Preparation method of probiotic fermented fruit juice beverage

Abstract

A preparation method of a probiotic fermented fruit juice beverage belongs to the technical field of food fermentation processing, and comprises the following steps: peeling and denucleating fruits, crushing, adding ascorbic acid, homogenizing, filtering and sterilizing to obtain fruit juice; the fruit juice is subjected to enzymolysis treatment, fructo-oligosaccharide is added, lactobacillus paracasei S-NB is used for fermentation, the pH value is dynamically adjusted in the fermentation process so that the pH value can be larger than 4, and fermented fruit juice is obtained; the fermented fruit juice is subjected to centrifugal treatment, supernate is taken to be subjected to ultrafiltration concentration, and the probiotic fermented fruit juice beverage is obtained through ultrahigh pressure treatment and sterile cold filling. Wherein the lactobacillus paracasei S-NB is preserved in the China Center for Type Culture Collection, and the preservation number of the lactobacillus paracasei S-NB is CCTCC M2021461. The probiotic fermented fruit juice beverage prepared by the method disclosed by the invention has relatively high EPS content and probiotic survival rate.

Description

Technical Field

[0001] The invention belongs to the technical field of food fermentation processing, and particularly relates to a method for preparing a probiotic fermented fruit juice beverage. Background Art

[0002] Fruits and vegetables, as essential foods for human life, are rich in carbohydrates, proteins, minerals, vitamins, and other nutrients, providing the body with the energy it needs to maintain normal life. However, because fruits and vegetables are rich in water and nutrients, these conditions easily promote the growth and reproduction of various microorganisms, leading to fruit and vegetable rot. This characteristic makes it more difficult to maintain the good quality of fruits and vegetables during long-term storage and transportation, resulting in a significant waste of fruit and vegetable resources.

[0003] Fermentation, as a processing method, is widely used in the production and processing of fruit and vegetable juices. Fermented fruit and vegetable juices not only have excellent antibacterial effects but also maintain good sensory quality, enhancing the product's market competitiveness. Furthermore, fermentation technology offers environmental and energy-saving advantages, reducing pollutant emissions during food processing and improving energy efficiency, thereby enhancing the overall efficiency of the food industry.

[0004] In the existing technology, fruit and vegetable juice fermented by lactic acid bacteria has shown significant advantages in improving the nutrition and functionality of juice and is widely cited in the field of juice fermentation. However, lactic acid bacteria fermentation still has many problems, such as: low survival rate of probiotics in fruit and vegetable juice fermented beverages, insufficient production of extracellular polysaccharides (EPS) (usually <200 mg / L), easy polyphenol oxidation leading to severe browning, and sour taste of the fermented product requiring additional acidification. Summary of the Invention

[0005] In view of the above situation, in order to overcome at least some of the defects of the above-mentioned prior art, the present invention provides a method for preparing a probiotic fermented fruit juice beverage.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The present invention provides a method for preparing a probiotic fermented fruit juice beverage, comprising: The fruit is peeled and cored, crushed, and ascorbic acid is added, followed by homogenization, filtration, and sterilization to obtain juice; The juice is subjected to enzymatic hydrolysis, oligofructose is added, and fermented using Lactobacillus paracasei S-NB, and the pH value is dynamically adjusted during the fermentation process to be greater than 4, to obtain fermented juice; The fermented juice is centrifuged, the supernatant is ultrafiltered and concentrated, and then subjected to ultrahigh pressure treatment and aseptic cold filling to obtain a probiotic fermented juice beverage; Among them, the classification name of the Lactobacillus paracasei S-NB is Lactobacillus paracasei S-NB, which is preserved in the China Center for Type Culture Collection with the preservation number CCTCC M2021461, the preservation date is April 27, 2021, and the preservation address is Wuhan University, Wuhan, China.

[0007] In some embodiments, the amount of ascorbic acid added is 0.45% (v / v)-0.6% (v / v).

[0008] In some embodiments, the homogenization process is performed at a pressure of 20-30 MPa, a temperature of 60-70° C., and is repeated 2-3 times.

[0009] In some embodiments, in the enzymatic hydrolysis treatment, pectinase and cellulase are used for enzymatic hydrolysis, the enzymatic hydrolysis temperature is 43-47° C., the time is 2-3 h, wherein the amount of the pectinase added is 0.03% (w / v), and the amount of the cellulase added is 0.01% (w / v); And / or, an enzyme inactivation treatment is performed after the enzymatic hydrolysis treatment, the time of the enzyme inactivation treatment is 5-8 minutes, and the temperature is 85-90°C.

[0010] In some embodiments, the added amount of the oligofructose is 0.5% (w / v)-2% (w / v).

[0011] In some embodiments, the fermentation includes primary fermentation and secondary fermentation, wherein the primary fermentation includes: inoculating 1% (v / v)-3% (v / v) of Lactobacillus paracasei S-NB strains, and fermenting at a constant temperature of 36-38° C. for 2-6 hours, and the secondary fermentation includes: adjusting the pH value to 5.0, gradually cooling to 28-32° C., and fermenting for 12-18 hours; Wherein, the number of viable bacteria in the Lactobacillus paracasei S-NB strain is ≥1×10 9 CFU / mL.

[0012] In some embodiments, the dynamic adjustment of pH value includes: monitoring pH value in real time during the fermentation process, and automatically adding 0.5 mol / L NaOH solution when the pH value is less than 4.0.

[0013] In some embodiments, the centrifugation treatment uses low-temperature centrifugation, with a temperature of 3-5°C, a centrifugal force of 7000-9000g, and a time of 10-20 minutes.

[0014] In some embodiments, the ultrafiltration concentration uses ultrafiltration using a 10 kDa membrane package.

[0015] In some embodiments, the ultrahigh pressure treatment is performed at a pressure of 400-500 MPa and for a time of 4-6 minutes.

[0016] The beneficial effects achieved by the present invention are as follows: Lactobacillus paracasei S-NB was used for juice fermentation, and oligofructose was added to promote the synthesis of extracellular polysaccharides (EPS), which increased the EPS synthesis amount by 143%. EPS has good compatibility and adaptability with Lactobacillus paracasei S-NB, and can be used as a prebiotic to promote the proliferation of Lactobacillus paracasei S-NB. At the same time, EPS has significant biofilm formation ability and can improve the survival rate of Lactobacillus paracasei S-NB.

[0017] By adding ascorbic acid to the juice before fermentation, browning caused by polyphenol oxidation can be prevented. Moreover, by dynamically adjusting the pH value during the fermentation process, the pH value can be kept in a range suitable for Lactobacillus paracasei S-NB fermentation, thereby promoting the synthesis of extracellular polysaccharides (EPS). DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only and are not intended to limit the scope of this application.

[0020] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0021] Lactobacillus paracasei is a facultative anaerobic, homofermentative, Gram-positive lactic acid bacterium with benefits such as inhibiting the growth and reproduction of enteric pathogens, lowering cholesterol, combating hypertension, preventing diabetes, and alleviating obesity. Lactobacillus paracasei S-NB (L. paracasei S-NB), isolated from Xinjiang yogurt, exhibits excellent exopolysaccharide formation. Furthermore, this strain partially converts dietary fiber into active oligosaccharides, resulting in products with high viscosity and high quality without the addition of food additives or additional nutrients. Therefore, utilizing L. paracasei S-NB to ferment fruit juice is highly desirable to enhance the economic value of fruit and to develop a healthy, safe, and probiotic-fermented fruit juice beverage with excellent fermentation performance, high yield of active polysaccharides, and a high yield of active polysaccharides.

[0022] An embodiment of the present invention provides a method for preparing a probiotic fermented fruit juice beverage, comprising: After peeling and core-removing the fruit, it is crushed and ascorbic acid is added. The fruit is then homogenized, filtered, and sterilized to produce the juice. The crushing process mechanically breaks down the fruit cell walls to produce pulp, releasing the juice and increasing the yield. Adding ascorbic acid during the crushing process prevents browning caused by oxidation of polyphenols in the fruit. A high-pressure homogenizer is used for homogenization, which uses high pressure and shear forces to refine the particles in the pulp, promoting pectin exudation and allowing the pectin to fully combine with water to produce the juice. Filtration removes residual impurities in the juice and prevents precipitation. Sterilization removes bacteria from the juice, increasing its shelf life. The fruit is preferably apple or pear, which are rich in nutrients and highly adaptable to processing, making them suitable for preparing fermented juice. Preferably, the pear is of the Qiuyue or Xuehua varieties.

[0023] The juice is enzymatically hydrolyzed, oligofructose is added, and fermented using Lactobacillus paracasei S-NB. During the fermentation process, the pH value is dynamically adjusted to a value greater than 4 to obtain the fermented juice. Since pectin and cellulose are the main components of plant cell walls, they naturally hinder the release of juice. Enzymatic hydrolysis degrades pectin and cellulose, breaking down the dense structure of the cell wall and allowing the intracellular fluid to flow out more easily, thereby increasing the juice yield. At the same time, enzymatic hydrolysis breaks the pectin molecular chains, reducing the viscosity of the juice, enhancing its fluidity, and evenly dispersing the pulp particles to form a stable suspension system. Fermentation can convert the sugars in the juice into bioactive substances such as lactic acid, short-chain fatty acids, and peptides, giving the juice a soft fruity and frankincense aroma, forming the unique flavor of the fermented beverage, and promoting the hydrolysis of glycosidic bonds of phenolic substances, releasing free phenols and enhancing antioxidant activity. Using Lactobacillus paracasei S-NB as a strain can increase the production of extracellular polysaccharides (EPS). Furthermore, adding oligofructose as a carbon source before fermentation can promote the growth of S-NB, further increasing EPS production. EPS has good compatibility and adaptability with S-NB, acting as a prebiotic to promote its proliferation. Furthermore, EPS has significant biofilm-forming ability, which can enhance the survival rate of S-NB. Furthermore, fermented juice carrying active S-NB can enter the human intestine, where it has the potential to inhibit the growth and reproduction of intestinal pathogens, lower cholesterol, combat hypertension, prevent diabetes, and improve obesity. Furthermore, by dynamically adjusting the pH during fermentation, the pH can be maintained within the optimal range for S-NB fermentation, thereby promoting EPS production and increasing EPS production. Among them, the classification name of Lactobacillus paracasei S-NB is Lactobacillus paracasei S-NB, preserved in the China Center for Type Culture Collection, with the preservation number CCTCC M2021461, the preservation date is April 27, 2021, and the preservation address is Wuhan University, Wuhan, China.

[0024] The fermented juice is centrifuged, and the supernatant is ultrafiltered and concentrated. After ultrahigh pressure treatment and aseptic cold filling, the probiotic fermented juice beverage is obtained. Centrifugation separates impurities and bacteria, improving the purity of the juice. Ultrafiltration and concentration selectively intercept large EPS molecules, thereby increasing the EPS concentration in the juice. Ultrahigh pressure treatment destroys the cell membrane structure of harmful microorganisms by applying high pressure. Lactobacillus paracasei S-NB has a stronger ability to withstand pressure and survives, thus achieving selective sterilization.

[0025] In some embodiments, the amount of ascorbic acid added is 0.45% (v / v)-0.6% (v / v). The amount of ascorbic acid added affects the antioxidant effect of the juice. If the amount of ascorbic acid added is too little, the antioxidant effect of the juice will be poorer, causing the juice to produce browning. If the amount of ascorbic acid added is too much, it will affect the fermentation effect of Lactobacillus paracasei S-NB and reduce the synthesis of EPS. Therefore, by setting the amount of ascorbic acid added to 0.45% (v / v)-0.6% (v / v), the juice can have a good antioxidant effect, avoid browning of the juice, and at the same time, the fermentation effect of Lactobacillus paracasei S-NB can be better, thereby increasing the synthesis of EPS.

[0026] In some embodiments, the homogenization process is performed at a pressure of 20-30 MPa and a temperature of 60-70°C, and is repeated 2-3 times. The pressure and temperature of the homogenization process affect the homogenization effect. Setting the homogenization pressure to 20-30 MPa and the temperature to 60-70°C can improve the homogenization effect. Furthermore, multiple homogenization processes can enhance the homogenization effect, further refine the pulp particles, and improve the clarity of the juice.

[0027] In some embodiments, the sterilization process uses ultra-high temperature instantaneous sterilization at a temperature of 120-130°C for 2-4 seconds. Ultra-high temperature instantaneous sterilization can instantly destroy the cell membrane and nucleic acid structure of microorganisms, and can eliminate more than 99% of bacteria.

[0028] In some embodiments, during the enzymatic hydrolysis treatment, pectinase and cellulase are used for enzymatic hydrolysis, the enzymatic hydrolysis temperature is 43-47°C, and the time is 2-3 hours. The amount of pectinase added is 0.03% (w / v) and the amount of cellulase added is 0.01% (w / v). Pectinase converts insoluble pectin into water-soluble substances by hydrolyzing pectin molecules, effectively destroying the plant cell wall structure, releasing intracellular juice, and increasing the juice yield. Cellulase releases polysaccharides by hydrolyzing cellulose chains, thereby increasing the nutritional value of the juice. By controlling the amount of pectinase and cellulase added, the enzymatic hydrolysis can be sufficient.

[0029] In some embodiments, an enzyme inactivation treatment is performed after the enzymatic hydrolysis treatment, and the enzyme inactivation treatment lasts for 5-8 minutes at a temperature of 85-90° C. The high temperature of 85-90° C. can inactivate pectinase and cellulase, preventing residual pectinase and cellulase from continuing to decompose juice components during subsequent processing or storage, thereby ensuring the nutritional and flavor of the juice.

[0030] In some embodiments, the amount of oligofructose added is 0.5% (w / v)-2% (w / v). The addition of oligofructose as a carbon source can promote the growth and reproduction of Lactobacillus paracasei S-NB during its growth and reproduction phase. At the same time, it can also promote the synthesis of EPS by Lactobacillus paracasei S-NB after the growth and reproduction phase is completed, thereby increasing the synthesis of EPS. Too low an amount of oligofructose added cannot provide a sufficient carbon source, which will reduce the synthesis of EPS. Too much oligofructose added will affect the fermented flavor of the juice.

[0031] In some embodiments, the fermentation includes primary fermentation and secondary fermentation. The primary fermentation includes: inoculating 1% (v / v)-3% (v / v) of Lactobacillus paracasei S-NB strain, and fermenting at a constant temperature of 36-38°C for 2-6 hours. The secondary fermentation includes: adjusting the pH to 5.0, gradually cooling to 28-32°C, and fermenting for 12-18 hours. The number of viable cells in the Lactobacillus paracasei S-NB strain is ≥1×10 9 CFU / mL. During the primary fermentation process, Lactobacillus paracasei S-NB is in the growth and reproduction stage. If the primary fermentation temperature is too low or too high, it will reduce the growth and reproduction rate of Lactobacillus paracasei S-NB. Therefore, the primary fermentation temperature needs to be set at 36-38°C. During the secondary fermentation process, Lactobacillus paracasei S-NB is in the stage with the highest EPS synthesis rate. If the pH value is too high or too low, or the temperature is too high or too low, it will reduce the EPS synthesis rate of Lactobacillus paracasei S-NB. Therefore, the pH value needs to be adjusted to 5.0 and the temperature needs to be maintained at 28-32°C.

[0032] In some embodiments, dynamically adjusting the pH includes: monitoring the pH in real time during the fermentation process, and automatically adding a 0.5 mol / L NaOH solution when the pH is less than 4.0. Since Lactobacillus paracasei S-NB continuously produces lactic acid during the fermentation process, causing the pH of the fermentation system to gradually decrease, by monitoring the pH in real time during the fermentation process and automatically adding a 0.5 mol / L NaOH solution when the pH is less than 4.0, the Lactobacillus paracasei S-NB is maintained in an optimal pH environment, thereby increasing the rate of EPS synthesis.

[0033] In some embodiments, the centrifugation is performed at a low temperature of 3-5°C, a centrifugal force of 7000-9000g, and a time of 10-20 minutes. Excessively high temperatures can destroy Lactobacillus paracasei S-NB, resulting in a decrease in the viable count of Lactobacillus paracasei. Therefore, a low temperature of 3-5°C is required. By setting the centrifugal force to 7000-9000g and the time to 10-20 minutes, the miscellaneous bacteria and impurities can be fully separated.

[0034] In some embodiments, ultrafiltration concentration uses a 10kDa membrane package for ultrafiltration. Ultrafiltration concentration can selectively retain large molecules of EPS, thereby increasing the concentration of EPS in the juice. By using a 10kDa membrane package for ultrafiltration, it can retain molecules with a molecular weight of ≥10 4 Da EPS, thereby achieving EPS concentration.

[0035] In some embodiments, the ultrahigh pressure treatment is performed at a pressure of 400-500 MPa for 4-6 minutes. Ultrahigh pressure treatment can destroy the cell membrane structure of harmful microorganisms by applying high pressure, while Lactobacillus paracasei S-NB has a stronger pressure tolerance and can survive and be retained, thereby achieving selective sterilization. However, too low a pressure will not fully eliminate harmful microorganisms, while too high a pressure will eliminate Lactobacillus paracasei S-NB. Therefore, it is necessary to set the pressure of ultrahigh pressure treatment to 400-500 MPa to achieve a viable Lactobacillus paracasei S-NB bacteria retention rate in the juice of more than 95%.

[0036] The present invention will be further described below by way of specific embodiments.

[0037] The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0038] Before fermentation, the Lactobacillus paracasei S-NB strain needs to be activated and expanded as follows: The freeze-dried Lactobacillus paracasei S-NB strain was inoculated into 30 ml of sterile MRS liquid medium and cultured anaerobically at 37°C for 18-24 h until OD600 = 3.0 to prepare live bacterial solution 1. Then, the strain was aseptically inoculated into new MRS liquid medium at a 2% (v / v) inoculation ratio and cultured anaerobically at 37°C for 10-12 h to prepare live bacterial solution 2. The strain was activated and expanded in this way to obtain a viable bacterial count of ≥1×10 9 CFU / mL of Lactobacillus paracasei S-NB strain.

[0039] Example 1 Fully ripe, mildew-free, disease- and insect-free Qiuyue pears were selected, peeled and cored, and then crushed with a stainless steel crusher. 0.45% (v / v) ascorbic acid was added during the crushing process to obtain pear pulp, which was homogenized with a high-pressure homogenizer at a pressure of 20 MPa and a temperature of 60°C for 2 times. After filtration, the pulp was sterilized at an ultra-high temperature of 120°C for 2 seconds to obtain juice.

[0040] 0.03% (w / v) pectinase and 0.01% (w / v) cellulase were added to the juice, and enzymatic hydrolysis was carried out at 43°C for 2 h. After the enzymatic hydrolysis, the juice was inactivated at 85°C for 5 min. 0.5% (w / v) oligofructose was added and 1% (v / v) Lactobacillus paracasei S-NB was inoculated. The juice was fermented at a constant temperature of 36°C for 2 h, and then the pH value was adjusted to 5.0, and the temperature was gradually decreased to 28°C. The juice was fermented for 12 h. The pH value was monitored in real time during the fermentation process. When the pH value was less than 4.0, 0.5 mol / L NaOH solution was automatically added to obtain the fermented juice.

[0041] The fermented juice was subjected to low-temperature centrifugation at 3°C ​​with a centrifugal force of 7000g for 10 minutes. The supernatant was concentrated by ultrafiltration using a 10kDa membrane package, and then subjected to ultrahigh pressure treatment at 400MPa for 4 minutes. The probiotic fermented juice beverage was obtained by reflux filling in glass bottles.

[0042] Example 2 Fully ripe, mildew-free, disease- and insect-free Qiuyue pears were selected, peeled and cored, and then crushed with a stainless steel crusher. 0.6% (v / v) ascorbic acid was added during the crushing process to obtain pear pulp, which was homogenized with a high-pressure homogenizer at a pressure of 30 MPa and a temperature of 70°C for three times. After filtration, the pulp was sterilized at an ultra-high temperature of 130°C for 4 seconds to obtain juice.

[0043] 0.03% (w / v) pectinase and 0.01% (w / v) cellulase were added to the juice, and enzymatic hydrolysis was carried out at 47°C for 3 h. After the enzymatic hydrolysis, the juice was inactivated at 90°C for 8 min, 2% (w / v) oligofructose was added, and 3% (v / v) Lactobacillus paracasei S-NB was inoculated. The juice was fermented at a constant temperature of 38°C for 6 h, and then the pH value was adjusted to 5.0, and the temperature was gradually decreased to 32°C. The juice was fermented for 18 h. The pH value was monitored in real time during the fermentation process. When the pH value was less than 4.0, 0.5 mol / L NaOH solution was automatically added to obtain the fermented juice.

[0044] The fermented juice was subjected to low-temperature centrifugation at 5°C with a centrifugal force of 9000g for 20 minutes. The supernatant was concentrated by ultrafiltration using a 10kDa membrane package, and then subjected to ultrahigh pressure treatment at 500MPa for 6 minutes. The probiotic fermented juice beverage was obtained by reflux filling in glass bottles.

[0045] Example 3 Fully ripe, mildew-free, disease- and insect-free Qiuyue pears were selected, peeled and cored, and then crushed with a stainless steel crusher. 0.5% (v / v) ascorbic acid was added during the crushing process to obtain pear pulp, which was homogenized with a high-pressure homogenizer at a pressure of 25 MPa and a temperature of 60°C for 2 times. After filtration, the pulp was sterilized at an ultra-high temperature of 121°C for 3 seconds to obtain juice.

[0046] 0.03% (w / v) pectinase and 0.01% (w / v) cellulase were added to the juice, and enzymatic hydrolysis was carried out at 45°C for 2 h. After the enzymatic hydrolysis, the juice was inactivated at 85°C for 5 min. 1% (w / v) oligofructose was added and 2% (v / v) Lactobacillus paracasei S-NB was inoculated. The juice was fermented at a constant temperature of 37°C for 4 h, and then the pH value was adjusted to 5.0, and the temperature was gradually decreased to 30°C. The juice was fermented for 15 h. The pH value was monitored in real time during the fermentation process. When the pH value was less than 4.0, 0.5 mol / L NaOH solution was automatically added to obtain the fermented juice.

[0047] The fermented juice was subjected to low-temperature centrifugation at 4°C with a centrifugal force of 8000g for 15 minutes. The supernatant was concentrated by ultrafiltration using a 10kDa membrane package, and then subjected to ultrahigh pressure treatment at 500MPa for 5 minutes. The probiotic fermented juice beverage was obtained by reflux filling in glass bottles.

[0048] Comparative Example 1 The same as Example 1, except that Lactobacillus casei Shirota was used instead of Lactobacillus paracasei S-NB.

[0049] Comparative Example 2 The same as Example 1, except that the inoculation of Lactobacillus paracasei S-NB strain and subsequent fermentation were not performed.

[0050] Key index tests were performed on Examples 1-3. The test items included the viable count of Lactobacillus paracasei S-NB, EPS content, DPPH clearance rate, and Escherichia coli content. The test method was as follows. The test results are shown in Table 1.

[0051] The viable bacterial count test follows GB 4789.35-2016: Add 25 mL of the sample to be tested to a sterile Erlenmeyer flask containing 225 mL of normal saline. Shake thoroughly. Using a 1 mL sterile pipette or micropipette, draw 1 mL of a 1:10 sample homogenate. Slowly pour along the tube wall into a sterile test tube containing 9 mL of normal saline (taking care not to let the pipette tip touch the diluent). Shake the test tube or use a sterile pipette to repeatedly pipette and mix thoroughly to make a 1:100 sample homogenate. Using another 1 mL sterile pipette or micropipette tip, make 10-fold incremental dilutions of the sample homogenate, switching to a 1 mL sterile pipette or tip for each incremental dilution. Based on the estimated viable bacterial count of the sample to be tested, select two to three appropriate serial dilutions. Pipette 1 mL of the sample homogenate for each dilution into a sterile plate, making two plates for each dilution. After the dilution has been transferred to the plate, pour approximately 15 mL of MRS agar, cooled to 48°C, onto the plate and swirl the plate to mix thoroughly. Incubate anaerobically at 36°C ± 1°C for 72 h ± 2 h. The process from sample dilution to plate pouring should be completed within 15 min. If the colony count on only one dilution plate is within the appropriate counting range, calculate the average of the colony counts on both plates and multiply this average by the corresponding dilution factor to obtain the total number of colonies per gram or milliliter. If the colony counts on two consecutive dilution plates are within the appropriate counting range, calculate the total number of colonies per gram or milliliter using the following formula: N = ΣC / [(n1 + 0.1n2)d], where N is the number of colonies in the sample, ΣC is the sum of the colony counts on the plates, n1 is the number of plates at the first dilution (lowest dilution factor), n2 is the number of plates at the second dilution (highest dilution factor), and d is the dilution factor.

[0052] EPS content is determined using the phenol-sulfuric acid method: 20 mg of standard dextran (or glucose) is placed in a 500 ml volumetric flask. Water is added to the mark. 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, and 1.8 ml of each aliquot are pipetted and made up to 2.0 ml with distilled water. Then, 1.0 ml of 6% phenol and 5.0 ml of concentrated sulfuric acid are added. After shaking and allowing to stand for 30 minutes, the absorbance is measured at 490 nm. A 2.0 ml water aliquot is used as a blank and the same color development procedure is repeated. The abscissa represents micrograms of polysaccharide, and the ordinate represents the optical density. A standard curve is generated. 1.0 ml of the sample to be tested is added to 1.0 ml of distilled water, followed by 1.0 ml of 6% phenol. 5.0 ml of concentrated sulfuric acid is quickly added. The mixture is vortexed and thoroughly mixed. The mixture is allowed to stand for 30 minutes. The absorbance is measured at 490 nm. The measured absorbance is then applied to the standard curve to determine the EPS concentration.

[0053] DPPH scavenging rate test: Take 3.5mg DPPH powder, dissolve it with anhydrous ethanol and dilute to 10mL, store in the dark and refrigerate, add 4mL DPPH solution and the sample to be tested in a 10mL colorimetric tube in sequence, add anhydrous ethanol to the scale, mix well and immediately use a 1cm colorimetric dish to measure the absorbance value (A1) at a wavelength of 517nm, keep it in the dark at room temperature for 30min and measure the absorbance value (A2). The control test is the ethanol solution with only DPPH added, and its absorbance value is recorded as A0. Calculate the DPPH scavenging rate (K) according to the following formula: K=[1-(A 1- A 2) / A0]×100%.

[0054] E. coli content testing follows the procedures outlined in GB4789.3-2016: Use a sterile pipette to draw 25 mL of sample into a sterile Erlenmeyer flask (pre-filled with an appropriate number of sterile glass beads) or other sterile container containing 225 mL of phosphate buffered saline or normal saline. Shake thoroughly or place on a mechanical shaker to mix thoroughly to prepare a 1:10 sample solution. Select two to three appropriate serial dilutions and inoculate two sterile plates with 1 mL of each dilution. Simultaneously, add 1 mL of normal saline to a sterile plate as a blank control. Promptly pour approximately 15 mL to 20 mL of melted and maintained at 46°C crystal violet neutral red bile agar (VRBA) into each plate. Carefully swirl the plate to thoroughly mix the medium and sample solution. After the agar solidifies, add 3 mL to 4 mL of VRBA to cover the plate. Invert the plate and incubate at 36°C ± 1°C for 18 to 24 hours. Select plates with colony counts between 15 and 150 CFU and count typical and suspected coliform bacteria (e.g., colony diameters smaller than typical colonies) on each plate. Typical colonies are purple-red with a red bile salt precipitate ring around them. Colony diameters are 0.5 mm or larger. For plates with the lowest dilution, record the specific colony count for those with fewer than 15 CFU.

[0055] Table 1

[0056] It can be seen from Table 1 that the probiotic fermented fruit juice beverages of Examples 1-3 have a high viable bacteria count, EPS content and DPPH clearance rate, and the E. coli count is <1 CFU / ml, meeting the national standards.

[0057] A comparative experimental test was conducted on Example 3 and Comparative Examples 1 and 2. The test items included EPS content, fermentation acidity and sensory score. The test method was as follows. The test results are shown in Table 2.

[0058] The EPS content test is the same as above.

[0059] Fermentation acidity test: Weigh 10 g of the mixed sample and place it in a 150 mL conical flask. Add 2.0 mL of phenolphthalein indicator solution and titrate to neutrality with 0.1 mol / L sodium hydroxide standard solution. Record the volume of sodium hydroxide solution consumed and calculate the acidity (X) according to the following formula: X = [c2 × (V2 - V0) × 100] / (m2 × 0.1), where c2 is the molar concentration of the sodium hydroxide standard solution, V2 is the volume of the sodium hydroxide solution consumed, V0 is the volume of the sodium hydroxide solution consumed in the blank experiment, and m2 is the mass of the sample.

[0060] Sensory evaluation: 30 people were randomly selected to conduct sensory evaluation of the juice drinks. The evaluation indicators included the sweet-sour ratio (ideal range 15-20), aroma characteristics and taste. The score range was 0-10 points, and the average of the 30 scores was finally taken.

[0061] Table 2

[0062] Table 2 shows that the probiotic fermented fruit juice beverage of Example 3 has a higher EPS content and a higher sensory score than Comparative Examples 1 and 2. In the sensory score test, Example 3 has a sour-sweet ratio of 18.5 and fully retains the pear aroma and fermented frankincense, resulting in a higher sensory score.

[0063] The physical and chemical indicators of Example 3 were tested, including polysaccharide molecular weight, total acid (lactic acid meter), browning index (A420) and viable bacterial survival rate after treatment with simulated gastrointestinal fluid. The test method is as follows, and the test results are shown in Table 3.

[0064] Survival of viable bacteria after treatment with simulated gastrointestinal fluid: Gastric fluid buffer was prepared with the following components: 2.758g NaCl, 0.5144g KCl, 0.225g KH2PO4, 2.1g NaHCO3, 0.203g MgCl2, 0.0786g (NH4)2CO3, and 1L deionized water. Before the experiment, 15mg of gastric mucin and 6.25mg of pepsin were weighed and dissolved in 10mL of gastric buffer solution. The mixture was mixed and incubated in a 37°C water bath for 20 minutes to activate their activity, thus creating artificial gastric fluid. The artificial gastric fluid was inoculated at 10% of the inoculum volume, shaken to mix thoroughly, and 1mL of the mixture was aspirated for plate count, recorded as N0 (the number of viable bacteria at 0 hours). The mixture was incubated in a 37°C incubator for 3 hours, shaken to mix thoroughly, and 1mL of the mixture was aspirated for plate count, recorded as N1 (the number of viable bacteria at 3 hours). The survival rate was calculated as follows: survival rate (%) = (N1 / N0) × 100%.

[0065] Table 3

[0066] As can be seen from Table 3, the probiotic fermented fruit juice beverage prepared in Example 3 has a lower browning index (A420), and because EPS has significant biofilm-forming ability, Example 3 has a higher viable bacteria survival rate after treatment with simulated gastrointestinal fluid.

[0067] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0068] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, and all of these should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a probiotic fermented fruit juice beverage, characterized in that: include: The fruit is peeled and cored, crushed, and ascorbic acid is added, followed by homogenization, filtration, and sterilization to obtain juice; The juice is subjected to enzymatic hydrolysis, oligofructose is added, and fermented using Lactobacillus paracasei S-NB, and the pH value is dynamically adjusted during the fermentation process to be greater than 4, to obtain fermented juice; The fermented juice is centrifuged, the supernatant is ultrafiltered and concentrated, and then subjected to ultrahigh pressure treatment and aseptic cold filling to obtain a probiotic fermented juice beverage; Among them, the classification name of the Lactobacillus paracasei S-NB is Lactobacillus paracasei S-NB, which is preserved in the China Center for Type Culture Collection with the preservation number CCTCC M2021461, the preservation date is April 27, 2021, and the preservation address is Wuhan University, Wuhan, China.

2. The preparation method according to claim 1, characterized in that The added amount of ascorbic acid is 0.45% (v / v)-0.6% (v / v).

3. The preparation method according to claim 1, characterized in that The homogenization process is performed at a pressure of 20-30 MPa, a temperature of 60-70° C., and is repeated 2-3 times.

4. The preparation method according to claim 1, characterized in that In the enzymatic hydrolysis treatment, pectinase and cellulase are used for enzymatic hydrolysis, the enzymatic hydrolysis temperature is 43-47° C., and the time is 2-3 h. The amount of the pectinase added is 0.03% (w / v), and the amount of the cellulase added is 0.01% (w / v); And / or, an enzyme inactivation treatment is performed after the enzymatic hydrolysis treatment, the time of the enzyme inactivation treatment is 5-8 minutes, and the temperature is 85-90°C.

5. The preparation method according to claim 1, characterized in that The added amount of the oligofructose is 0.5% (w / v)-2% (w / v).

6. The preparation method according to claim 1, characterized in that The fermentation comprises a primary fermentation and a secondary fermentation, wherein the primary fermentation comprises: inoculating 1% (v / v)-3% (v / v) of Lactobacillus paracasei S-NB strains, and fermenting at a constant temperature of 36-38° C. for 2-6 hours; and the secondary fermentation comprises: adjusting the pH value to 5.0, gradually cooling to 28-32° C., and fermenting for 12-18 hours; Wherein, the number of viable bacteria in the Lactobacillus paracasei S-NB strain is ≥1×10 9 CFU / mL.

7. The preparation method according to claim 1, characterized in that The dynamic adjustment of pH value includes: monitoring pH value in real time during the fermentation process, and automatically adding 0.5 mol / L NaOH solution when the pH value is less than 4.

0.

8. The preparation method according to claim 1, characterized in that The centrifugation treatment adopts low-temperature centrifugation, the temperature is 3-5°C, the centrifugal force is 7000-9000g, and the time is 10-20min.

9. The preparation method according to claim 1, characterized in that The ultrafiltration concentration adopts 10kDa membrane ultrafiltration.

10. The preparation method according to claim 1, characterized in that The ultra-high pressure treatment is performed at a pressure of 400-500 MPa and for a time of 4-6 minutes.

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