Application of polyphenol thallus leavening agent and modifier thereof in acidic fruit juice

Abstract

The invention provides a polyphenol thallus leavening agent for improving the antioxidant activity of acidic fruit juice, and belongs to the technical field of microbial fermentation. Lactic acid bacteria are wrapped by a network structure generated after metal polyphenol reaction to form a polyphenol thallus fermentation agent, and polysaccharide substances are adopted for modification to form a polyphenol thallus fermentation agent modifier. The polyphenol thallus leavening agent and the modifier thereof are used for fermenting the acidic fruit juice, so that a good protection effect on lactic acid bacteria is achieved, the viability of the lactic acid bacteria is enhanced, and the viable count in the acidic fruit juice is remarkably increased; key nutritional ingredients such as total phenols and anthocyanin in the acidic fruit juice are also maintained; the oxidation resistance of the acidic fruit juice is improved, and the quality of the fruit juice is effectively improved. Therefore, the fermentation method has important significance for enhancing the flavor, the nutritional value and the physiological function of the acidic fruit juice.

Description

Technical Field

[0001] The invention relates to the field of food science and biotechnology, in particular to a composite fermentation agent capable of improving the antioxidant capacity of fruit juice rich in organic acids and phenols and application thereof. Background Art

[0002] As a natural healthy drink, fruit juice is rich in organic acids, polyphenols, vitamins, minerals and antioxidants, which have many benefits to human health, such as anti-oxidation, anti-aging, immunity enhancement and metabolism regulation. However, during the production, processing and storage of fruit juice, its quality is easily degraded due to oxidation reactions, and its antioxidant capacity is weakened, which not only affects the taste and color of the juice, but also reduces its nutritional value. In order to solve this problem, the traditional method is usually to delay the oxidation process of the juice by adding antioxidants, but these antioxidants are often artificially synthesized and have certain safety risks. Therefore, it is particularly important to develop a technology that can effectively improve the antioxidant capacity of fruit juice and is safe and harmless.

[0003] In recent years, the research on improving food quality and functionality by using microbial fermentation technology has been increasing. Lactic acid bacteria fermentation has the characteristics of safety and efficiency and has attracted wide attention. As a common probiotic, lactic acid bacteria exist in fermented dairy products, sourdough bread, fruits, and the gastrointestinal tract of mammals. Lactic acid bacteria not only have good fermentation performance, can effectively improve the composition and stability of active ingredients in juice, but also can produce a variety of metabolites that are beneficial to the human body, can improve intestinal microorganisms, regulate the immune system, anti-oxidation, anti-cancer, anti-obesity and other health-promoting effects, which is conducive to improving the added value of juice. However, many lactic acid bacteria do not form spores and have poor stress resistance. For juice rich in organic acids and polyphenols, directly adding them to such juice for fermentation will be affected by organic acids and phenols, inhibiting the growth activity of lactic acid bacteria and affecting the fermentation process, thereby limiting the use of lactic acid bacteria in juice rich in organic acids and phenols. Therefore, traditional lactic acid bacteria fermentation technology still has limitations in improving the antioxidant capacity of juice, and there is a lack of methods that can protect lactic acid bacteria from effective fermentation in juice rich in organic acids and phenols, which requires further exploration and optimization.

[0004] Metal-polyphenol network (MPN) is formed by chelating polyphenols with metal ions to form films or coatings of different shapes and functions, and is used in drug delivery, biomedicine, sewage purification, and food industry. After the probiotics are wrapped with the metal-polyphenol network coating, they can help the probiotics resist the harsh physiological environment of the gastrointestinal tract, thereby improving the stability of the probiotics. Therefore, using the metal-polyphenol coating as a protective agent to form a protective film on the surface of the bacteria to improve the stability of the bacteria provides a new idea for the direction of bacteria encapsulation. Summary of the invention

[0005] The present invention aims to solve the problem that high organic acid and polyphenol in fruit juice easily inhibit lactic acid bacteria fermentation, and provides a polyphenol bacterial fermentation agent and its modified product which can protect lactic acid bacteria to effectively ferment in fruit juice rich in organic acid and phenolic substances, and an application method thereof for improving the antioxidant capacity of fermented fruit juice.

[0006] A polyphenol bacterial fermentation agent and its modified product, and its application in improving the antioxidant capacity of fruit juice rich in organic acids and phenols, characterized in that:

[0007] (1) The fruit juice rich in organic acids and phenols is characterized in that the fruit juice is obtained by discarding the pomace sediment after squeezing the fruit and extracting the supernatant juice, wherein the pH range of the clear juice is 2.0 to 6.5, and specific types of fruit juice include but are not limited to blackberry juice, blueberry juice, strawberry juice, grape juice and wolfberry juice.

[0008] (2) The polyphenol bacterial fermentation agent described herein mainly comprises lactic acid bacteria, specifically classified and named as Lactobacillus plantrum, with strain number FM-LP-BC17, which was deposited in the General Microbiology Center of the China National Microbiological Culture Collection on August 29, 2022, with a deposit number of CGMCC No. 25603; Lactobacillus paracasei, with strain number FM-LP-PC18, which was deposited in the General Microbiology Center of the China National Microbiological Culture Collection on August 29, 2022, with a deposit number of CGMCC No. 25604.

[0009] (3) The polyphenol bacterial fermentation agent is characterized in that the lactic acid bacteria are wrapped by a metal polyphenol network structure, wherein the polyphenols include proanthocyanidins, caffeic acid, quercetin, ellagic acid and resveratrol; and the metals include iron ions, magnesium ions, calcium ions, manganese ions, aluminum ions and zinc ions.

[0010] (4) The modified substance is characterized in that the polyphenol bacterial fermentation agent is modified by using sodium heparin, chondroitin sulfate, β-cyclodextrin, blackberry polysaccharide and blueberry polysaccharide as modified substances.

[0011] 2. The preparation of the polyphenol bacterial fermentation agent according to claim 1, characterized in that:

[0012] (1) The metal concentration range is 1 mM to 10 mM, and the polyphenol concentration range is 1 mM to 10 mM;

[0013] (2) The addition ratio of polyphenols to metals is 1:10-10:1;

[0014] (3) The ratio of metal polyphenol network structure (in terms of polyphenol concentration) to lactic acid bacteria is 1 mM: 6 Log CFU / mL to 5 mM: 10 Log CFU / mL;

[0015] (4) The concentration range of its modifier is 1-5 mM.

[0016] 3. A method for improving the antioxidant capacity of fruit juice rich in organic acids and phenols by using the polyphenol bacterial fermentation agent and its modified product according to claim 1, characterized in that it comprises the following steps:

[0017] (1) Activating the Lactobacillus plantarum FM-LP-BC17 strain, the method comprising: thawing the Lactobacillus plantarum FM-LP-BC17 strain frozen at -20°C or -80°C at room temperature, inoculating it into MRS culture medium at a 1-5% v / v inoculation amount, and culturing and activating it at 36-38°C for 40-54 hours.

[0018] (2) The activated bacteria in step (1) are inoculated into a new MRS culture medium to obtain a bacterial solution, wherein the inoculation amount is 1-5% v / v and the culture conditions are: culture at 36-38° C. for 40-54 hours.

[0019] (3) The bacterial liquid obtained in step (2) is inoculated into the fruit juice pretreated with cold sterilization using the polyphenol bacterial fermentation agent and its modified product described in claim 1 for fermentation, and then centrifuged to obtain the fermented juice, wherein the inoculation amount of the fermentation agent is 3% v / v, and the fermentation conditions are: fermentation at 37° C. for 40-54 hours.

[0020] 4. The application effect of the polyphenol bacterial fermentation agent and its modified products as described in claim 1 in fruit juice rich in organic acids and phenols, characterized in that the antioxidant capacity of the fruit juice fermented with the fermentation agent and its modified products is at least 10% higher than that of the fermentation liquid of unprotected strains.

[0021] 5. The application effect of the polyphenol bacterial fermentation agent and its modified products as described in claim 1 in specific fruit juices is characterized in that when the fermentation agent and its modified products are used to ferment fruit juice containing anthocyanins, the stability of anthocyanins can be maintained, and the anthocyanin content in the fruit juice after fermentation is at least 20% higher than that of the fermentation liquid of unprotected strains.

[0022] Compared with the prior art, the advantages of the present invention are as follows:

[0023] 1. The polyphenol bacterial fermentation agent prepared by the present invention and its modified products can be used for the fermentation of acidic fruit juice.

[0024] 2. The polyphenol bacterial fermentation agent prepared by the present invention and its modified products can increase the antioxidant level by at least 10% during the acidic fruit juice fermentation process.

[0025] 3. The polyphenol bacterial fermentation agent prepared by the present invention and its modified product can alleviate the massive degradation of anthocyanins during the fermentation process of anthocyanin-containing juice, and the content of anthocyanin juice is at least 20% higher than that of the fermentation liquid of unprotected strains.

[0026] In summary, the present invention effectively solves the technical problem of improving the antioxidant capacity of fruit juice through the unique design of polyphenol bacterial fermentation agent and its modified products and its application method, and provides a new technical means for product innovation and quality upgrade of the fruit juice industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and other advantages of the present invention will become more clear.

[0028] Figure 1 Preparation of polyphenol bacterial fermentation agents containing different polyphenols

[0029] Figure 2 Growth curves of polyphenol bacterial fermentation cultures with different polyphenol species

[0030] Figure 3 The number of viable bacteria in blackberry juice fermented by polyphenol bacterial culture and its modifications

[0031] Figure 4 Reducing sugar content of blackberry juice fermented by polyphenol bacterial starter and its modified products

[0032] Figure 5 Anthocyanin content in blackberry juice fermented by polyphenol bacterial starter culture and its modified products

[0033] Figure 6 DPPH scavenging rate of blackberry juice fermented by polyphenol bacterial culture and its modified products DETAILED DESCRIPTION

[0034] The acidic juice fermented by the present invention takes blackberry juice as an example, and the fermentation agent used is Lactobacillus plantarum BC17 (strain number FM-LP-BC17, deposited in the General Microbiological Culture Collection Management Center on August 29, 2022, with a deposit number of CGMCC No. 25603) and Lactobacillus paracasei PC18 (strain number FM-LP-PC18, deposited in the General Microbiological Culture Collection Management Center on August 29, 2022, with a deposit number of CGMCC No. 25604) as an example. According to the following examples, the present invention can be better understood, and the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0035] Example 1 Preparation of polyphenol bacterial fermentation agents of different polyphenol types and determination of growth activity

[0036] A solution of proanthocyanidins (Pc), resveratrol (Re), caffeic acid (Ca) and ellagic acid (EA) with a concentration of 3 mM and a 2.5 mM FeCl3 solution are prepared and mixed in a ratio of 1:1 (v:v). The solution is a metal polyphenol solution.

[0037] Inoculate the frozen BC17 into MRS liquid medium at a 3% inoculation volume, culture at 37°C for 48 hours, use an inoculation loop to take liquid and streak on MRS solid medium, culture at 37°C for 48 hours, pick a typical single colony and inoculate it into fresh MRS liquid medium for 24 hours, repeat this three times to obtain the bacterial solution, which is the working solution. 600 =1.3) Place in a centrifuge at 5000 rpm for 10 min, use 0.1 M PBS to wash off the culture medium on the surface of the bacteria. Use the prepared metal polyphenol solution to resuspend the washed bacterial sludge, incubate for 10 min, centrifuge at 5000 rpm for 10 min, discard the supernatant, resuspend with 0.1 M PBS and mix evenly to obtain the polyphenol bacterial fermentation agent.

[0038] Different polyphenol bacterial starter cultures were added to MRS fermentation medium at an inoculation ratio of 3% and fermented at 37°C for 48 h. Samples were taken at 0, 2, 4, 6, 8, 10, 12, 24, 36 and 48 h, and the number of viable bacteria in the fermentation broth was determined to draw a growth curve.

[0039] The results showed that after adding metal polyphenol solution and centrifuging, the supernatant was basically clear and the bacteria in the precipitate were colored, indicating that the metal polyphenol network structure wrapped the bacteria and formed a polyphenol bacteria fermentation agent that can be used for fermentation (attached Figure 1 ). By detecting the growth curve of polyphenol fermentation, it can be found that different polyphenols have no inhibitory effect on the growth of lactic acid bacteria. Metal polyphenols within this range of conditions are safe for lactic acid bacteria (see Appendix Figure 2 ).

[0040] Example 2 Characterization of polyphenol bacterial fermentation agent and its modified products

[0041] 3mM heparin sodium (Hs) and β-cyclodextrin (CD) were added to the polyphenol bacterial fermentation medium, incubated at room temperature for 30 minutes, centrifuged at 5000rpm for 10 minutes and the supernatant was discarded to form BC17@Pc-Fe@Hs and BC17@Pc-Fe@CD polyphenol bacterial fermentation medium modifications, respectively. The polyphenol bacterial fermentation medium and its modifications were diluted to a suitable concentration with PBS, and the particle size and potential of the bacteria were analyzed using a laser particle size analyzer.

[0042] The results of particle size and potential were: BC17 particle size: 2.46±0.19μm, potential: -13.52±1.18mV; BC17@Pc-Fe particle size: 8.83±0.90μm, potential: -4.09±1.21mV; BC17@Pc-Fe@Hs particle size: 10.73±0.76μm, potential: -3.06±0.51mV; and BC17@Pc-Fe@CD particle size: 11.01±0.39μm, potential: -6.28±1.54mV. The increase in particle size and the change in potential indicate that the polyphenol bacterial fermentation agent was prepared and modified.

[0043] Example 3 Protective effect of polyphenol bacterial fermentation agent and its modified products on its acidity

[0044] According to the 2020 edition of the Chinese Pharmacopoeia, take 10.0g of pepsin and 16.4mL of dilute hydrochloric acid, add ultrapure to 1000mL to obtain simulated gastric fluid (SGF). According to the 2020 edition of the Chinese Pharmacopoeia, use a balance to weigh 0.3g of pig bile salt, add ultrapure to 1000mL to obtain simulated bile salt. The prepared BC17@Pc-Fe, BC17@Pc-Fe@Hs, PC18@Pc-Fe@Hs and PC18@Pc-Fe were resuspended in simulated gastric fluid and bile salts, with BC17 and PC18 as controls, respectively. Samples were taken after 2h and 4h at room temperature, and the number of viable bacteria was detected by plate counting method.

[0045] The results of acid resistance and bile salt resistance are shown in Table 1 and Table 2 respectively:

[0046] Table 1 Protective effect of polyphenol bacterial fermentation agent and its modified products on bacterial acidity

[0047]

[0048] Table 2 Protective effect of polyphenol bacterial fermentation agent and its modified products on bacterial bile salt

[0049]

[0050] Example 4 Effect of polyphenol bacterial fermentation agent and its modified products on blackberry juice fermentation

[0051] The blackberries were thawed at room temperature, washed and cleaned, drained and crushed, centrifuged at 5000rpm for 20min, and the precipitate was removed to obtain blackberry juice, which was sterilized at 85°C for 20min. After cooling, 3% volume fraction of BC17, PC18, BC17@Pc-Fe, PC18@Pc-Fe, BC17@Pc-Fe@Hs and BC17@Pc-Fe@CD inoculation liquid was added, and fermented at 37°C for 48h. Samples were taken at 0, 2, 4, 6, 12, 24, 36 and 48h, and the number of viable bacteria in the fermented blackberry juice was determined by the plate count method.

[0052] The research results are attached Figure 3 During the fermentation of blackberry juice, the number of live bacteria showed an obvious upward trend within 2-12 hours. At 12 hours of fermentation, the number of live bacteria in the blackberry juice fermented by the polyphenol bacteria fermentation agent (BC17@Pc-Fe) reached 8.65±0.031logCFU / mL. There was no significant difference in the number of live bacteria in the fermentation process between the polyphenol bacteria fermentation agent (BC17@Pc-Fe@Hs) modified with sodium heparin and BC17@Pc-Fe, and the growth curve trend was basically the same; while the highest number of live bacteria in the fermentation of blackberry juice directly fermented with lactic acid bacteria (BC17) was only 7.55±0.050logCFU / mL, and the number of live bacteria decreased rapidly in the later stage of fermentation.

[0053] Example 5 Detection of reducing sugar and anthocyanin contents in blackberry juice fermented by polyphenol bacterial fermentation agent

[0054] (1) Reducing sugar content detection

[0055] The reducing sugar content in the sample was determined by 3,5-dinitrosalicylic acid (DNS) method using anhydrous glucose as the standard substance. Accurately weigh 0.1g of anhydrous glucose standard, dissolve it in water and dilute to 10mL. Use this solution as the stock solution to prepare a gradient solution of 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1mg / mL. Take 0.5mL of gradient solution, add 0.5mL DNS, boil it in a boiling water bath for 5min, cool it down, add water to 5mL, and then measure OD540nm. When measuring the sample, dilute the sample 100 times first, and the rest of the operation is the same as above. Draw a glucose standard curve with anhydrous glucose mass concentration (x) as the horizontal axis and absorbance value (y) as the vertical axis. The regression equation of the standard curve is y=0.161x-0.1222, R2=0.9991.

[0056] (2) Anthocyanin content detection

[0057] Take 2 portions (0.1 mL) of the fermented blackberry juice to be tested, add 0.2 mol / L KCl (pH 1.0) and 0.9 mL of 0.2 mol / L sodium acetate (pH 4.5), respectively. After reacting at 4 °C for 2 h, measure the absorbance of the samples at 510 and 700 nm, respectively, and calculate the anthocyanin content using the following formula.

[0058] Anthocyanin content = ΔA × Wm × D × 1000 / ε

[0059] Where: ΔA is (A 510 -A 700 )pH 1.0-(A 510 -A 700 )pH 4.5, mg / L; Wm is the molecular weight of cyanidin-3-glucoside 449.2; D is the dilution factor; ε is the extinction coefficient of cyanidin-3-glucoside (26900).

[0060] The results of reducing sugar and anthocyanin in blackberry fermented juice are shown in the attached Figure 4 and 5 The highest reducing sugar contents of blackberry juice fermented with BC17@Pc-Fe, BC17@Pc-Fe@Hs and BC17@Pc-Fe@CD were 28.09±0.098g / L, 26.40±0.52g / L and 22.63±1.12g / L, respectively, which were significantly higher than the reducing sugar content of BC17 fermentation (20.74±0.47g / L). During the whole fermentation process, the highest anthocyanin contents of blackberry juice fermented with BC17@Pc-Fe and BC17@Pc-Fe@Hs were 34.31±1.77g / L and 23.38±4.49g / L, respectively, which were significantly higher than the anthocyanin content of blackberry juice fermented with BC17.

[0061] Example 6 Antioxidant Capacity Detection of Blackberry Fermented Juice

[0062] DPPH free radical scavenging rate determination

[0063] Dilute the fermented blackberry juice until it is colorless, put 0.5 mL of the dilution in a test tube, add 1 mL of 0.2 mmol / mL DPPH solution, add anhydrous ethanol to 2 mL, shake well and react in a dark place for 30 minutes, measure the absorbance of the sample at 517 nm, and calculate the DPPH free radical scavenging rate using the following formula.

[0064] DPPH clearance rate (%) = 1-A j / A0×100%

[0065] Where: A j is the absorbance of sample + DPPH solution; A0 is the absorbance of DPPH solution + anhydrous ethanol.

[0066] After DPPH free radical scavenging capacity determination (attached Figure 6 ), the antioxidant capacity of blackberry juice fermented with a composite fermentation agent is at least 10% higher than that of blackberry juice directly fermented with an unprotected strain.

[0067] The present invention provides a preparation of a composite leavening agent and its application in acidic juice to improve the antioxidant capacity of fermented juice. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A polyphenol bacterial fermentation agent and its modified product, and its application in fruit juice rich in organic acids and phenols, characterized in that: (1) The fruit juice rich in organic acids and phenols is characterized in that the fruit juice is obtained by discarding the pomace sediment after squeezing the fruit juice and extracting the supernatant juice, wherein the pH range of the clear juice is 2.0 to 6.5, and the specific types of fruit juice may include but are not limited to blackberry juice, blueberry juice, strawberry juice, grape juice and wolfberry juice. (2) The polyphenol bacterial fermentation agent described herein mainly comprises lactic acid bacteria, specifically classified and named as Lactobacillus plantrum, with strain number FM-LP-BC17, deposited in the China General Microbiological Culture Collection on August 29, 2022, with a deposit number of CGMCC No. 25603; Lactobacillus paracasei, with strain number FM-LP-PC18, deposited in the China General Microbiological Culture Collection on August 29, 2022, with a deposit number of CGMCC No. 25604. (3) The polyphenol bacterial fermentation agent is characterized in that the lactic acid bacteria are wrapped by a metal polyphenol network structure, wherein the polyphenols include proanthocyanidins, caffeic acid, quercetin, ellagic acid and resveratrol; and the metals include iron ions, magnesium ions, calcium ions, manganese ions, aluminum ions and zinc ions. (4) The modified substance is characterized in that the polyphenol bacterial fermentation agent is modified by using sodium heparin, chondroitin sulfate, β-cyclodextrin, blackberry polysaccharide and blueberry polysaccharide as modified substances.

2. The preparation of the polyphenol bacterial fermentation agent according to claim 1, characterized in that: (1) The metal concentration range is 1 mM to 10 mM, and the polyphenol concentration range is 1 mM to 10 mM; (2) The addition ratio of polyphenols to metals is 1:10-10:1; (3) The ratio of metal polyphenol network structure (in terms of polyphenol concentration) to lactic acid bacteria is 1 mM: 6 Log CFU / mL to 5 mM: 10 Log CFU / mL; (4) The concentration range of its modifier is 1-5 mM.

3. A method for improving the antioxidant capacity of fruit juice rich in organic acids and phenols by using the polyphenol bacterial fermentation agent and its modified product according to claim 1, characterized in that The steps include: (1) Activating the Lactobacillus plantarum FM-LP-BC17 strain, the method comprising: thawing the Lactobacillus plantarum FM-LP-BC17 strain frozen at -20°C or -80°C at room temperature, inoculating it into MRS culture medium at a 1-5% v / v inoculation amount, and culturing and activating it at 36-38°C for 40-54 hours. (2) The activated bacteria in step (1) are inoculated into a new MRS culture medium to obtain a bacterial solution, wherein the inoculation amount is 1-5% v / v and the culture conditions are: culture at 36-38° C. for 40-54 hours. (3) The bacterial liquid obtained in step (2) is inoculated into the fruit juice pretreated with cold sterilization using the polyphenol bacterial fermentation agent and its modified product described in claim 1 for fermentation, and then centrifuged to obtain the fermented juice, wherein the inoculation amount of the fermentation agent is 3% v / v, and the fermentation conditions are: fermentation at 37° C. for 40-54 hours.

4. The application effect of the polyphenol bacterial fermentation agent and its modified products as claimed in claim 1 in fruit juice rich in organic acids and phenols is characterized in that The antioxidant capacity of the juice fermented by using the starter and its modified product is at least 10% higher than that of the fermentation liquid of the unprotected strain.

5. The application effect of the polyphenol bacterial fermentation agent and its modified product in specific fruit juice according to claim 1 is characterized in that When the fermentation agent and its modified product are used to ferment anthocyanin-containing juice, the stability of anthocyanin can be maintained, and the anthocyanin content in the fermented juice is at least 20% higher than that of the fermentation liquid of the unprotected strain.

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