A strain of Lactobacillus reuteri with high β-glucosidase activity and its application in fermenting seabuckthorn juice

Abstract

The present invention belongs to the technical field of fermentation engineering, and specifically relates to a strain of Lactobacillus reuteri with high β-glucosidase activity and its application in fermented seabuckthorn juice. The strain is deposited with CGMCC NO: 33026; the strain is an adaptive evolution probiotic, which can improve the activity and tolerance of probiotics, has a good application effect in fermented seabuckthorn juice, can maintain a high bacterial activity, and its viable count is maintained at 9.12~9.37 log (cfu / mL), and compared with unfermented seabuckthorn juice, fermented seabuckthorn juice has a higher total phenol content and total flavonoid content, and the antioxidant activity of the fermented seabuckthorn juice is improved, and the total phenol content can reach 545 mg / 100 mL, which is 95% higher than that of unfermented seabuckthorn juice. It is also a safe method without introducing exogenous genes and can be used in food, and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation engineering, and in particular relates to a strain of Lactobacillus reuteri with high beta-glucosidase activity and application thereof in fermenting seabuckthorn juice. Background Art

[0002] Lactobacillus reuteri ( Lactobacillus reuteri ) is a lactic acid bacterium that has been reported to exist naturally in the intestines of almost all vertebrates and mammals. Lactobacillus reuteri has a strong ability to adhere to the intestinal mucosa, which can improve the distribution of intestinal flora, antagonize the colonization of harmful bacteria, and avoid intestinal diseases; Lactobacillus reuteri can produce a non-protein broad-spectrum antibacterial substance called "Reuterin", which can widely inhibit the growth of Gram-positive bacteria, Gram-negative bacteria, yeast, fungi and pathogens.

[0003] Sea buckthorn (Hippophae rhamnoides L.) is a deciduous shrub that is drought-resistant, sand-resistant, and can survive on salinized land. Sea buckthorn is a plant that is both medicinal and edible. Its fruit is rich in vitamins, organic acids, phenolic acids, sugars, fatty acids, amino acids, carotenoids, proanthocyanidin alkaloids, flavonoids, tannins, triterpenes and steroids. It has anti-inflammatory, antioxidant, blood sugar-lowering, anti-tumor, radiation protection, delayed photoaging, anti-atherosclerosis, prevention of liver damage, and prevention of thrombosis.

[0004] Lactic acid bacteria itself has the function of producing β-glucosidase. By applying lactic acid bacteria to seabuckthorn fermentation, seabuckthorn polyphenols are deglycosylated, thereby achieving the purpose of improving the body's bioavailability. β-glucosidase participates in the metabolism and transformation of phenolic substances in the fermentation broth, changing the content and existence form of phenolic substances. The activity of β-glucosidase affects the transformation of bound polyphenols to free polyphenols, thus affecting the polyphenol yield.

[0005] Fermenting seabuckthorn juice with lactic acid bacteria can not only retain the nutrients of seabuckthorn itself to the maximum extent, but also improve the overall taste and flavor of seabuckthorn juice, and greatly increase the total flavonoids and total phenol content of seabuckthorn juice, and improve the antioxidant capacity of seabuckthorn juice. The patent CN115024452B on fermented seabuckthorn juice discloses a strain of plant lactobacillus and its application in fermented seabuckthorn juice, CN113908182B discloses fermented seabuckthorn juice and its preparation method, and CN118266581A discloses a method for preparing seabuckthorn enzyme, and respectively studies the application of strains in fermented seabuckthorn juice, the preparation method of fermented seabuckthorn juice, and the preparation method of seabuckthorn enzyme. When fermenting seabuckthorn juice, additional carbon and nitrogen sources are added, which increases the economic cost; seabuckthorn is rich in nutrients and can be used as a natural culture medium to meet the needs of probiotics and maintain natural characteristics; however, various acids in seabuckthorn have a stress effect on probiotics, affecting the growth and metabolism of probiotics. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention aims to provide a strain of Lactobacillus reuteri with high β-glucosidase activity and its application in fermenting seabuckthorn juice. The strain is isolated from pulp water collected in Beipiao City, Chaoyang City, Liaoning Province, and is identified as Lactobacillus reuteri by 16S rRNA method (16S ribosomal RNA gene sequencing technology); the strain is an adaptively evolved probiotic, which can improve the activity and tolerance of probiotics. It is also a safe method without introducing exogenous genes and can be used in food, and has good application prospects.

[0007] In order to achieve the above technical objectives, the technical solution provided by the present invention is as follows:

[0008] In a first aspect of the present invention, a strain of Lactobacillus reuteri ( Lactobacillus reuteri )LY-1, which was deposited in the General Microbiology Center of China Culture Collection Administration (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​December 11, 2024, with the deposit number CGMCC NO: 33026.

[0009] The second aspect of the present invention provides a microbial agent, wherein the microbial agent contains the Lactobacillus reuteri described in the first aspect ( Lactobacillus reuteri ) LY-1 or its fermentation products or its metabolites.

[0010] The third aspect of the present invention provides a fermented seabuckthorn juice, the raw materials of which include the Lactobacillus reuteri described in the first aspect ( Lactobacillus reuteri ) LY-1 or the microbial agent described in the second aspect.

[0011] Preferably, the fermented seabuckthorn juice has at least one of the following functions (1) to (3):

[0012] (1) Increase the content of flavonoids;

[0013] (2) Increase the content of polyphenols;

[0014] (3) Improve antioxidant capacity.

[0015] The fourth aspect of the present invention provides a method for preparing the fermented sea buckthorn juice described in the third aspect, specifically: adding pectinase to the sea buckthorn pulp obtained after sea buckthorn pretreatment for enzymolysis, mixing the sea buckthorn pulp after enzymolysis with water evenly and then sterilizing it, and then inoculating Lactobacillus reuteri LY-1 for fermentation culture to obtain the juice.

[0016] Preferably, the pretreatment operation is to sequentially wash, pulp, filter and centrifuge the seabuckthorn fruits to obtain seabuckthorn pulp.

[0017] Preferably, the dosage ratio of the seabuckthorn puree to pectinase is 8-16 mg:10 L; the enzymolysis time is 2-4 h; and the volume ratio of the seabuckthorn puree after enzymolysis to water is 1-1.5:3-6.

[0018] Preferably, the sterilization condition is 115-125° C. and the time is 10-20 min.

[0019] Preferably, the inoculation amount of Lactobacillus reuteri LY-1 is 4-6%.

[0020] Preferably, the fermentation culture conditions are: culture at 33-39° C. for 4-10 days.

[0021] A fifth aspect of the present invention provides the Lactobacillus reuteri ( Lactobacillus reuteri ) Use of the microbial agent described in LY-1 or the second aspect in preparing food.

[0022] In a sixth aspect of the present invention, there is provided the Lactobacillus reuteri ( Lactobacillus reuteri ) Use of LY-1 or the microbial agent described in the second aspect in the preparation of food that helps the fermentation of sea buckthorn juice.

[0023] A seventh aspect of the present invention provides the Lactobacillus reuteri ( Lactobacillus reuteri ) Use of LY-1 or the microbial agent described in the second aspect in the preparation of a product for improving the activity of sea buckthorn juice fermentation bacteria.

[0024] Preferably, the product is a medicine.

[0025] Preferably, the drug has the effect of increasing the activity of β-glucosidase.

[0026] Beneficial technical effects of one or more of the above technical solutions:

[0027] (1) In the present invention, Lactobacillus reuteri YL-1 is obtained by adaptively evolving Lactobacillus reuteri YL. The obtained Lactobacillus reuteri YL-1 has stronger activity and tolerance in seabuckthorn puree than the original strain, and the β-glucosidase activity is greatly improved, which is increased by 2 times in 48 hours.

[0028] (2) The application effect of Lactobacillus reuteri YL-1 provided by the present invention in fermenting sea buckthorn juice is good. By using this strain in sea buckthorn juice, the bacterial activity can be maintained at a high level, and the number of viable bacteria is always maintained at 9.12~9.37 log (cfu / mL).

[0029] (3) Compared with unfermented seabuckthorn juice, fermented seabuckthorn juice has higher total phenol content and total flavonoid content. The antioxidant activity of fermented seabuckthorn juice is improved, and the total phenol content can reach 545 mg / 100 mL, which is 95% higher than that of unfermented seabuckthorn juice.

[0030] (4) The present invention provides a new seabuckthorn processing method, enriches the food market, and increases the added value of the seabuckthorn industry. The seabuckthorn's own nutritional active substances are fully utilized, so that the prepared product contains high levels of flavonoids and polyphenols and has strong antioxidant capacity. The fermented seabuckthorn juice has a simple preparation process, and the product is convenient and directly drinkable, which has a good market prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the drawings required for use in the description of the embodiment will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0032] Figure 1 is the growth curve of Lactobacillus reuteri YL and Lactobacillus reuteri YL-1 in seabuckthorn puree in Example 2 of the present invention;

[0033] Figure 2 is the gallic acid standard curve of Example 11 of the present invention;

[0034] Figure 3 is the total phenol content at the fermentation termination stage of Example 11 of the present invention;

[0035] Figure 4 is the rutin standard curve of Example 12 of the present invention;

[0036] Figure 5is the total flavonoids content of seabuckthorn juice at the fermentation termination stage of Example 12 of the present invention;

[0037] Figure 6 is the ABTS free radical scavenging rate of seabuckthorn juice at the fermentation termination stage in Example 13 of the present invention;

[0038] Figure 7 is the DPPH radical scavenging rate of seabuckthorn juice at the end of fermentation in Example 13 of the present invention;

[0039] Figure 8 The changes in pH value of seabuckthorn juice at different fermentation stages in Example 14 of the present invention;

[0040] Fig. 9 The figure shows the change of viable bacteria count at different fermentation stages in Example 15 of the present invention. DETAILED DESCRIPTION

[0041] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0042] In view of the fact that various acids in seabuckthorn have a stress effect on probiotics and affect the growth and metabolism of probiotics, the present invention provides a Lactobacillus reuteri LY-1 with high β-glucosidase activity.

[0043] Specifically, in a first typical embodiment of the present invention, a strain of Lactobacillus reuteri ( Lactobacillus reuteri )LY-1, the strain was deposited in the General Microbiology Center of China Culture Collection Administration (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​December 11, 2024, with the deposit number CGMCCNO: 33026.

[0044] In some embodiments, the Lactobacillus reuteri ( Lactobacillus reuteri ) The culture medium components involved in the LY-1 strain activation process include: peptone 8~12 g / L, beef powder 6~10 g / L, yeast powder 2~6 g / L, glucose 16~24 g / L, dipotassium hydrogen phosphate 1~3 g / L, diammonium hydrogen citrate 1~3 g / L, sodium acetate 4~6 g / L, magnesium sulfate 0.1~0.3 g / L, manganese sulfate 0.02~0.06 g / L, Tween 80 0.5~2 mL / L, and the pH of the culture medium is 6.0~6.4.

[0045] In some embodiments, the conditions for activating the strains are: activating at 33-39°C for 22-50 h until the bacterial density of the bacterial suspension reaches 10 6 ~10 8CFU / mL.

[0046] In a second typical embodiment of the present invention, a microbial agent is provided, wherein the microbial agent contains the Lactobacillus reuteri ( Lactobacillus reuteri ) LY-1 or its fermentation products or its metabolites.

[0047] In a third typical embodiment of the present invention, a fermented seabuckthorn juice is provided, wherein the raw materials include the above-mentioned Lactobacillus reuteri ( Lactobacillus reuteri )LY-1 or the above-mentioned microbial agents.

[0048] In some embodiments, the fermented seabuckthorn juice has at least one of the following functions (1) to (3):

[0049] (1) Increase the content of flavonoids;

[0050] (2) Increase the content of polyphenols;

[0051] (3) Improve antioxidant capacity.

[0052] In a fourth typical specific embodiment of the present invention, a method for preparing the above-mentioned fermented sea buckthorn juice is provided, specifically: pectinase is added to the sea buckthorn pulp obtained after sea buckthorn pretreatment for enzymolysis, the enzymolysis product is evenly mixed with water and then sterilized, and then Lactobacillus reuteri LY-1 is inoculated for fermentation culture to obtain the juice.

[0053] In some embodiments, the pretreatment operation specifically involves washing, pulping, filtering, and centrifuging the seabuckthorn fruits in sequence to obtain seabuckthorn puree.

[0054] In some embodiments, the dosage ratio of the seabuckthorn puree to pectinase is 8-16 mg:10 L; the enzymolysis time is 2-4 h; and the volume ratio of the seabuckthorn puree after enzymolysis to water is 1-1.5:3-6.

[0055] In some embodiments, the sterilization condition is 115-125° C. and the time is 10-20 min.

[0056] In some embodiments, the inoculation amount of Lactobacillus reuteri LY-1 is 4-6%.

[0057] In some embodiments, the fermentation culture conditions are: culture at 33-39° C. for 4-10 days.

[0058] In a fifth typical embodiment of the present invention, there is provided a Lactobacillus reuteri ( Lactobacillus reuteri ) Use of LY-1 or the above-mentioned microbial agents in food preparation.

[0059] In a sixth typical embodiment of the present invention, there is provided a Lactobacillus reuteri ( Lactobacillus reuteri ) Application of LY-1 or the above-mentioned microbial agent in the preparation of food that helps to ferment sea buckthorn juice.

[0060] In a seventh typical embodiment of the present invention, there is provided a Lactobacillus reuteri ( Lactobacillus reuteri ) Application of LY-1 or the above-mentioned microbial agents in the preparation of products for improving the activity of sea buckthorn juice fermentation bacteria.

[0061] In some embodiments, the product is a pharmaceutical.

[0062] In some embodiments, the drug has the effect of increasing the activity of β-glucosidase.

[0063] The present invention is further described below in conjunction with examples. The present invention is further described below by way of examples, but the present invention is not limited to the scope of the embodiments described. Based on the embodiments in the present invention, any variation of the present invention by those skilled in the art without making a creative premise all belongs to the protection scope of the present invention. Meanwhile, in the embodiments of the present invention, unless otherwise specified, all preparation raw materials are commercially available products well known to those skilled in the art.

[0064] Example 1: Isolation, adaptive evolution and identification of bacterial strains

[0065] (1) Isolation of strains: Lactobacillus reuteri LY (original strain) was isolated from pulp water collected in Beipiao City, Chaoyang City, Liaoning Province. Specific isolation process: First, use a 1 mL sterile pipette to draw 1 mL of pulp water and add it to a large test tube containing 9 mL of sterile water to mix thoroughly. Then use a sterile pipette to draw 1 mL from this test tube and add it to another test tube containing 9 mL of sterile water and mix well. Repeat this process to make 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Solutions with different dilution factors.

[0066] Use a sterile pipette to take 50 μL of slurry with different concentration gradients and evenly spread it on the surface of the MRS solid culture dish, put it in a 37℃ constant temperature incubator for 48 hours, and separate a pure single colony. It was identified as lactic acid bacteria through catalase test, and further determined by gene sequence to be Lactobacillus reuteri.

[0067] (2) Adaptive evolution of strains: The strains isolated above were inoculated into a conical flask containing sterile liquid culture medium at a 5% inoculation rate, sealed, and placed in a 37°C constant temperature incubator for 24 h. The bacterial growth was counted based on the number of colonies grown. When the OD value of the cultured strain reached 1.2, 10 mL was taken and transferred into 100 mL of sterilized seabuckthorn puree. When the OD value reached 1.2 again, 10 mL was taken and transferred into a new 100 mL of sterilized seabuckthorn puree. The culture was continued for multiple times, and 50 μL was taken each time and applied to seabuckthorn solid culture medium. The best single colony was selected based on colony size, shape, edge, protuberance, color and transparency to obtain Lactobacillus reuteri YL-1. The strain was identified as Lactobacillus reuteri YL-1 by the 16S rRNA method. The 16S rDNA sequence of the strain is shown in SEQ ID NO: 1.

[0068] The components of the sterile liquid culture medium include: 10.0 g / L peptone, 8.0 g / L beef powder, 4.0 g / L yeast powder, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L diammonium hydrogen citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, and 1.0 mL / L Tween 80, and the pH of the culture medium is 6.2.

[0069] The above-screened Lactobacillus reuteri ( Lactobacillus reuteri )LY-1, which was deposited in the General Microbiology Center of China Culture Collection Administration (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​December 11, 2024, with the deposit number CGMCC NO: 33026.

[0070] Example 2: Growth characteristics of original strains and evolved strains in seabuckthorn puree

[0071] The evolved Lactobacillus reuteri YL-1 and the original strain were inoculated at 2% in 100 mL of sterilized seabuckthorn puree and cultured for 48 h. Samples were taken every 2 h to determine the growth curves of the original strain and the evolved strain in seabuckthorn puree.

[0072] pass Figure 1 It can be seen that the OD value of the evolved strain in sea buckthorn puree culture medium reached 4.53, which was 2.1 times higher than that of the original strain (OD value was 2.14), and had higher activity and tolerance.

[0073] Example 3: Flavonoid and polyphenol content in fermentation broth of original strains

[0074] Pour 4% inoculum of Lactobacillus reuteri LY into a conical flask filled with sterile liquid culture medium, seal it, and place it in a 37°C constant temperature incubator to activate it for 24 h to obtain a bacterial suspension. Count the bacteria under a microscope using a hemocytometer until the bacterial density of the bacterial suspension reaches 10 6 ~10 8 CFU / mL. Pour 10 mL of bacterial solution into 100 mL of sterilized MRS liquid medium and ferment at constant temperature for 48 h; after fermentation, determine the content of total flavonoids and total polyphenols.

[0075] Example 4: Flavonoid and polyphenol content in fermentation broth of evolved strains

[0076] Pour 4% inoculum of Lactobacillus reuteri LY-1 into a conical flask filled with sterile liquid culture medium, seal it, and place it in a 37°C constant temperature incubator to activate it for 24 h to obtain a bacterial suspension. Count the bacteria under a microscope using a hemocytometer until the bacterial density of the bacterial suspension reaches 10 6 ~10 8 CFU / mL. Pour 10 mL of bacterial solution into 100 mL of sterilized MRS liquid medium and ferment at constant temperature for 48 h; after fermentation, determine the content of total flavonoids and total polyphenols.

[0077] Example 5: β-glucosidase activity assay

[0078] Experimental process: In this example, Lactobacillus reuteri LY and Lactobacillus reuteri LY-1 were activated, and the β-glucosidase activity was measured after culturing for 48 h.

[0079] The pNPG (p-nitrophenyl-β-D-pyranoglucoside) method was used to determine the β-glucosidase activity. pNPG reacts to generate pNP (p-nitrophenol) under the action of β-glucosidase. pNP appears yellow in an alkaline environment and has a maximum absorption peak at 400 nm. The specific method is as follows:

[0080] The substrate reaction solution is 100 mL of Na 2 HPO 4 -Dissolve 150.65 mg pNPG in citric acid buffer. Add 100 μL of moderately diluted probiotic bacterial solution (obtained after activation of the above strains) to 200 μL of substrate reaction solution, heat in a 50°C water bath for 30 min, add 2 mL of 1 mol / L Na 2 CO 3; Stop the reaction and develop color, and measure the absorbance at 400 nm with a UV spectrophotometer. Calibrate with a blank tube of buffer without sample. Define the amount of enzyme required to hydrolyze the substrate to produce 1 μg pNP per minute as 1U, and the unit of β-glucosidase activity is U / L. The specific results of β-glucosidase activity are shown in Table 1:

[0081] Table 1

[0082]

[0083] As can be seen from Table 1, the β-glucosidase activity of Lactobacillus reuteri LY-1 is much higher than that of Lactobacillus reuteri LY, which also shows that Lactobacillus reuteri LY-1 promotes the release of more polyphenols from seabuckthorn juice and further converts bound polyphenols into free polyphenols.

[0084] Example 6: Preparation of unfermented seabuckthorn juice

[0085] Follow the steps below:

[0086] (1) Take 10 L of sea buckthorn puree, add 10 mg of pectinase and perform enzymatic hydrolysis for 3 h, then add 40 L of purified water and mix well.

[0087] (2) Place the above mixture in a fermentation tank and sterilize it at 115℃ for 20 min. After sterilization is completed, allow it to cool naturally.

[0088] (3) After natural cooling, move to other containers for testing.

[0089] The above steps are strictly required to ensure that all processes are in a sterile state.

[0090] In this embodiment, the total polyphenol content of the prepared unfermented seabuckthorn juice is 279 mg / 100 mL, which is the total polyphenol content of the unfermented seabuckthorn juice.

[0091] Example 7: Fermentation of seabuckthorn juice with original strains

[0092] Follow the steps below:

[0093] (1) Take 10 L of sea buckthorn puree, add 10 mg of pectinase and perform enzymatic hydrolysis for 3 h, then add 40 L of purified water and mix well.

[0094] (2) Place the above mixture in a fermentation tank and sterilize it at 115℃ for 20 min. After sterilization is completed, allow it to cool naturally.

[0095] (3) Pour Lactobacillus reuteri YL into MRS liquid medium and culture at 37°C until the viable count reaches 5×10 8 cfu / mL.

[0096] (4) Inoculate the fermentation bacteria and ferment at 37°C and 100 r / min for 7 days. The inoculation amount of the bacteria is 5% of the material.

[0097] (5) After fermentation is completed, transfer the fermentation liquid to other containers for testing.

[0098] The above steps are strictly required to ensure that all processes are in a sterile state.

[0099] In this embodiment, the total polyphenol content of the fermented seabuckthorn juice of the prepared original strain is 266 mg / 100 mL.

[0100] Example 8: Fermentation of seabuckthorn juice with evolved strains

[0101] Follow the steps below:

[0102] (1) Take 10 L of sea buckthorn puree, add 10 mg of pectinase and perform enzymatic hydrolysis for 2 h, then add 40 L of purified water and mix well.

[0103] (2) Place the above mixture in a fermentation tank and sterilize it at 115℃ for 20 min. After sterilization is completed, allow it to cool naturally.

[0104] (3) Pour Lactobacillus reuteri YL-1 into MRS liquid medium and culture at 37°C until the viable count reaches 5×10 8 cfu / mL.

[0105] (4) The fermentation bacteria were inoculated and fermented at 37°C and 100 r / min for 7 days. The inoculation amount of the bacteria was 5% of the material.

[0106] (5) After fermentation is completed, transfer the fermentation liquid to other containers for testing.

[0107] The above steps are strictly required to ensure that all processes are in a sterile state.

[0108] In this embodiment, the total polyphenol content of the fermented seabuckthorn juice of the prepared evolved strain is 545 mg / 100 mL.

[0109] Example 9: Chr. Hansen probiotic LGG fermented sea buckthorn juice

[0110] In this example, Chr. Hansen probiotics LGG was purchased for fermenting sea buckthorn juice, and the specific steps were as follows:

[0111] (1) Take 10 L of sea buckthorn puree, add 10 mg of pectinase and perform enzymatic hydrolysis for 2 h, then add 40 L of purified water and mix well.

[0112] (2) Place the above mixed material in a fermentation tank and sterilize it at 115°C for 20 min. After sterilization is completed, allow it to cool naturally.

[0113] (3) Pour the Chr. Hansen probiotic LGG strain into MRS liquid culture medium and culture at 37°C until the viable count reaches 5×10 8 cfu / mL.

[0114] (4) The fermentation bacteria were inoculated and fermented at 37°C and 100 r / min for 7 days. The inoculation amount of the bacteria was 5% of the material.

[0115] After fermentation is completed, the fermentation liquid is transferred to other containers for testing.

[0116] The above steps are strictly required to ensure that all processes are in a sterile state.

[0117] In this embodiment, the total polyphenol content of the fermented sea buckthorn juice prepared by Chr. Hansen probiotics LGG is 433 mg / 100 mL.

[0118] Example 10: Sensory Evaluation

[0119] In this example, sensory evaluation was performed on the unfermented seabuckthorn juice and the fermented seabuckthorn juice prepared in Examples 6 to 9, respectively.

[0120] The sensory evaluation scoring standard of seabuckthorn juice mainly includes four indicators: color, aroma, taste and state. The score of each indicator is different, among which, color: 0~2 points; aroma: 0~3 points; taste: 0~3 points; state: 0~2 points. The specific evaluation results are shown in Table 2:

[0121] Table 2

[0122]

[0123] As can be seen from Table 2, compared with Examples 6-7 and Example 9, the sensory evaluation score of the fermented sea buckthorn juice prepared by the evolved strains obtained in the present invention (Example 8) is the highest, reaching 9.1±0.5 points, indicating that the evolved strains are more prominent in improving the sensory evaluation of sea buckthorn juice.

[0124] Example 11: Determination of total polyphenol content

[0125] Experimental process:

[0126] (1) Sample preparation: Accurately measure 40 mL of sample (the sample is the bacterial liquid after fermentation in Examples 3-4, and the sea buckthorn juice prepared in Examples 6-9) into a 50 mL stoppered centrifuge tube, centrifuge, and take the supernatant for later use. When the supernatant matrix is ​​complex, it needs to be filtered to a clear solution for later use.

[0127] (2) Drawing of standard curve: Accurately pipette 0.0, 0.2, 0.4, 0.6, 1.0, 1.5 mL of 200 mg / L gallic acid standard stock solution into 10 mL volumetric flasks, dilute with 60% ethanol solution to obtain gallic acid working solution. Then pipette 1.0 mL of gallic acid working solution into 10 mL colorimetric tubes, add 2.5 mL of Folin phenol reagent, shake well, add 2.5 mL of 15% sodium carbonate solution, dilute to the mark with water, shake well. Place in a 40℃ water bath for 60 min, and let stand to cool for 20 min. Prepare a standard series with concentrations of 0 mg / L, 4 mg / L, 8 mg / L, 12 mg / L, 20 mg / L, 30 mg / L, and measure their absorbance values. Draw a standard curve with concentration as the horizontal axis and absorbance as the vertical axis.

[0128] (3) Sample determination: Pipette 1.0 mL of filtrate (the filtrate is obtained by filtering the bacterial liquid after fermentation in Examples 3-4 and the sea buckthorn juice prepared in Examples 6-9) into a 10 mL colorimetric tube, add 2.5 mL of Folin phenol reagent, shake well, add 2.5 mL of 15% sodium carbonate solution, add water to the scale, and shake well. Place in a 40°C water bath for 60 min, let stand and cool for 20 min, and measure its absorbance value. Calculate the content of total polyphenols in the test solution based on the standard curve. The content of total polyphenols is calculated according to the following formula:

[0129]

[0130] In the formula, X: the content of total polyphenols in the sample, in milligrams per liter (mg / L); c: the content of total polyphenols in the test solution calculated from the standard curve, in milligrams per liter (mg / L); 10: the dilution factor of the filtrate; n: the dilution factor of the sample

[0131] Depend on Figure 3 It can be seen that the total polyphenol content of the original strain and the evolved strain after growth in the culture medium in Examples 3 and 4 is 52.65 mg / 100 mL and 78.91 mg / 100 mL respectively; Example 6 is unfermented seabuckthorn juice, and its total polyphenol content is 279 mg / 100 mL; the total polyphenol content of the fermented seabuckthorn juice obtained in Examples 7, 8 and 9 is 266 mg / 100 mL, 545 mg / 100 mL and 433 mg / 100 mL respectively. Among them, the total phenol content in Example 8 can reach up to 545 mg / 100 mL, which is 95% higher than that of the unfermented seabuckthorn juice in Example 6.

[0132] Example 12: Determination of total flavonoid content

[0133] (1) Sample pretreatment: Take 5-10 mL of sample (the sample is the bacterial solution after fermentation in Examples 3-4, or the sea buckthorn juice prepared in Examples 6-9), weigh it accurately, add about 30 mL of anhydrous ethanol, and perform ultrasonic extraction for 30-60 min. Filter the extract and make up to 50 mL with anhydrous ethanol.

[0134] (2) Drawing of standard curve: Accurately pipette 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL of 1 g / L rutin standard working solution, add anhydrous ethanol to make up to 15 mL, add 1 mL of aluminum nitrate solution (100 g / L) and 1 mL of potassium acetate solution (98 g / L) in sequence, add water to make up to 50 mL, and let stand in the dark for 1 hour. After the reaction is completed, read the absorbance at 420 nm with 30% ethanol solution as the control, and draw a standard curve of absorbance and working solution volume, such as Figure 4 shown.

[0135] (3) Sample determination: Accurately measure 1 mL of the sample to be tested (the sample is the bacterial solution prepared in Examples 3-4, or the sea buckthorn juice prepared in Examples 6-9), perform the experiment according to the standard curve processing method, and obtain the content of flavonoid compounds in the solution by checking the standard curve or calculating by regression equation. The flavonoid content in the sample is calculated according to the following formula:

[0136] ×100%

[0137] Where, X: the content of flavonoids; m: the mass of rutin in the sample colorimetric solution found from the standard curve or calculated by the linear regression equation, in milligrams (mg); W: the mass of the sample, in grams (g); d: the dilution ratio

[0138] Depend on Figure 5 It can be seen that the flavonoid contents of the original strain and the evolved strain after growth in the culture medium in Examples 3 and 4 are 8.79 mg / 100 mL and 12.45 mg / 100 mL respectively; Example 6 is unfermented seabuckthorn juice, and its flavonoid content is 53.22 mg / 100 mL; the flavonoid contents of the fermented seabuckthorn juice obtained in Examples 7, 8 and 9 are 56.63 mg / 100 mL, 77.24 mg / 100 mL and 67.25 mg / 100 mL respectively. Among them, the flavonoid content of the seabuckthorn juice fermented by the evolved strain in Example 8 is the best, and the flavonoid content of the unfermented seabuckthorn juice in Example 6 is increased by 45%.

[0139] Example 13: Determination of the antioxidant capacity of seabuckthorn juice

[0140] 1) Determination of ABTS free radical scavenging ability

[0141] Mix 5 mL of 7 mmol / L ABTS [2,2-azino-di(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt] and 88 μL of 140 mmol / L potassium persulfate, and let stand overnight at room temperature in the dark to form a free radical stock solution. Dilute it with anhydrous ethanol to a working solution before use, and its absorbance at 30°C and 734 nm wavelength is required to be 0.7.

[0142] Take 100 μL of sample ethanol solution of different concentrations, add 2 mL of ABTS solution, shake, and measure the absorbance of the reaction solution at 734 nm, using VC as the positive control.

[0143] The calculation formula of ABTS free radical scavenging rate is as follows:

[0144] Clearance = (Ac-As) / Ac×100%

[0145] Where: As is the absorbance of the sample solution mixed with ABTS solution; Ac is the absorbance of the anhydrous ethanol mixed with ABTS solution

[0146] Test results such as Figure 6 As shown, the ABTS free radical scavenging rates of Example 6, Example 7 and Example 9 are 64.79%, 71.29% and 85.66% respectively, while the ABTS free radical scavenging rate of Example 8 is the highest, reaching 93.41%, indicating that the ABTS free radical scavenging rate of sea buckthorn juice fermented with evolved strains can be greatly improved.

[0147] 2) Determination of DPPH free radical scavenging ability

[0148] (1) Sample treatment: Take an appropriate sample and centrifuge it at 10,000 r / min for 3 min. Take the supernatant and dilute it to 1 / 64 of the initial concentration according to a 2-fold concentration gradient.

[0149] (2) Sample determination: Take 100 μL of each sample solution of the concentration gradient and add it to a centrifuge tube. Add 3.9 mL of 0.1 mmol / L DPPH ethanol solution to each tube. The blank experimental group is 100 μL of anhydrous ethanol and 3.9 mL of 0.1 mmol / L DPPH ethanol solution. Mix well and place in a water bath at 37°C for 1 h in the dark. Adjust to zero with distilled water. Use the blank control tube as a control and measure the absorbance at 517 nm. Repeat the measurement three times.

[0150] Test results such as Figure 7As shown, the DPPH radical scavenging rates of Example 6, Example 7 and Example 9 are 52.33%, 59.78% and 82.61% respectively, while the DPPH radical scavenging rate of Example 8 is the highest, reaching 82.61%. The DPPH radical scavenging rate of sea buckthorn juice fermented with the evolved strains is increased by 30.28% compared with that of the fermented sea buckthorn juice in Example 6.

[0151] Example 14: Changes in pH value of fermented seabuckthorn juice at different fermentation stages

[0152] The pH value of sea buckthorn juice at different fermentation stages was tested in accordance with the national standard “National Food Safety Standard - Determination of Food pH” (GB5009.237-2016).

[0153] The results are as follows Figure 8 As shown, the pH of the unfermented seabuckthorn juice in Example 6 is basically stable. From the changes in the pH values ​​of the fermented seabuckthorn juice in Examples 7, 8 and 9, it can be seen that the pH in Example 8 is stable only on the 6th and 7th days, indicating that the probiotic activity is relatively high and the growth and metabolism are relatively vigorous.

[0154] Example 15: Changes in viable counts at different fermentation stages

[0155] The present invention refers to the national standard "National Food Safety Standard Food Microbiology Test Lactic Acid Bacteria Test" (GB4789.35-2016) to determine the content of active lactic acid bacteria in fermented seabuckthorn juice at different fermentation stages. Fig. 9 As shown, in Example 8, the number of viable bacteria was always between 9.12 log (cfu / mL) and 9.37 log (cfu / mL) within 7 days of fermentation. The results showed that the strain was always at a high level of activity before and after fermentation, and was suitable for subsequent fermentation research. The number of viable bacteria after fermentation (7d, 9.23 log (cfu / mL)) increased compared with that before fermentation (0d, 9.12 log (cfu / mL)).

[0156] The 16S rDNA sequence of the strain is as follows (SEQ ID NO: 1):

[0157] TGGGGGGGGTGCTATACATGCAAGTCGAACGCACTGGCCCAACTGATTGATGGTGCTTGCACCTGATTGACGATGGATCACCAGTGATTGGCGGACGGGTGAGTAACACGTACGTAACCTGCCCCGGAGCGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAACAACAAAAGCCACATGGCTTTTGTTTGAAAG ATGGCTTGGCTATCACTCTGGGATGGACCTGCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGACACTGATCGGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCG TGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTGGAGAACAACGTGCGTGAGAGCAACTGTTCACGCATTGACGGTATCCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTACGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATG TGAAATCCTTCGGCTTAACCGAAGAAATGCATCGGAAACCGGGCGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGCTGGAATGCGTACATACATGGAAAACACCAGTGGCGAAGGGGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTACCGAACAGGATTTAAATACCCCTGGTAGT

[0158] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A strain of Lactobacillus reuteri ( Lactobacillus reuteri )LY-1, the Lactobacillus reuteri ( Lactobacillus reuteri ) LY-1 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 11, 2024, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO: 33026.

2. A microbial agent, characterized in that: The microbial agent contains the Lactobacillus reuteri described in claim 1 ( Lactobacillus reuteri )LY-1.

3. A fermented seabuckthorn juice, characterized in that: The raw material includes the Lactobacillus reuteri described in claim 1 ( Lactobacillus reuteri )LY-1; The fermented seabuckthorn juice has at least one of the following functions (1) to (3): (1) Increase the content of flavonoids; (2) Increase the content of polyphenols; (3) Improve antioxidant capacity.

4. A method for preparing fermented seabuckthorn juice as claimed in claim 3, characterized in that: Specifically, pectinase is added to the sea buckthorn pulp obtained after the sea buckthorn pretreatment for enzymolysis, the sea buckthorn pulp after enzymolysis is evenly mixed with water and then sterilized, and then Lactobacillus reuteri LY-1 is inoculated for fermentation culture to obtain the product.

5. The preparation method according to claim 4, characterized in that: The pretreatment operation specifically involves washing, pulping, filtering and centrifuging the seabuckthorn fruits in sequence to obtain seabuckthorn pulp.

6. The preparation method according to claim 4, characterized in that: The dosage ratio of the seabuckthorn puree to the pectinase is 8-16 mg:10 L; the enzymatic hydrolysis time is 2-4 h; The volume ratio of the seabuckthorn puree after enzymatic hydrolysis to water is 1-1.5:3-6; The sterilization condition is 115-125°C and the time is 10-20 min; The inoculation amount of the Lactobacillus reuteri LY-1 is 4-6%; The fermentation culture conditions are: culture at 33-39° C. for 4-10 days.

7. A Lactobacillus reuteri according to claim 1 ( Lactobacillus reuteri ) Use of LY-1 or the microbial agent described in claim 2 in the preparation of fermented sea buckthorn juice.

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