Application of macroporous resin assisted ginkgo biloba juice fermented by probiotics in attenuating and enhancing efficacy

Abstract

The application discloses a method for reducing toxicity and increasing efficiency of ginkgo biloba juice fermented by macroreticular resin assisted probiotics, and the strains are ginkgo endogenous lactobacillus, and the names are lactobacillus plantarum TY-HYB-SYY-Y2 and lactobacillus pentosus TY-HYB-SYY-Y3 , and the preservation numbers of the China General Microbiological Culture Collection Center are CGMCC No. 25128 and CGMCC No. 25127 respectively; the macroreticular resin is a macroreticular adsorption resin. The application finds that the ginkgo endogenous lactobacillus can promote the reduction of the content of toxic substances such as ginkgoic acid and ginkgo poison during the fermentation of ginkgo biloba juice, and at the same time, the content of ginkgo lactone is increased, and it is found that the content of ginkgo acid is further reduced during the macroreticular resin assisted fermentation process, and a more obvious reduction effect is achieved.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, specifically to the application of *Lactobacillus endophyticus* (TY-HYB-SYY-Y2, TY-HYB-SYY-Y3) in fermenting ginkgo juice to reduce ginkgolic acid and ginkgolic toxicity, and increase the content of ginkgolides; and to the further reduction of ginkgolic acid content when macroporous resin is added. Background Technology

[0002] Ginkgo, known as a living fossil, has existed on Earth for 200 million years. Its seeds, ginkgo nuts, are a traditional food source due to their rich content of carbohydrates, proteins, lipids, vitamins, and various other functional substances. Key components of ginkgo nuts include flavonoids, terpene lactones, bifidobacteria, proanthocyanidins, and ginkgolic acid. Clinical studies have shown that consuming ginkgo nuts can enhance memory and alleviate symptoms of Alzheimer's disease; among these, ginkgolic acid terpene lactones are effective and selective platelet-activating factor antagonists.

[0003] However, due to the presence of potentially toxic components such as ginkgolic acid, 4′-O-methylpyridoxine, hydrocyanic acid, and allergenic proteins in ginkgo seeds, only a small portion of ginkgo seeds are used in the food industry. Ginkgoolic acid is a series of n-alkylphenolic acid compounds with similar and regular structures. The alkyl side chain in the molecular structure is between 13 and 17 carbons long, with 0-2 double bonds. Patent 201611101062.7 discloses a method to significantly reduce the ginkgolic acid content in ginkgo through a combination of heat treatment, mechanical core removal, and soaking in a low-concentration alkaline solution. 4′-O-methylpyridoxine (MPN) is a vitamin B6 analog and is known as ginkgotoxin. It is the main toxic substance in ginkgo. MPN can disrupt the balance between γ-aminobutyric acid and glutamate, thereby causing convulsions. Its cytotoxicity, allergenicity, immunotoxicity, and mutagenic potential should not be ignored.

[0004] Lactic acid bacteria are widely distributed in the environment, and in recent years, more and more lactic acid bacteria have been used in food to improve food flavor and promote digestion and absorption. During fermentation, lactic acid bacteria can promote the metabolism of phenolic substances, thereby enriching the nutrients in food and ensuring food safety. Patent 201811273902.7 describes a method using *Aspergillus cristatus* to solid-state ferment ginkgo biloba powder, effectively degrading ginkgolic acid and MPN while retaining most of the nutritional and functional components, and significantly improving the flavor. Plant endophytes are normal colonies in the micro-ecosystem of plant growth, rich in species, adapting to the plant environment and forming mutually beneficial symbiotic relationships with plants, possessing significant economic and ecological value. For example, patent 202110435091.1 screened two types of fruit-derived endophytic lactobacilli to ferment blueberry juice, achieving a viable count as high as 10.0 log CFU / ml and significantly increasing the flavonol content, antioxidant activity, and phenolic acid decarboxylase activity of the blueberry juice. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention primarily utilizes screening of endogenous bacteria from ginkgo biloba to reduce ginkgolic acid and MPN content and increase ginkgolides content during the fermentation of ginkgo juice. Furthermore, with the assistance of macroporous resin, the ginkgolic acid content can be further reduced. This method aims to improve food safety, enhance food flavor, increase nutritional value, and produce fermented ginkgo juice with probiotic activity.

[0006] The technical solution adopted by this invention to solve its technical problem is:

[0007] In the first aspect, this invention protects a type of endophytic Lactobacillus plantarum TY-HYB-SYY-Y2, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on June 20, 2022, with accession number CGMCC NO.25128.

[0008] This invention also protects a species of endophytic Lactobacillus pentosus TY-HYB-SYY-Y3 from Ginkgo biloba, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on June 20, 2022, with accession number CGMCC NO.25127.

[0009] Through physiological and biochemical experiments and 16S rDNA sequence comparison, this invention identified the endophytic lactobacilli TY-HYB-SYY-Y2 and TY-HYB-SYY-Y3 from Ginkgo biloba as Lactobacillus plantarum and Lactobacillus pentosus, respectively. Both strains have excellent toxicity reduction and efficacy enhancement effects.

[0010] Secondly, this invention protects the use of *Lactobacillus plantarum* TY-HYB-SYY-Y2 and / or *Lactobacillus pentosus* TY-HYB-SYY-Y3 in the preparation of live-culture fermented white fruit juice.

[0011] This invention also protects the application of the aforementioned *Lactobacillus plantarum* TY-HYB-SYY-Y2 in reducing ginkgolic acid content, and / or reducing ginkgolic acid content, and / or increasing ginkgolide content in fermented white fruit juice.

[0012] This invention also protects the application of the aforementioned Lactobacillus pentosolicus TY-HYB-SYY-Y3 in reducing ginkgolic acid content, and / or reducing ginkgolic toxin content, and / or increasing ginkgolide content in fermented white fruit juice.

[0013] This invention also protects the use of the aforementioned *Lactobacillus plantarum* TY-HYB-SYY-Y2 and the aforementioned *Lactobacillus pentosaccharide* TY-HYB-SYY-Y3 in the combined use of these two strains in fermented white fruit juice to reduce ginkgolic acid content, and / or reduce ginkgolic acid content, and / or increase ginkgolide content.

[0014] Preferably, the ginkgolic acid is ginkgolic acid C15:1 (ginkgolic acid) or ginkgolic acid C13:0 (ginkgolic acid), the ginkgotoxin is 4′-O-methylpyridoxine (MPN), and the ginkgolides refer to ginkgolide A, ginkgolide B, and ginkgolide C.

[0015] Specifically, this invention protects the application of *Lactobacillus plantarum* TY-HYB-SYY-Y2 and / or *Lactobacillus pentosus* TY-HYB-SYY-Y3, as described above, in reducing the content of ginkgolic acid C15:1 and ginkgolic acid C13:0 in fermented white fruit juice.

[0016] Although ginkgolic acid is a series of n-alkylphenolic acid compounds with similar and regular structures, ginkgolic acid and ginkgolic neonic acid account for more than 80% of them. The two strains of the present invention significantly reduce the content of ginkgolic acid and ginkgolic neonic acid, that is, significantly reduce the content of ginkgolic acid in ginkgolic juice.

[0017] Specifically, this invention protects the application of *Lactobacillus plantarum* TY-HYB-SYY-Y2 and / or *Lactobacillus pentosus* TY-HYB-SYY-Y3, as described above, in reducing the content of 4′-O-methylpyridoxine (MPN) in fermented white fruit juice.

[0018] The two strains of the present invention can significantly reduce the content of MPN in ginkgo biloba juice. MPN is the main toxic substance in ginkgo toxin, and reducing the content of MPN has a significant effect on reducing the toxicity of ginkgo biloba juice.

[0019] Specifically, this invention protects the use of *Lactobacillus plantarum* TY-HYB-SYY-Y2 and / or *Lactobacillus pentosus* TY-HYB-SYY-Y3, as described above, in increasing the content of ginkgolide A, ginkgolide B, and ginkgolide C in fermented white fruit juice.

[0020] Thirdly, this invention also protects the application of Lactobacillus plantarum TY-HYB-SYY-Y2 in combination with macroporous resin to reduce the content of ginkgolic acid in fermented white fruit juice.

[0021] This article also protects the application of Lactobacillus pentosolicus TY-HYB-SYY-Y3 in combination with macroporous resin in reducing the ginkgolic acid content in fermented white fruit juice.

[0022] Macroporous resins, with their network-like pore structure, large particle surface area, and certain polar groups, possess a large adsorption capacity to achieve separation, purification, and concentration. When lactic acid bacteria are combined with macroporous resins, they can further reduce the content of ginkgolic acid.

[0023] Preferably, the macroporous resin is a macroporous adsorption resin, and more preferably macroporous resin D101.

[0024] The present invention also protects the culture conditions of Lactobacillus plantarum TY-HYB-SYY-Y2 and Lactobacillus pentosus TY-HYB-SYY-Y3 as follows: 37±1℃, pH6.2±0.2.

[0025] Preferably, the culture conditions for Lactobacillus plantarum TY-HYB-SYY-Y2 and Lactobacillus pentosus TY-HYB-SYY-Y3 are: 37℃, pH 6.2.

[0026] Beneficial effects

[0027] (1) In this invention, two strains of *Lactobacillus plantarum*, TY-HYB-SYY-Y2 and TY-HYB-SYY-Y3, were screened, with accession numbers CGMCC NO.25128 and CGMCC NO.25127, respectively. Both strains showed good detoxification and efficacy enhancement effects against ginkgo biloba.

[0028] (2) After fermentation with Lactobacillus plantarum TY-HYB-SYY-Y2, the total ginkgolic acid content of white juice decreased by 68.82%, the MPN content decreased by 56.1%, and the ginkgolide content increased by 117.2%. After adding macroporous resin to assist fermentation, the total ginkgolic acid content further decreased to 85.40%.

[0029] (3) After fermentation with Lactobacillus pentosus TY-HYB-SYY-Y3, the total ginkgolic acid content of the white juice decreased by 60.13%, the MPN content decreased by 53.5%, and the ginkgolide content increased by 62.63%. After adding macroporous resin to assist fermentation, the total ginkgolic acid content further decreased to 84.74%.

[0030] (4) After the above treatment, the content of toxic substances in white juice is significantly reduced, which improves the safety of white juice; the flavor of white juice is improved, the operation is simple, and it is easy to promote industrially. Attached Figure Description

[0031] Figure 1 , Figure 2 Gram staining morphology of Lactobacillus plantarum TY-HYB-SYY-Y2 and Lactobacillus pentosaccharide TY-HYB-SYY-Y3;

[0032] Figure 3 , Figure 4 A comparison of 16S rDNA sequencing results between Lactobacillus plantarum TY-HYB-SYY-Y2 and Lactobacillus pentosus TY-HYB-SYY-Y3;

[0033] Figure 5 The effect of macroporous resin D101 combined with Lactobacillus plantarum TY-HYB-SYY-Y2 and Lactobacillus pentosaccharide TY-HYB-SYY-Y3 on ginkgolic acid content;

[0034] Figure 6 The effect of macroporous resin D101 combined with Lactobacillus plantarum TY-HYB-SYY-Y2 and Lactobacillus pentosacchari TY-HYB-SYY-Y3 on MPN content;

[0035] Figure 7 The effect of macroporous resin D101 combined with Lactobacillus plantarum TY-HYB-SYY-Y2 and Lactobacillus pentosacchari TY-HYB-SYY-Y3 on ginkgolide content;

[0036] Figure 8 The effect of macroporous resin D101 combined with Lactobacillus plantarum TY-HYB-SYY-Y2 and Lactobacillus pentosus TY-HYB-SYY-Y3 on total phenol content. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to the embodiments. Reagents or instruments used without a specified manufacturer are considered to be conventional products that can be purchased on the market.

[0038] This invention screened a strain of *Lactobacillus plantarum* TY-HYB-SYY-Y2, an endophytic plant-derived from ginkgo, which is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on June 20, 2022, with accession number CGMCC NO.25128.

[0039] This invention also screened a strain of *Lactobacillus pentosus* TY-HYB-SYY-Y3, an endophytic *Lactobacillus pentosus*, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on June 20, 2022, with accession number CGMCC NO.25127.

[0040] Example 1: Isolation and Identification of Endophytic Strains from Ginkgo Biloba

[0041] 1. Isolation of endophytic strains from Ginkgo biloba

[0042] Five whole, plump, and pest-free ginkgo nuts (variety, harvested from Jiangsu Ocean University campus in October 2021 and stored at -20℃) were selected and homogenized in an appropriate amount of physiological saline. The enrichment culture was serially diluted with sterile physiological saline and then spread onto MRS agar medium at appropriate gradients. The culture was incubated at 37℃ for 36-48 hours, and round, plump colonies were picked. The selected single colonies were further isolated and purified using the "three-zone streak" method.

[0043] 2. Identification of strains

[0044] After the above strains were fixed, they were subjected to primary staining, enzyme staining, decolorization, and counterstaining for Gram staining. The morphology, color, and size of the strains were observed under a microscope. Gram-positive bacteria were selected, inoculated into white juice, and fermented at 37°C for 24 hours to determine the following indicators.

[0045] (1) Select fermented white juice and dilute it with physiological saline gradient. Select 2-3 appropriate dilution gradients, take 0.1 ml of diluted white juice into MRS agar medium and shake well. Incubate at 37℃ for 36-48 h. Select colony counts between 30-300.

[0046] (2) Physiological and biochemical experiments

[0047] Peroxidase assay: Select one loop of fermentation broth, spread it evenly on a clean glass slide, add an appropriate amount of 10% hydrogen peroxide solution, and observe whether bubbles are produced. If bubbles are produced, it is recorded as positive (+), and if no bubbles are produced, it is recorded as negative (-).

[0048] Starch hydrolysis experiment: Take an appropriate amount of fermentation broth on a glass slide, add an appropriate amount of Lugol's iodine solution, and compare it with the uninoculated fermentation broth. If no color is developed, it indicates that the starch has been hydrolyzed (+). If a blue-black or blue-purple color is developed, it indicates that the starch has not been hydrolyzed or has not been completely hydrolyzed (-).

[0049] The results of strain identification are shown in Table 1.

[0050] Table 1 Results of Gram staining experiment, catalase assay, and starch hydrolysis experiment

[0051]

[0052] 16S rDNA sequencing: Genomic DNA of lactic acid bacteria was extracted as an amplification template, and its concentration and purity were detected. PCR amplification was then performed using the template and fixed primers, and the samples were sent to Shanghai Baiqu Biomedical Technology Co., Ltd. for sequencing. The sequencing results were compared with those in the GenBank database. The sequencing results are as follows: Figure 3 As shown in Figure 4.

[0053] The above experiments have proven that TY-HYB-SYY-Y2 in this invention is Lactobacillus plantarum, and TY-HYB-SYY-Y3 is Lactobacillus pentosus.

[0054] Example 2: Study on the fermentation characteristics of the above-mentioned strains in white fruit juice

[0055] 1. Pretreatment of macroporous resin D101

[0056] The macroporous resin was soaked in ethanol for 24 hours, and after removing the ethanol, it was thoroughly washed with distilled water. Then, it was soaked in 6 times its volume of 4% HCl solution for 3 hours, and washed with distilled water until neutral. Next, it was soaked in 6 times its volume of 5% NaOH solution for 3 hours, and washed thoroughly with distilled water until neutral. Finally, the pretreated resin was dried at 30°C to constant weight.

[0057] 2. Preparation of Seed Liquid

[0058] Place the pre-preserved lactic acid bacteria in 50 ml of sterilized MRS broth medium and incubate at 37°C for 24 h until the colony count reaches 9.0 log CFU / ml for later use.

[0059] 3. Preparation of white juice

[0060] Select plump ginkgo nuts, wash them, add water at a ratio of 1:5 and blend them into a pulp. Then add α-amylase (20 U / g) and saccharifying enzyme (30 U / g), and enzymatically hydrolyze at 60℃ for 2 hours. After that, centrifuge at 5000 rpm for 20 minutes, and finally sterilize in a water bath at 90℃ for 20 minutes. Store in a refrigerator at -4℃.

[0061] 4. White fruit juice fermentation

[0062] Sterilized macroporous resin D101 was added to white juice cooled to room temperature (20 g / L). Seed culture was then inoculated into the white juice containing macroporous resin at a 2% inoculum. The mixture was cultured at 30°C and 150 rpm for 48 h with shaking. The culture conditions were 37°C and pH = 7.0. After centrifugation at 10000 rpm for 10 min, the mixture was ready for use.

[0063] 5. Study on fermentation characteristics

[0064] (1) Determination of Ginkgo Biloba Acid Content

[0065] Take 50 ml of the fermented sample, rotary evaporate to 5 ml, add the same volume of methanol, and sonicate at 30℃, 250W for 30 min, centrifuge at 10000 rpm for 10 min to remove the precipitate. Take the supernatant, rotary evaporate to remove methanol, add 10 ml of n-hexane for extraction three times, combine the organic phases, rotary dry, dissolve in methanol and make up to 1 ml. After the sample passes through a 0.45 μm organic filter membrane, ginkgolic acid is analyzed by HPLC-UV. The chromatographic column is YMC Carotenoid (4.6×150 mm, 3 μm); the mobile phase is 3% glacial acetic acid:methanol (8:92 v / v); the flow rate is 0.8 ml / min; the detection wavelength is 310 nm; and the column temperature is 30℃. The results are as follows. Figure 5 As shown.

[0066] The results showed that after fermentation with TY-HYB-SYY-Y2 and TY-HYB-SYY-Y3 for 48 hours, the ginkgolic acid content decreased by 68.82% and 60.13%, respectively. Under the same fermentation conditions, the addition of macroporous resin D101 to assist fermentation reduced the ginkgolic acid content by 85.40% and 84.74%, respectively. Furthermore, after 48 hours of fermentation with D101, ginkgolic acid was not detected. This indicates that macroporous resin D101, as a styrene-type nonpolar copolymer, has a large internal pore structure and strong adsorption capacity. During probiotic fermentation, it can help reduce the ginkgolic acid content, further improving the safety of ginkgo juice.

[0067] 6. MPN content determination

[0068] MPN was analyzed using HPLC-UV. An Agilent TC-C18 column (4.6 × 250 mm, 5 μm) was used. Mobile phase A consisted of 5 mM potassium phosphate solution containing 5 mM sodium pentanesulfonate (pH 2.5), and mobile phase B consisted of acetonitrile. Gradient conditions were as follows: 0 min, 4% B, 96% A (v / v); 0–10 min, 8% B–92% A (v / v); 10–15 min, 10% B–90% A (v / v); 15–20 min, 8% B–92% A (v / v); 20–40 min, 4% B and 96% A (v / v); column temperature: 30℃; flow rate: 1 ml / min. MPN content changes were shown below. Figure 6 As shown.

[0069] Depend on Figure 6 It can be seen that the MPN content decreased significantly after fermentation with the two types of Ginkgo biloba endophytic bacteria, from 5.85±0.40 mg / L before fermentation to 2.57±0.17 mg / L (TY-HYB-SYY-Y2) and 2.72±0.77 mg / L (TY-HYB-SYY-Y3). Meanwhile, the macroporous resin D101 had no significant effect on the MPN content during fermentation, indicating that it does not adsorb MPN.

[0070] 7. Determination of Ginkgolide Content

[0071] Take 50 ml of the fermented sample, rotary evaporate to 5 ml, add the same volume of methanol, sonicate at 30℃ and 250W for 30 min, centrifuge to remove the precipitate, take the supernatant, rotary evaporate to remove methanol, add 10 ml of ethyl acetate to extract three times, combine the organic phases and rotary dry, dissolve in methanol and make up to 1 ml, the sample is filtered through a 0.45 μm organic filter membrane and then used for HPLC detection.

[0072] HPLC-ELSD was used for detection: the chromatographic column was a Krosmail 100-C18 (250×4.6mm, 5μm); the mobile phase was water:methanol:tetrahydrofuran (65:25:10 v / v); the flow rate was 1 ml / min; the column temperature was 30℃; the injection volume was 20 μl; the nitrogen flow rate of the evaporative light detector was approximately 3.5 bar; and the evaporation temperature was 50℃. The results are as follows: Figure 7 As shown.

[0073] Mass spectrometry results showed that ginkgo biloba contains three ginkgolides: ginkgolide A, ginkgolide B, and ginkgolide C. After fermentation with *Lactobacillus plantarum* TY-HYB-SYY-Y2 and *Lactobacillus pentosus* TY-HYB-SYY-Y3, the total ginkgolide content increased by 117.23% and 62.63%, respectively. However, after fermentation with macroporous resin D101, the ginkgolide content decreased due to the adsorption effect of the macroporous resin, but it was still higher than that of unfermented ginkgo juice.

[0074] 8. Determination of total phenol content

[0075] The total phenol content was determined using the Folin-Ciocalteu method. 0.2 ml of appropriately diluted fermented sample solution was taken, and 1.5 ml of Folin-Ciocalteu reagent diluted 10 times was added. After mixing, 1.5 ml of 7.5% Na₂CO₃ solution was added and mixed again. The mixture was then reacted at room temperature in the dark for 2 hours. The absorbance was measured at 765 nm. A standard curve was established using gallic acid as the standard. The unit is mg gallic acid / L. The total phenol content is as follows: Figure 8 As shown.

[0076] The phenolic compounds in ginkgo have antioxidant properties, which can regulate the cardiovascular system and reduce the incidence of cardiovascular diseases. The total phenol content increased under the fermentation of two types of ginkgo endophytic bacteria, possibly because enzymes produced during lactic acid bacteria growth hydrolyze bound phenols into free phenols, thus increasing the total phenol content. Similarly, the addition of macroporous resin reduced the total phenol content, but it was still higher than before fermentation.

[0077] This invention only protects the ability of macroporous resin D101 to assist probiotics in reducing the ginkgolic acid content in white fruit juice, and does not target other macroporous resins that also have this characteristic.

[0078] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A type of Lactobacillus pentosus TY-HYB-SYY-Y3, endogenous Lactobacillus pentosus, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 25127.

2. A microbial inoculant comprising Lactobacillus pentosus TY-HYB-SYY-Y3 as described in claim 1.

3. The application of Lactobacillus pentosolicus TY-HYB-SYY-Y3 as described in claim 1 in reducing ginkgolic acid content and / or reducing ginkgolic toxicity content and / or increasing ginkgolide content in fermented white fruit juice, characterized in that, The ginkgolic acid is ginkgolic acid C15:1 or ginkgolic acid C13:0, the ginkgotoxin is 4′-O-methylpyridoxine, and the ginkgolides refer to ginkgolide A, ginkgolide B, and ginkgolide C.

4. The application of Lactobacillus pentosolicus TY-HYB-SYY-Y3 as described in claim 1, in combination with macroporous resin to reduce ginkgolic acid content, is characterized in that... The macroporous resin is a macroporous adsorption resin, and the ginkgolic acid is ginkgolic acid C15:1 or ginkgolic acid C13:

0.

5. The application according to claim 4, characterized in that, The macroporous resin is macroporous resin D101.

Images