Preparation method for enhancing inhibitory activity of lonicera caerulea juice xanthine oxidase through fermentation of plant lactobacillus

Abstract

According to the preparation method disclosed by the invention, the whole fruit juice of the lonicera caerulea (which retains fruit residues) is fermented by virtue of the plant lactobacillus KLDS1.0391, KLDS1.0320 or KLDS1.0368, so that the xanthine oxidase (XO) inhibition activity is remarkably improved. After fermentation, the XO inhibition rate is increased to 48-50.87% (the content of unfermented juice is 31.21%), the total phenol content is increased by 15-24.3%, and the contents of main XO inhibitors in the lonicera caerulea: cyanidin-3-O-glucoside (IC50 = 115.28 [mu] M), cyanidin-3, 5-diglucoside (IC50 = 116.34 [mu] M), peonidin-3-glucoside (IC50 = 105.34 [mu] M), quercetin (IC50 = 88.75 [mu] M), catechin (IC50 = 110 [mu] M) and caffeoylquinic acid (IC50 = 250.35 [mu] M) are remarkably increased. Wherein the quercetin content is increased by 73.8% at most (KLDS1.0320 strain fermentation is performed for 24 hours), and the XO inhibition rate is increased from 31.21% to 50.87%. Technical support is provided for development of a natural XO inhibitor and a functional fermented beverage, so that a new thought is provided for functional fermented juice with lonicera caerulea as a key component.

Description

Technical Field

[0001] The present invention relates to the fields of functional foods, biofermentation technology, and deep processing of agricultural products. Specifically, it relates to a method for preparing indigo fruit juice by fermenting it with a specific probiotic (Lactobacillus plantarum) to significantly enhance its xanthine oxidase (XO) inhibitory activity. This method aims to develop a functional beverage with potential health benefits, such as lowering uric acid and assisting in the prevention or relief of gout. Background Art

[0002] Blue loquat is a small, deciduous berry belonging to the genus Lonicera, Caprifoliaceae, and a deciduous shrub. Native to cold temperate regions and possessing strong frost resistance, it is widely cultivated in Heilongjiang Province, China. The rich abundance of bioactive compounds in blue loquat, particularly anthocyanins and phenolic acids, makes it a functional berry that has garnered significant research interest in contemporary science. In 2018, it was approved as a novel food (EU 2015 / 2283EU), further enhancing its commercial potential. However, its extremely thin peel and high juice content make the fresh fruit highly perishable, limiting direct sales and thus driving the production of processed products such as juice.

[0003] Blue loquat fruit has significant health-promoting properties, including antioxidant and antiproliferative activities, as well as α-amylase and α-glucosidase inhibition. Notably, its anthocyanins (400-457.5 mg / 100 g dry weight) have been shown to inhibit the activity of xanthine oxidase (XO), a key enzyme in uric acid synthesis, through in vitro experiments, animal models, and clinical studies. Furthermore, fermentation of the fruit juice with Lactobacillus plantarum can promote the release of phenolic active ingredients from the pomace, and its biotransformation can degrade anthocyanins into quercetin and phenolic acids, thereby increasing the content of active ingredients. Therefore, fermenting blue loquat fruit juice with Lactobacillus plantarum to enhance its functional activity and achieve high-value utilization of blue loquat fruit juice is of great significance. Summary of the Invention

[0004] The present invention uses Lonicera caerulea L. juice as raw material and ferments it with three strains of Lactobacillus plantarum to significantly enhance xanthine oxidase inhibitory activity and total phenol content. LC / MS targeted quantification of key phenolic compounds with XO inhibitory activity is performed: Table 1 shows the main active substances in Lonicera caerulea L. that inhibit XO, accounting for 89.75% of the phenolic substances identified by LC / MS. Its IC value for XO inhibition is 2.34. 50 :Cyanidin-3-O-glucoside (IC 50 =115.28 μM), cyanidin-3,5-diglucoside (IC 50 =116.34 μM), paeoniflorin-3-glucoside (IC50 =105.34 μM), quercetin (IC 50 =88.75μM), catechin (IC 50 =110 μM) and caffeoylquinic acid (IC 50 =250.35μM). Molecular docking revealed the site of action and mechanism of action of the target phenolic compounds in edulis. This study will provide a theoretical basis for the use of edulis as a natural XO inhibitor, thereby offering new ideas for functional fermented juices using edulis as a key ingredient. Table 1 Main XO inhibitors in Lonicera edulis and their IC values ​​for XO inhibition 50

[0005] In order to achieve the above object, the present invention is implemented through the following technical solutions: (1) Strain activation and preparation of indigo juice. The plant lactobacillus KLDS1.0368, KLDS1.0320 or KLDS1.0391 selected in this test was first inoculated into a sterile MRS medium at a 1% (v / v) inoculum size and cultured at 37°C for 24 hours, repeated twice to obtain activated plant lactobacillus. Then, the three plant lactobacillus strains were inoculated into fresh sterile MRS medium at an inoculum ratio of 3% (v / v), and cultured at 37°C for 14 hours (KLDS1.0368 / KLDS1.0320) or 11 hours (KLDS1.0391) to the end of the logarithmic phase. The preparation method of blue indigo juice comprises the following steps: washing blue indigo fruit, mixing distilled water and blue indigo fruit at a ratio of 1:1.5, crushing the fruit using a juicer, adding 5% (w / v) glucose, adjusting the pH to 6.5±0.1 with NaHCO3, pasteurizing at 85°C for 15 minutes, and cooling to 4°C for later use. (2) Fermentation process optimization: The optimal process was determined through experiments: fermentation temperature 37°C, fermentation time 16-24 h (KLDS1.0368 fermentation 16 h; KLDS1.0320 fermentation 24 h; KLDS1.0391 fermentation 20 h). (3) Preparation of functional fermented blue indigo fruit juice. After fermentation under optimized conditions, the reaction is terminated to obtain highly active blue indigo fruit fermented juice; the product can be freeze-dried for storage or directly used in functional beverages. (4) The functional components of the fermented blue indigo juice prepared by the present invention were significantly improved. The total phenol content increased by 15-24.3% (up to 1781.72 μg GAE / gDW); the content of key active substances (cyanidin-3-O-glucoside, cyanidin-3,5-diglucoside, paeoniflorin-3-glucoside, quercetin, catechins, and caffeoylquinic acid) increased by 8.7-73.8%; (5) The xanthine oxidase inhibition rate of the fermented indigo juice prepared by the present invention is increased to 48-50.87% (31.21% for unfermented juice). (6) Strain-specific advantages. The KLDS1.0368 strain simultaneously achieved the highest increase in active ingredients, with quercetin increasing by 24.3% and cyanidin-3-glucoside increasing by 73.8%. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 This is a comparison chart of the effects of fermentation with different strains on the inhibitory activity of xanthine oxidase (XO) in blue loquat fruit juice.

[0007] Figure 2 This is a comparison chart of the effects of fermentation with different strains on the total phenol content of blue loquat fruit juice.

[0008] Figure 3 This is a diagram showing the molecular docking interactions between xanthine oxidase and key XO inhibitors in blue indigo juice before and after fermentation: cyanidin-3-O-glucoside (A), cyanidin-3,5-diglucoside (B), paeoniflorin-3-glucoside (C), quercetin (D), catechin (E), and caffeoylquinic acid (F). DETAILED DESCRIPTION The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0009] Example 1: Fermentation of whole Indigo Carica fruit juice (without pomace) with Lactobacillus plantarum KLDS1.0320

[0010] Step 1: Strain activation: Lactobacillus plantarum KLDS1.0320 was removed from a -80°C storage tube and first inoculated into sterile MRS medium at a 1% (v / v) inoculum size, and cultured at 37°C for 24 hours, repeated twice to obtain activated KLDS1.0320. The activated KLDS1.0320 was then inoculated into fresh sterile MRS medium at an inoculum ratio of 3% (v / v), and cultured at 37°C for another 14 hours until the end of the logarithmic phase;

[0011] Step 2: Preparation of whole blue honeysuckle juice (retaining the pomace): After washing the blue honeysuckle, distilled water and blue honeysuckle were mixed in a ratio of 1:1.5, crushed with a juicer, and 5% (w / v) glucose was added. NaHCO3 was used to adjust the pH to 6.5±0.1, and the mixture was pasteurized at 85°C for 15 minutes and cooled to 4°C for later use.

[0012] Step 3: Fermentation process: The KLDS1.0320 bacterial suspension at the end of the logarithmic phase was centrifuged at 8000 g for 10 minutes at 4°C to collect the bacteria, washed twice with sterile phosphate buffered saline (PBS), and then resuspended in sterile PBS. The bacteria were then added to pasteurized blue indigo juice, and the initial bacterial concentration of the blue indigo juice was adjusted to approximately 1.0 × 10 8 All juices were fermented at 37°C for 24 h; samples were taken every 4 h to determine the xanthine oxidase (XO) inhibition rate and total phenolic content.

[0013] Step 4: The key indicator detection method is as follows

[0014] XO inhibition rate determination: Reaction system: 50 μL fermentation supernatant + 60 μL PBS buffer (0.075 mol / L, pH 7.5) + 30 μL XO solution (0.1 U / mL), pre-incubated at 25°C for 15 minutes; 60 μL xanthine substrate (0.05 mol / L) was added to initiate the reaction, and the absorbance change was monitored at a wavelength of 295 nm for 5 minutes. The calculation formula is: Results: As Figure 1 The XO inhibition rate reached 50.87% after 24 hours of fermentation (31.21% for unfermented juice).

[0015] Determination of total phenolic content: 100 μL of fermented or unfermented juice supernatant was quantitatively mixed with freshly prepared 10% Folin-Ciocalteu solution in a strict 1:1 ratio and then heat-incubated at 37°C for 10 minutes. Subsequently, 100 μL of 10% sodium carbonate solution was added, and the reaction mixture was maintained at 37°C in the absence of light for 60 minutes. Absorbance readings were obtained at a wavelength of 750 nm. A calibration curve was established using gallic acid standard solutions of varying concentrations prepared in distilled water. TPC was quantified and expressed as milligrams of gallic acid equivalents (GAE) per gram of dry weight (DW). Results: As Figure 2 After 24 h of fermentation, the total phenolic level increased to 1719.94 ± 2.82 μg GAE / g (1472.24 μg GAE / g in unfermented juice).

[0016] Quantification of Phenolic Compounds by HPLC-ESI-QTOF-MS2: Phenolic compounds were analyzed in the supernatants of unfermented and 24-h fermented L. edulis juices using KLDS1.0320. Anthocyanin and non-anthocyanin phenols were analyzed using high-performance liquid chromatography with diode array detection (HPLC-DAD) coupled to HPLC with electrospray ionization quadrupole time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS2) on an AB Sciex (CA, USA) system. Compound separation was achieved using a C18 column (Waters 1.7 μm, 100 mm × 2.1 mm, ACQUITY UPLC BEH). Anthocyanin detection was set at a wavelength of 520 nm, and non-anthocyanin phenols at a wavelength of 280 nm. The column temperature was maintained at 40°C, and the mobile phase flow rate was set at 0.4 mL / min. The eluents were water (formic acid, 0.1%) (A) and acetonitrile (formic acid, 0.2%) (B) with a gradient of 98-1% A (0-25 min), 1% A (25-30 min) and 98% A (31-35 min). The mass spectrometer settings included a full scan range of m / z 100-2000 (MS1) and 50-2000 (MS2). Anthocyanins were analyzed in positive ion mode, while non-anthocyanin phenolic compounds were detected in negative ion mode. In negative ion electrospray ionization (ESI), 4500 V was applied to the capillary, while positive polarity required 5500 V, and the drying gas was maintained at 550°C. As shown in Table 2, the content of the key active ingredient cyanidin-3-O-glucoside increased by 13.59% compared with the unfermented sample, the content of cyanidin-3,5-diglucoside increased by 33.45%, the content of paeoniflorin-3-glucoside increased by 8.99%, the content of quercetin increased by 73.84%, the content of catechin increased by 30.59% and the content of caffeoylquinic acid increased by 1.68%. Table 2 Key phenolic compounds increased before and after fermentation of blue indigo juice by KLDS1.0320

[0017] Example 2: Fermentation of Indigofera edulis whole juice (with pomace retained) with Lactobacillus plantarum KLDS1.0368

[0018] Step 1: Strain activation: Take out Lactobacillus plantarum KLDS1.0368 stored at -80°C, inoculate it into sterile MRS medium at an inoculum ratio of 1% (v / v), and culture it at 37°C for 24 hours. Repeat twice for activation. Then, inoculate the activated KLDS1.0368 into fresh sterile MRS medium at an inoculum size of 3% (v / v), and continue to culture it at 37°C for 14 hours until the end of the logarithmic phase;

[0019] Step 2: Preparation of whole Lonicera edulis juice (retaining the pomace): Wash the Lonicera edulis berries, mix them with distilled water in a ratio of 1:1.5, and crush them in a juicer. Add sugar: 5% (w / v) glucose; adjust pH: 0.1 mol / L NaHCO3 solution to pH 6.5 ± 0.05; sterilize: in an 85°C water bath for 15 minutes, then cool to 4°C.

[0020] Step 3: Fermentation process: The KLDS1.0368 bacterial suspension at the end of the logarithmic phase was centrifuged at 8000 g for 10 minutes at 4°C. The bacteria were collected, washed twice with sterile phosphate buffered saline (PBS), and then resuspended in sterile PBS. The bacteria were then added to pasteurized blue indigo juice, and the initial bacterial concentration of the blue indigo juice was adjusted to approximately 1.0 × 10 8 All juices were fermented at 37°C for 24 h; samples were taken every 4 h to determine the xanthine oxidase (XO) inhibition rate and total phenolic content.

[0021] Step 4: Key indicator detection

[0022] XO inhibitory activity was determined as described in

[0016] . Results: As Figure 1 The XO inhibition rate was 48.93% after 16 h of fermentation (31.21% for unfermented juice).

[0023] The total phenol content was determined as described in

[0017] Results: As Figure 2 The total phenolic content reached 1781.72±0.68 μg GAE / g after 16 h of fermentation (1472.24 μg GAE / g in unfermented juice).

[0024] The HPLC-ESI-QTOF-MS2 method was used to identify and quantify phenolic compounds as described in

[0018] . As shown in Table 3, the content of the key active ingredient cyanidin-3-O-glucoside increased by 24.26%, the content of cyanidin-3,5-diglucoside increased by 41.26%, the content of paeoniflorin-3-glucoside increased by 4.23%, the content of quercetin increased by 31.85%, the content of catechin increased by 53.21% and the content of caffeoylquinic acid increased by 16.03% compared with the unfermented sample. Table 3 Key phenolic compounds increased before and after fermentation of blue indigo juice by KLDS1.0368

[0025] Example 3: Fermentation of Indigofera edulis whole juice (with pomace retained) with Lactobacillus plantarum KLDS1.0391

[0026] Step 1: Time for the late logarithmic phase of bacterial strain activation: 11 hours; other steps are the same as those in Example 1.

[0027] Step 2: Preparation of whole blueberry juice (retaining pomace): same as in Example 1

[0028] Step 3: Fermentation process: The KLDS1.0391 bacterial suspension at the end of the logarithmic phase was centrifuged at 8000 g for 10 minutes at 4°C to collect the bacteria, washed twice with sterile phosphate-buffered saline (PBS), and then resuspended in sterile PBS. The bacteria were then added to pasteurized blue indigo juice, and the initial bacterial concentration of the blue indigo juice was adjusted to approximately 1.0 × 10 8 All juices were fermented at 37°C for 24 h; samples were taken every 4 h to determine the xanthine oxidase (XO) inhibition rate and total phenolic content.

[0029] Step 4: Key indicator detection

[0030] XO inhibitory activity was determined as described in

[0016] . Results: As Figure 1 As shown, the XO inhibition rate reached 48.07% after 16 hours of fermentation (31.21% for unfermented juice).

[0031] The total phenol content was determined as described in

[0017] Results: As Figure 2 The total phenolic content reached 1695.96±2.55 μg GAE / g after 16 hours of fermentation (1472.24 μg GAE / g in unfermented juice).

[0032] The HPLC-ESI-QTOF-MS2 method was used to identify and quantify phenolic compounds as described in

[0018] . As shown in Table 4, the content of the key active ingredient, cyanidin-3-O-glucoside, increased by 8.72% compared to the unfermented sample; the content of cyanidin-3,5-diglucoside increased by 23.38%; the content of paeoniflorin-3-glucoside increased by 29.07%; the content of quercetin increased by 30.95%; the content of catechin increased by 21.09%; and the content of caffeoylquinic acid increased by 13.19%. Table 4 Key phenolic compounds increased before and after fermentation of KLDS1.0391 blue indigo juice

Claims

1. A method for preparing Lactobacillus plantarum fermented blue indigo juice to enhance the xanthine oxidase inhibitory activity, characterized by the following steps: (1) Lactobacillus plantarum is KLDS1.0391, KLDS1.0320 or KLDS1.0368; (2) Indigo Fruit Juice The fermentation process uses whole fruit juice of Indigo Fruit (retaining the pomace) as raw material.

2. The method according to claim 1, wherein: Fermentation time is optimized according to the strain: KLDS1.0391 fermentation for 20h KLDS1.0320 fermentation for 24 hours KLDS1.0368 was fermented for 16 h.

3. The method according to claim 1, wherein: After fermentation with Lactobacillus plantarum, the xanthine oxidase (XO) inhibition rate of blue indigo juice increased to 45-51%, and the total phenol content increased by 15%-25%.

4. The method according to claims 1-3, characterized in that: Key active ingredients increase after fermentation with Lactobacillus plantarum: The content of cyanidin-3-glucoside increased by 8.7%-24.3% (KLDS1.0391 / KLDS1.0368), and the content of quercetin increased by 31%-74% (KLDS1.0320).

Images