate of fermented hawthorn pulp. For specific determination experiments and result data, see Figure 4 .

[0081] Depend on Figure 4 It can be seen that with the increase of inoculation size, the total phenol content and DPPH clearance rate showed a trend of first increasing and then decreasing. When the inoculation size was 4×10 6 CFU / mL (4%), the total phenolic content and DPPH scavenging rate of fermented hawthorn pulp were the highest.

[0082] 7. Material-liquid ratio

[0083] The initial pH of hawthorn pulp was 6.0, and the amount of glucose added was 4×10 6 CFU / mL (4%), the amount of soy protein isolate added was 0.3%, the fermentation temperature was 37°C, and the fermentation time was 48h. The effects of different solid-liquid ratios (1:2, 1:3, 1:4, 1:5, 1:6) on the number of viable bacteria, total phenol content, and DPPH clearance rate of fermented hawthorn pulp were investigated. For specific determination experiments and result data, see Figure 5 .

[0084] Depend on Figure 5 It can be seen that with the increase of the solid-liquid ratio, the fermented hawthorn pulp was diluted. When the solid-liquid ratio was 1:3, it was suitable for the growth of Lactobacillus rhamnosus and could better maintain a higher total phenol content and DPPH clearance rate.

[0085] 8. Initial pH

[0086] The material-liquid ratio was adjusted to 1:3, the amount of glucose added was 4%, the amount of soy protein isolate added was 0.3%, and the inoculation amount of Lactobacillus rhamnosus was 4×10 6 CFU / mL (4%), fermentation temperature was 37 ° C, fermentation time was 48 h, and the effects of different initial pH (5.0, 5.5, 6.0, 6.5, 7.0) on the number of viable bacteria, total phenol content, and DPPH clearance rate of fermented hawthorn pulp were investigated. For specific determination experiments and result data, see Figure 6 .

[0087] Depend on Figure 6 It can be seen that when the pH is between 5 and 6.5, the total phenol content and DPPH clearance first decrease and then increase. Although the total phenol content and DPPH clearance rate are the highest at pH 5, it is not too suitable for the growth of Lactobacillus rhamnosus. When the pH is 6.5, the number of viable bacteria is the highest, and the total phenol content and DPPH clearance rate are also improved.

[0088] 9. Amount of glucose added

[0089] The material-liquid ratio was adjusted to 1:3, the initial pH of hawthorn pulp was 6.5, the amount of soy protein isolate added was 0.3%, and the inoculation amount of Lactobacillus rhamnosus was 4×10 6 CFU / mL (4%), fermentation temperature was 37 ° C, fermentation time was 48h, and the effect of different initial glucose addition amounts (2%, 4%, 6%, 8%, 10%) on the viable bacterial count, total phenol content, and DPPH clearance rate of fermented hawthorn pulp was investigated. For specific determination experiments and result data, see Figure 7 .

[0090] Depend on Figure 7 It can be seen that with the increase of glucose addition, the total phenol content and DPPH scavenging in fermented hawthorn pulp first increased and then decreased, and reached the highest when the addition amount was 6%.

[0091] 10. Addition amount of soy protein isolate

[0092] The material-liquid ratio was adjusted to 1:3, the initial pH of hawthorn pulp was 6.5, the amount of glucose added was 6%, and the inoculation amount of Lactobacillus rhamnosus was 4×10 6 CFU / mL (4%), fermentation temperature was 37 ° C, fermentation time was 48h, and the effect of different soy protein isolate addition amounts (0.1%, 0.2%, 0.3%, 0.4%, 0.5%) on the viable bacterial count, total phenol content, and DPPH clearance rate of fermented hawthorn pulp was investigated. For specific determination experiments and result data, see Figure 8 .

[0093] Depend on Figure 8 It can be seen that with the increase in the amount of soy protein isolate added, the number of live bacteria first increased and then leveled off, and the total phenol content decreased, which may be due to the decrease caused by the combination with the soy protein isolate. DPPH clearance first increased and then decreased. Adding 0.3% soy protein isolate can meet the growth requirements of Lactobacillus rhamnosus.

[0094] Through this single factor experiment, it can be concluded that the optimal conditions for improving polyphenol content and DPPH scavenging activity in the process of fermentation of hawthorn pulp by Lactobacillus rhamnosus are fermentation time of 48h, fermentation temperature of 37℃, inoculation amount of 4%, solid-liquid ratio of 1:3, initial pH of 6.5, glucose addition of 6%, and soy protein isolate addition of 0.3%. Under these conditions, the number of viable bacteria, total phenol content and DPPH scavenging rate in fermented hawthorn pulp are the highest.

[0095] This condition is used in subsequent embodiments.

[0096] Example 2: Changes in viable bacterial count during fermentation.

[0097] This example explores the changes in the number of viable Lactobacillus rhamnosus in hawthorn pulp during the 48h fermentation process. For specific determination experiments and result data, see Fig. 9 .

[0098] The specific experimental method is to use the standard plate count method to determine the number of viable bacteria.

[0099] Depend on Fig. 9 It can be seen that 0-30h is the logarithmic growth period of Lactobacillus rhamnosus in hawthorn pulp, 30-48h is the stable period, and during the fermentation process, the number of live bacteria increased significantly from 6.70±0.10Log CFU / mL to 8.28±0.03Log CFU / mL (p<0.05). When the number of live lactic acid bacteria exceeds 8.0Log CFU / mL, the fermented hawthorn pulp has probiotic functional characteristics.

[0100] Example 3: Changes in the contents of free phenol and bound phenol during fermentation.

[0101] This example explores the changes in the content of phenolic compounds in hawthorn pulp during the 48h fermentation process, including the content of free phenol and bound phenol. The specific measurement experiments and result data are shown in Table 3.

[0102] The specific experimental method is to mix the freeze-dried fermented hawthorn pulp sample with 70% ethanol at a solid-liquid ratio of 1:25, ultrasonically extract for 30 minutes, centrifuge at 6000rpm for 8 minutes to collect the supernatant, extract the remaining precipitate twice, then combine the supernatant and rotary evaporate to dryness at 45°C, and dilute to 10mL with ultrapure water. Get the free phenol extract. The obtained precipitate was dried at 37°C for 20 hours, 4M NaOH solution was added at a solid-liquid ratio of 1:40, oscillated for 2 hours (200rpm, 37°C), and then the pH was adjusted to 2.0 with 6M HCl, centrifuged at 6000rpm for 20 minutes, and the supernatant was extracted 3 times with an equal volume of ethyl acetate, rotary evaporated to dryness at 45°C, and diluted to 10mL with ultrapure water. Get the bound phenol extract. The total phenol content was determined by the above-mentioned Folin phenol colorimetric method. Three parallel samples were set for each group of experiments, and three independent parallel operations were repeated.

[0103] As shown in Table 3, with the change of fermentation time, the total phenol content increased significantly from 9.93±0.12mg GAE / g to 11.25±0.12mg GAE / g (p<0.05), the free phenol content increased significantly from 9.77±0.12mg GAE / g to 11.04±0.12mg GAE / g (p<0.05), and the bound phenol content increased significantly from 0.15mg GAE / g to 0.21mg GAE / g (p<0.05). The increase in free phenol content may be due to the increase in acidity of the fermentation system, which releases free phenol through acid hydrolysis, and the increase in the activity of metabolic enzymes such as phenolesterase, glycosidase, reductase, decarboxylase and dehydrogenase, which leads to the hydrolysis of cell walls and the release of soluble conjugated or insoluble bound phenols. The content of bound phenols also increased significantly, which may be because the ether bond or ester bond connecting bound phenols and cell walls was broken under the action of lactic acid bacteria, making bound phenols easier to extract. The experiment showed that Lactobacillus rhamnosus fermentation can effectively increase the content of phenolic compounds in hawthorn pulp and promote the conversion of phenolic compounds.

[0104] Table 3 Changes in total phenol, free phenol and bound phenol content in hawthorn pulp during lactic acid bacteria fermentation

[0105]

[0106]

[0107] Example 4: Changes in antioxidant activity during fermentation.

[0108] This example explores the changes in the antioxidant activity of hawthorn pulp during the 48h fermentation process. For specific determination experiments and result data, see Figure 10-12 , are the changes in DPPH scavenging rate, hydroxyl radical scavenging rate and FRAP of hawthorn pulp during 48h fermentation.

[0109] The specific experimental method is the same as above for DPPH determination; the hydroxyl radical scavenging rate determination method is: 1 mL of diluted sample and 1 mL of 9 mM FeSO 4 , 1 mL 9 mM salicylic acid and 1 mL 30% hydrogen peroxide were mixed and reacted at 37°C for 30 min, and the absorbance was measured at 510 nm. The FRAP assay method is as follows: acetate buffer (0.3 M, pH 3.6), 2,4,6-tripyridyl triazine solution (10 mM) prepared with HCl (40 mmol / L) and FeCl 3 The solution (20mM) was mixed in a ratio of 10:1:1 as the FRAP working solution. Then 30μL of the diluted sample was mixed with 900μL of the FRAP working solution, reacted at 37°C for 20min, and the absorbance was measured at 593nm. Three parallel samples were set for each group of experiments, and the operation was repeated three times independently.

[0110] Depend on Figure 10-12 It can be seen that after 48 hours of fermentation, the DPPH scavenging rate of hawthorn pulp increased significantly by 30.80% (p<0.05), the hydroxyl radical scavenging rate increased significantly by 21.04% (p<0.05), and FRAP gradually increased from 369.70±5.26mmolTrolox / L to 418.54±1.06mmolTrolox / L. The experiment showed that Lactobacillus rhamnosus fermentation increased the antioxidant activity of hawthorn pulp, and it may be related to the production of phenolic metabolites with antioxidant activity under the action of Lactobacillus rhamnosus.

[0111] Example 5: Metabolomics was used to investigate the biotransformation pathways of phenolic compounds in hawthorn pulp before and after fermentation.

[0112] In order to further explore the changes of phenolic compounds during fermentation, this example combines LC / MS non-targeted metabolomics to explore the transformation pathways of hawthorn pulp phenolic compounds during fermentation. For specific determination experiments and result data, see Fig.13 .

[0113] The specific experimental method is to take 1mL of sample through the SPE solid phase column, collect 3mL of methanol eluent, blow it with nitrogen blower, add 300μL of pre-cooled methanol-water (V:V=4:1, containing mixed internal standard, 4μg / mL) after drying, vortex for 1min, ultrasonicate in ice water bath for 10min, stand at -40℃ for 2h, centrifuge for 10min (13000rpm, 4℃), draw 150μL of supernatant with syringe, transfer to LC injection vial, and store at -80℃. Waters ACQUITY UPLC I-Class plus / Thermo QE ultra-high performance liquid phase tandem high-resolution mass spectrometry was used for ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry (UPLC-MS / MS) analysis. The chromatographic conditions are: using ACQUITY UPLC HSS T3 (100mm×2.1mm,1.8μm) chromatographic column, mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is acetonitrile. The elution gradient was as follows: 0-4min, 5-30% B; 4-8min, 30-50% B; 8-10min, 50-80% B; 10-14min, 80-100% B; 14-15.1min, 100-5% B; 15.1-16min, 5-5% B, the injection volume was 2μL, the flow rate was 0.4mL / min, and the column temperature was 45°C. The mass spectrometry conditions were: the scanning range was 70-1050m / z, and positive ion (3800V) and negative ion scanning modes (-3200V were used. The auxiliary gas temperature was 350°C, the capillary temperature was 320°C, the auxiliary gas flow rate was 8arb, and the sheath gas flow rate was 35arb. Three parallel samples were set up for each group of experiments.

[0114] Depend on Fig.13 (a) It can be seen that the PCA graph of hawthorn pulp before and after fermentation shows that the points of hawthorn pulp before and after fermentation are relatively scattered, indicating that fermentation will have a significant impact on the composition of hawthorn pulp. Fig.13 (b) As can be seen from the volcano plot, the differential metabolites are widely distributed on the horizontal axis, indicating that fermentation gives hawthorn pulp rich metabolic characteristics. Fig.13 (c) It can be seen that a total of 26 phenolic compounds were identified as key differential metabolites, of which 18 compounds were upregulated, mainly including free flavonoids and phenolic acids, and 8 compounds were downregulated, mainly including polymers and bound polyphenols. KEGG pathway enrichment analysis showed that the main metabolic pathway enriched in the fermentation group was the flavonoid biosynthesis pathway compared with the non-fermented group. Metabolome analysis showed that 8 differential metabolites existed in the flavonoid biosynthesis pathway, including phenylalanine, naringenin, eriodictyol, apigenin, luteolin, triticum tiliaceum, myricetin and dihydromyricetin. Compared with the control group, except for phenylalanine, the other 7 metabolites were significantly upregulated in the fermented hawthorn pulp (p<0.05). This indicates that the activity of phenylalanine ammonia lyase may be increased during the fermentation of Lactobacillus rhamnosus, so that phenylalanine is metabolized to trans-cinnamic acid, thereby entering the flavonoid production pathway and causing the conversion of phenolic compounds. At the same time, the results of the metabolomics group showed that compared with the control group, the relative contents of myricetin 3-O-robinoside, apigenin 7-O-glucoside and phloridzin were down-regulated, and the relative contents of their corresponding aglycone forms, including myricetin, apigenin and phloridzin, were up-regulated. After fermentation, the relative content of catechin gallate was significantly down-regulated, and the relative contents of dimers of catechins and their derivatives, such as prodelphinidin B and proanthocyanidin C1, were significantly down-regulated, while the relative content of catechin was up-regulated. The above results show that the activity of glycosidase and esterase is enhanced during the fermentation process, causing the conversion of macromolecular phenolic compounds to small molecular phenolic compounds, thereby improving the bioavailability of phenolic compounds, making them easier to be digested and absorbed by the human body, and achieving health benefits.

[0115] Compared with the control group, caffeic acid was significantly downregulated (p<0.05). Caffeic acid can be reduced to dihydrocaffeic acid or decarboxylated to other substances such as 4-vinylguaiacol, 4-vinylphenol or 4-vinylcatechol by the action of phenolic acid reductase. At the same time, cryptochlorogenic acid and rosmarinic acid were significantly upregulated in fermented hawthorn pulp (p<0.05), which may be related to caffeic acid metabolism. Caffeic acid and quinine acid are esterified to form cryptochlorogenic acid, and 3,4-dihydroxyphenyllactic acid generated by tyrosine metabolism combines with caffeic acid to form rosmarinic acid. Urolithin C was significantly upregulated (p<0.05), which may be because ellagic acid undergoes lactone ring cleavage, decarboxylation and dehydroxylation under the action of Lactobacillus rhamnosus, thereby converting to urolithin C, which is more easily absorbed by the human body. In short, after fermentation by Lactobacillus rhamnosus, more phenolic metabolites will be produced in hawthorn pulp, thus giving the fermented hawthorn pulp higher biological activity.

[0116] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, can implement the present invention in a wider range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principle of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departing from the disclosed scope in the application and the changes made with conventional techniques known in the art.

Claims

1. A method for preparing a hawthorn pulp fermentation product by increasing the polyphenol content and antioxidant activity of hawthorn pulp by lactic acid bacteria fermentation, characterized in that: The steps include: (1) removing the stems and cores of hawthorn, adding water to crush and pulp, and obtaining hawthorn pulp; (2) adjusting the pH value of the hawthorn pulp, adding a carbon source and a nitrogen source, and sterilizing the mixture to obtain a hawthorn pulp formulation; (3) Inoculating lactic acid bacteria into the hawthorn pulp formulation for fermentation, and obtaining the hawthorn pulp fermentation product with high polyphenol content and high antioxidant activity after fermentation.

2. The method for preparing hawthorn pulp fermentation products by increasing the polyphenol content and antioxidant activity of hawthorn pulp by lactic acid bacteria fermentation according to claim 1, characterized in that: In the crushing and pulping step, the solid-liquid ratio of hawthorn to water is 1:(2-6).

3. The method for preparing a hawthorn pulp fermentation product by increasing the polyphenol content and antioxidant activity of hawthorn pulp by lactic acid bacteria fermentation according to any one of claims 1 to 2, characterized in that: Step (1) also includes the step of enzymatically hydrolyzing the slurry obtained by crushing and beating; preferably, the enzymatic hydrolysis step includes: adding 0.5 g / kg pectinase to the slurry and enzymatically hydrolyzing it at 50° C. for 2 hours.

4. The method for preparing a hawthorn pulp fermentation product by increasing the polyphenol content and antioxidant activity of hawthorn pulp by lactic acid bacteria fermentation according to any one of claims 1 to 3, characterized in that: In the step of adjusting the pH value of the hawthorn pulp, the pH value is adjusted to 5.0-7.0; and / or, In the step of adjusting the pH value of hawthorn pulp, food grade sodium carbonate, sodium bicarbonate or sodium hydroxide is used for adjustment.

5. The method for preparing a hawthorn pulp fermentation product by increasing the polyphenol content and antioxidant activity of hawthorn pulp by lactic acid bacteria fermentation according to any one of claims 1 to 4, characterized in that: The carbon source is glucose, and the amount of glucose added is 2% to 10% based on the mass of the hawthorn pulp after adjusting the pH value; and / or, The nitrogen source is soy protein isolate, and the added amount of the soy protein isolate is 0.1% to 0.5% based on the mass of the hawthorn pulp after adjusting the pH value.

6. The method for preparing a hawthorn pulp fermentation product by increasing the polyphenol content and antioxidant activity of hawthorn pulp by lactic acid bacteria fermentation according to any one of claims 1 to 5, characterized in that: The lactic acid bacteria is lactobacillus, preferably Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus paracasei or Lactobacillus casei; more preferably, the lactic acid bacteria is Lactobacillus rhamnosus, and the inoculation amount of Lactobacillus rhamnosus in the hawthorn pulp formulation is 5×10 5 CFU / mL~6×10 6 CFU / mL.

7. The method for preparing a hawthorn pulp fermentation product by increasing the polyphenol content and antioxidant activity of hawthorn pulp by lactic acid bacteria fermentation according to any one of claims 1 to 6, characterized in that: In the fermentation step, the fermentation temperature is 27°C to 47°C; and / or, In the fermentation step, the fermentation time is 18h to 66h.

8. The method for preparing a hawthorn pulp fermentation product by increasing the polyphenol content and antioxidant activity of hawthorn pulp by lactic acid bacteria fermentation according to any one of claims 1 to 7, characterized in that: In the crushing and pulping step, the solid-liquid ratio of hawthorn to water is 1:3; In the step of adjusting the initial pH value of the hawthorn pulp, the pH value is adjusted to 6.5; The added amount of glucose is 6% of the hawthorn pulp; The added amount of the soy protein isolate is 0.3% of the hawthorn pulp; Based on the initial viable count in the hawthorn pulp formulation, the inoculation amount of Lactobacillus rhamnosus was 4×10 6 CFU / mL; In the fermentation step, the fermentation temperature is 37°C; In the fermentation step, the fermentation time is 48 hours.

9. The method for preparing a hawthorn pulp fermentation product with high polyphenol content and high antioxidant activity by lactic acid bacteria fermentation according to claims 1-8, characterized in that: The effective viable bacteria count in the hawthorn pulp fermentation product is higher than 8×10 8 CFU / mL; and / or, Increasing the polyphenol content of hawthorn pulp includes increasing the free polyphenol content, bound polyphenol content and total phenol content of hawthorn pulp; and / or, The improvement of the polyphenol content of hawthorn pulp is achieved by mediating the flavonoid biosynthesis pathway through the action of lactic acid bacteria, while promoting the conversion of macromolecular phenolic compounds into small molecular phenolic compounds; and / or, The improvement of antioxidant activity is reflected in at least one of improving DPPH scavenging rate, hydroxyl radical scavenging rate and FRAP.

10. A hawthorn pulp fermentation product prepared by the method according to any one of claims 1 to 9.

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