Lactobacillus plantarum and application thereof in moistening intestines and defecating

Abstract

The present application relates to the technical field of microbial fermentation, and particularly relates to a lactobacillus plantarum and application thereof in moistening intestines and defecation. The strain is lactobacillus plantarum RX-261, which is preserved in China Center for Type Culture Collection, and is classified and named as Lactobacillus plantarum RX-261, with a preservation number of CCTCC NO: M 2026428, a preservation date of March 11, 2026, and a preservation address of China Center for Type Culture Collection, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The lactobacillus plantarum RX-261 provided by the present application can be used to improve the content of hesperidin in pericarpium citri reticulatae, and fermented pericarpium citri reticulatae powder can promote small intestinal movement of constipated mice, shorten the first defecation time, and has a good effect of moistening intestines and defecation.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a type of Lactobacillus plantarum and its applications. Background Technology

[0002] Dried tangerine peel is a type of tangerine (Citrus reticulata) belonging to the Rutaceae family. Citrus reticulata The dried, mature peel of *Citrus medica* (Blanco) and its cultivated varieties is a commonly used traditional Chinese medicine, possessing properties such as regulating qi and strengthening the spleen, drying dampness and resolving phlegm, and harmonizing the stomach and relieving nausea. Modern research shows that dried tangerine peel is rich in various flavonoids, with hesperidin being a representative active ingredient. Its content directly affects the quality and functional activity of the peel. Hesperidin is a dihydroflavonoid compound with various biological activities, including antioxidant, anti-inflammatory, intestinal flora regulation, and intestinal barrier protection, making it valuable in the food and pharmaceutical fields. However, traditional processing methods for dried tangerine peel mainly rely on natural aging, which is time-consuming and makes it difficult to precisely control the content of active ingredients, resulting in relatively low utilization of hesperidin.

[0003] In recent years, microbial fermentation technology has been widely used in the research on the functional enhancement of traditional Chinese medicine and natural products. Microorganisms can improve the cell wall breaking efficiency of plants through metabolism and the action of secreted enzymes, promote the release and transformation of effective components, and thus improve the comprehensive utilization rate of traditional Chinese medicine resources.

[0004] Therefore, developing a method to effectively increase hesperidin content is an urgent problem to be solved in this field. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides a *Lactobacillus plantarum* and its application.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A species of Lactobacillus plantarum, RX-261, has been deposited at the China Center for Type Culture Collection (CCTCC), with the classification name Lactobacillus plantarum RX-261, accession number CCTCC NO: M 2026428, deposit date March 11, 2026, and address CCTCC, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0007] This invention provides a microbial inoculant, the active ingredient of which is the above-mentioned Lactobacillus plantarum RX-261.

[0008] This invention does not impose any particular limitation on the dosage form of the microbial agent; it can be a solid dosage form, a liquid dosage form, or other types of dosage forms in the art. This invention can add excipients or other components to the microbial agent according to usage requirements.

[0009] This invention provides the application of Lactobacillus plantarum RX-261 in increasing the hesperidin content in dried tangerine peel.

[0010] This invention provides the application of microbial agents in increasing the hesperidin content in dried tangerine peel.

[0011] A method for increasing the hesperidin content in dried tangerine peel involves using the aforementioned *Lactobacillus plantarum* RX-261 or the aforementioned microbial agent to increase the hesperidin content in dried tangerine peel.

[0012] Furthermore, Lactobacillus plantarum RX-261 or the above-mentioned microbial agents are inoculated into the tangerine peel fermentation medium and fermented to obtain fermented tangerine peel powder.

[0013] Furthermore, the inoculum size of Lactobacillus plantarum RX-261 was 1-13%.

[0014] Furthermore, the fermentation temperature was 37℃, and the fermentation time was 2 days.

[0015] The present invention also provides a fermented tangerine peel powder, which is prepared by the above method.

[0016] This invention provides an application of fermented tangerine peel powder in the preparation of laxative products.

[0017] The products mentioned above include, but are not limited to, one or more of the following: pharmaceuticals, food, and health products.

[0018] The above technical solution offers the following benefits: This invention uses *Lactobacillus plantarum* RX-261 to ferment dried tangerine peel, and the changes in hesperidin content in the peel before and after fermentation are determined by liquid chromatography. The results show that after fermentation with *Lactobacillus plantarum* RX-261, the hesperidin content in the tangerine peel is significantly increased, with an increase rate reaching up to 112%, effectively overcoming the problem of slow and unstable increase in active ingredients during the traditional aging process of dried tangerine peel. Animal experiments using a constipated mouse model demonstrate that the fermented tangerine peel powder prepared in this invention can promote small intestinal motility in mice, shorten the time to first defecation, and exhibit a good laxative effect. This invention has promising application prospects in the field of intestinal health regulation. Attached Figure Description

[0019] Figure 1 This is a graph showing the change in hesperidin content in dried tangerine peel before and after fermentation according to the present invention. * in the graph indicates a comparison with the hesperidin content before fermentation. P <0.05, *** indicates a comparison with the hesperidin content before fermentation. P <0.001; Figure 2 The percentage increase in hesperidin content in tangerine peel before and after fermentation according to this invention; Figure 3This invention illustrates the effect of fermented tangerine peel powder on the propulsion rate of ink in the small intestine of mice. ** in the figure represent a comparison with the negative control group. P <0.01, # indicates comparison with the model group, P <0.05, ## indicates comparison with the model group, P <0.001; Figure 4 To illustrate the effect of fermented tangerine peel powder of this invention on the time of first black stool excretion in cases of constipation, ** in the figure indicates a comparison with the negative control group. P <0.01, # indicates comparison with the model group, P <0.05, ## indicates comparison with the model group, P <0.001. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Unless otherwise specified, all techniques or conditions used in the examples are conventional methods or performed according to techniques or conditions described in the literature in this field, or according to product instructions. Reagents used, unless otherwise specified, are all conventional products that can be purchased from legitimate channels or prepared according to conventional methods in this field. Instruments used, unless otherwise specified, are all conventional products that can be purchased from legitimate channels.

[0022] The dried tangerine peel provided in this invention was purchased from the Bozhou Traditional Chinese Medicine Market in China.

[0023] Example 1

[0024] The strain *Lactobacillus plantarum* RX-261 provided in this invention is a new strain screened by the applicant's laboratory. The depository institution is the China Center for Type Culture Collection (CCTCC), and the classification name is *Lactobacillus plantarum* RX-261. The accession number is CCTCC NO: M 2026428, the deposit date is March 11, 2026, and the deposit address is China Center for Type Culture Collection, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0025] 1. Preparation method of fermented tangerine peel powder (1) The dried tangerine peel was pulverized and passed through a 100-mesh sieve to obtain tangerine peel powder with uniform particle size. 60g of tangerine peel powder was accurately weighed and poured into a wide-mouth bottle, and 600 mL of sterile water was added and mixed well. The mixture was then autoclaved at 121℃ for 20 min and cooled to room temperature to obtain tangerine peel fermentation culture medium. (2) Activate Lactobacillus plantarum RX-261 and inoculate it into MRS liquid medium at an inoculation rate of 2% (V / V). Incubate at 37℃ for 24-36 h to obtain Lactobacillus plantarum RX-261 fermentation broth. (3) The fermentation broth obtained in step (2) was inoculated into the tangerine peel fermentation medium prepared in step (1) at inoculation amounts of 1%, 3%, 5%, 7%, 9%, 11% and 13% (V / V), respectively, and fermented at 37℃ for 2 days. After fermentation, the bacterial solution was freeze-dried, and the freeze-dried fermented material was pulverized and passed through a 40-mesh sieve to obtain fermented tangerine peel powder.

[0026] 2. The content of hesperidin in tangerine peel before and after fermentation was detected. (1) Preparation of test solution: Accurately weigh 0.5 g of dried tangerine peel powder and fermented dried tangerine peel powder respectively, place them in a 10 mL volumetric flask, add 10 mL of chromatographic grade methanol, sonicate for 20 min at a power of 250 W, cool to room temperature, add chromatographic grade methanol to make up to 10 mL, mix thoroughly, filter through a 0.22 μm microporous membrane, and take the filtrate to obtain the test solution. Each sample is tested in triplicate.

[0027] (2) Preparation of reference solution: Accurately weigh 10 mg of hesperidin reference standard, add an appropriate amount of dimethyl sulfoxide to dissolve it completely, transfer it to a 10 mL volumetric flask, and dilute to the mark with chromatographic grade methanol to prepare a stock solution with a concentration of 1.0 mg / mL. When using, dilute with chromatographic grade methanol to the required concentration.

[0028] (3) Liquid Chromatography (HPLC) conditions: A Waters 2695 high-performance liquid chromatograph (HPLC) was used for analysis. The chromatographic column was Ultimate XB-C18 (250nm×4.6nm, 5μm). Methanol was used as the mobile phase A and glacial acetic acid-water (v / v=1:300) was used as the mobile phase B for gradient elution. The injection volume was 10 μL, the flow rate was 0.8 mL / min, the column temperature was room temperature, and the detection wavelength was 284nm. The gradient elution program was as follows: 0-30 min, 38 (A): 62 (B); 30-50 min, 80 (A): 20 (B); 50-70 min, 38 (A): 62 (B).

[0029] (4) Hesperidin content determination: The hesperidin content in the tangerine peel powder before and after fermentation was calculated using the external standard method. The increase rate was also calculated using the following formula: Increase rate = (Hesperidin content in fermented tangerine peel powder - Hesperidin content in tangerine peel powder) / Hesperidin content in tangerine peel powder × 100%.

[0030] The results are as follows Figure 1 and Figure 2 As shown, compared with the hesperidin content in the tangerine peel powder before fermentation (i.e., when the inoculum amount was 0%), the hesperidin content in the fermented tangerine peel powder reached its highest level when the inoculum amount of *Lactobacillus plantarum* RX-261 fermentation broth was 9% (v / v), with an increase rate of 112%. This result indicates that fermentation treatment of tangerine peel with *Lactobacillus plantarum* RX-261 can significantly increase the hesperidin content in tangerine peel.

[0031] Experimental Example Fermented tangerine peel powder prepared under the condition of 9% (v / v) inoculum of Lactobacillus plantarum RX-261 was selected to evaluate its laxative effect; the obtained fermented tangerine peel powder was dissolved in physiological saline to prepare drug solutions of different concentrations required for the experiment.

[0032] Experimental animals: 120 male SPF-grade Kunming mice, weighing 18-22 g.

[0033] Animal grouping: After 1 week of acclimatization, 120 mice were randomly divided into a small intestinal motility test group and a defecation function test group. The two groups were further divided into a negative control group, a model group, a tangerine peel powder group (500 mg / kg), a low-dose fermented tangerine peel powder group (125 mg / kg), a medium-dose group (250 mg / kg), and a high-dose group (500 mg / kg), with 10 mice per group. After grouping, the mice were administered the corresponding concentration of fermented tangerine peel powder solution and tangerine peel powder solution by gavage at a dose of 20 mL / kg body weight. The negative control group and the model control group were administered physiological saline by gavage once a day.

[0034] (1) Small intestinal motility test: Mice were administered the test substance by gavage for 7 days and were fasted but allowed free access to water for about 16 hours. Except for the negative control group, the other groups were given 4 mg / kg phenobarbital hydrochloride solution to establish a mouse model of small intestinal motility inhibition. 0.5 h after modeling, each dose group of fermented tangerine peel powder and the tangerine peel powder group were given ink containing the corresponding test sample at 20 mL / kg body weight. The negative control group and the model control group were given the same volume of ink by gavage. The mice were sacrificed 25 min after the ink was administered, and the total length of the small intestine and the length of ink propulsion were measured to calculate the ink propulsion rate.

[0035] (2) Defecation function test: Mice were administered the test substance by gavage for 7 days and were fasted but allowed free access to water for approximately 16 hours. Except for the negative control group, the other groups were given 7 mg / kg phentermine hydrochloride solution to establish a mouse constipation model. 0.5 hours after modeling, each dose of fermented tangerine peel powder and the tangerine peel powder group were given 20 mL / kg body weight of ink containing the corresponding test sample. The negative control group and the model control group were given the same volume of ink by gavage. The time of excretion of the first black stool after ink administration was recorded for each mouse.

[0036] Experimental results (1) Small bowel motility test: Results are as follows Figure 3 As shown, compared with the negative control group, the ink propulsion rate of the model group mice was significantly reduced, and the difference was statistically significant. P <0.01), indicating that the mouse small intestinal peristalsis inhibition model was successfully established; compared with the model group, the ink propulsion rate of the medium-dose group of fermented tangerine peel powder increased ( P <0.05), the ink propulsion rate increased more significantly in the high-dose group of fermented tangerine peel powder ( P <0.01). The above results indicate that tangerine peel powder has no significant effect on promoting small intestinal motility in mice, while fermented tangerine peel powder can significantly enhance the peristaltic function of small intestines in mice and has a better effect on promoting intestinal motility.

[0037] (2) Bowel function test: Results are as follows Figure 4 As shown, compared with the negative control group, the time of excretion of the first black stool in the model group was significantly delayed, which was statistically significant. P <0.01), indicating that the mouse constipation model was successfully established; compared with the model group, the time of excretion of the first black stool in the medium dose group of fermented tangerine peel powder was earlier ( P <0.05%, the high-dose group of fermented tangerine peel powder had a shorter first black stool excretion time and the most significant effect. P <0.01). The results indicate that fermented tangerine peel powder promotes defecation in constipated mice.

[0038] Example 2

[0039] The fermented tangerine peel powder provided by this invention can be used in Sanren Tea, which is composed of the following components by weight: 40-60 parts of fructooligosaccharides, 5-10 parts of apricot kernel powder, 5-10 parts of radish seed powder, 5-10 parts of ophiopogon japonicus powder, 1-5 parts of licorice powder, 1-5 parts of jujube seed powder, 1-5 parts of coix seed powder, 1-5 parts of lotus leaf powder, 1-5 parts of red bean powder, 1-5 parts of resistant dextrin, 1-5 parts of hemp seed powder, 1-5 parts of lactitol, 1-5 parts of xylitol, 1-5 parts of polydextrose, and magnesium hydrogen phosphate. 1-5 parts, instant white tea powder 0.5-5 parts, fermented tangerine peel powder 0.5-2 parts, mulberry powder 0.5-2 parts, fermented hawthorn powder 0.1-1 parts, stachyose 0-0.5 parts, inulin 0-0.5 parts, xanthan gum 0-0.1 parts. Small amounts of jujube powder, gardenia powder, peach kernel powder, perilla seed powder, wheat flour, longan powder, fermented poria powder, gorgon fruit powder, white hyacinth bean powder, papaya powder, fish collagen peptide, yeast extract and snow lotus fruit powder can be added as needed. The amount of each component added is 0-0.1 parts.

[0040] Example 3

[0041] The fermented tangerine peel powder provided by this invention can be used to prepare a solid beverage for relieving constipation. By weight, the solid beverage includes: 25-30 parts fermented tangerine peel powder, 30-35 parts resistant dextrin, 12-18 parts fructooligosaccharides (sucrose source, content ≥95%), 12-18 parts hemp seed powder, 8-12 parts inulin, 1-3 parts citric acid, 0.1-0.3 parts sucralose, and 0.07-0.1 parts food flavoring.

[0042] The specific preparation method of the solid beverage is as follows: accurately weigh each component according to the above formula, wherein the fermented tangerine peel powder is pre-sieved through an 80-mesh sieve; add resistant dextrin, fructooligosaccharides, and inulin to a V-type mixer and mix at a speed of 20-25 rpm for 10-15 min; then add the sieved fermented tangerine peel powder and hemp seed powder, and mix at a speed of 25-30 rpm for 15-20 min to obtain a mixture; Wet granulation was adopted, using a 5% (w / v) maltodextrin solution as a binder, which was slowly added by spraying at a spraying speed of 3-5 mL / min. The granulator speed was controlled at 200-250 rpm, and the granulation time was 8-10 min, until uniform, loose and moderately loose wet granules were formed. The obtained wet granules were placed in a constant temperature hot air drying oven at 45-50℃ and dried for 3-4 hours, with the final moisture content of the finished product being ≤5.0% as the control standard. After drying, the granules were sized and passed through a 20-mesh sieve; then, they were quantitatively packaged in 5 g / bags to obtain the finished product of a laxative solid beverage.

[0043] To use, add 150-200mL of 70-80℃ hot water to each packet, stir well, and it is ready to drink.

[0044] Example 4

[0045] The fermented tangerine peel powder provided by this invention can be used to prepare medicines with laxative effects. By weight, the compressed candy comprises: 10-15 parts fermented tangerine peel powder, 15-18 parts erythritol (glucose source, content ≥99%), 1-3 parts fructooligosaccharides (sucrose source, content ≥95%), 1-1.5 parts citric acid, 0.5-1 part peppermint powder, and 0.1-0.5 parts magnesium stearate.

[0046] The main preparation method includes the following steps: Accurately weigh each component according to the above formula. Fermented tangerine peel powder, erythritol, fructooligosaccharides, citric acid, and peppermint powder are pulverized and passed through an 80-mesh sieve. The sieved fermented tangerine peel powder, erythritol, fructooligosaccharides, citric acid, and peppermint powder are added to a mixer and mixed at 20-25 rpm for 15-20 minutes. Subsequently, a wet granulation process is used, with a 5% (w / v) hydroxypropyl cellulose ethanol solution as a binder, slowly sprayed at 3%-5% of the total mass of the mixture, while simultaneously stirring at 250-300 rpm for 5-8 minutes to form uniform wet granules. The resulting wet granules are placed in a 45-50℃ constant temperature hot air drying oven and dried for 3-4 hours, controlling the final moisture content of the granules to ≤4.0%. After drying, the granules are lightly sized and passed through a 20-mesh sieve to obtain dried granules. Magnesium stearate is then added to the granules and mixed at 20 rpm for 3-5 minutes. After mixing, the granules are fed into the feed hopper of a tableting machine, and the tableting pressure is controlled at 8-12 kN to obtain compressed candy.

[0047] Example 5

[0048] The fermented tangerine peel powder provided by this invention can be used to prepare lactic acid bacteria beverages, which include: 45-55 parts milk, 35-45 parts strawberry juice, 4-8 parts white sugar, 3-8 parts fermented tangerine peel powder, 1-3 parts freeze-dried Lactobacillus plantarum powder, and 1-3 parts freeze-dried Bifidobacterium.

[0049] The main preparation method includes the following steps: accurately weigh each component according to the above formula, mix strawberry juice and milk, stir thoroughly, add white sugar and continue stirring until fully dissolved to obtain a mixture; place the obtained mixture in a high-pressure homogenizer and homogenize it 1-2 times at 15-20 MPa, then pasteurize for 5-10 min, and quickly cool it to 37-40℃ after sterilization; after the temperature drops to the appropriate inoculation range, inoculate with freeze-dried Lactobacillus plantarum powder and freeze-dried Bifidobacterium powder, wherein the viable count of both Lactobacillus plantarum and Bifidobacterium is controlled to be ≥1×10⁻⁶. 9After thoroughly mixing CFU / g, the mixture is allowed to ferment at 40℃ for 8-12 hours until the pH of the system drops to 4.2-4.6. After fermentation, 3-8 parts of fermented tangerine peel powder that has been passed through an 80-mesh sieve are added, and the mixture is stirred and homogenized again at 20 MPa. Finally, the mixture is filled under aseptic conditions, sealed, and stored at 4℃ to obtain a lactic acid bacteria beverage.

[0050] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for increasing the hesperidin content in dried tangerine peel, characterized in that, The content of hesperidin in tangerine peel can be increased by using Lactobacillus plantarum RX-261, preservation number CCTCC NO: M 2026428; or by using a microbial agent with Lactobacillus plantarum RX-261 as the active ingredient, preservation number CCTCC NO: M 2026428. The Lactobacillus plantarum RX-261 or the microbial agent was inoculated into the tangerine peel fermentation medium, and fermented to obtain fermented tangerine peel powder. The inoculum size of Lactobacillus plantarum RX-261 was 9%; the fermentation temperature was 37℃ and the fermentation time was 2 days.

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