Preparation method of probiotic carrot juice capable of improving intestinal health and resisting oxidation and metabiotics

Abstract

The invention discloses probiotic fermented carrot juice capable of improving intestinal health and resisting oxidation and a preparation method of a metagen. The carrot juice is prepared by fermenting carrot raw materials through a compound probiotic agent containing lactobacillus plantarum YZX21, lactobacillus casei YRL577, lactobacillus paracasei X11 and lactobacillus rhamnosus LR-C111. The preparation method comprises the following steps: cleaning, peeling, crushing and pulping carrots to prepare carrot primary pulp, then carrying out high-temperature instantaneous sterilization treatment on the carrot primary pulp, and carrying out vacuum concentration after sterilization; the concentrated carrot primary pulp is inoculated with the composite probiotic agent, the final inoculation concentration is 102-109 CFU / mL, standing fermentation is conducted for 9-15 hours at the temperature of 30-40 DEG C, the probiotic fermented carrot juice is obtained, and the probiotic fermented carrot juice is subjected to high-temperature sterilization treatment to obtain the postbiotics. The probiotic carrot juice and the metabiotics prepared by the invention have the functions of improving constipation, regulating intestinal flora and improving antioxidant capacity, and the preparation method is stable and efficient.

Description

Technical Field

[0001] This invention relates to the field of functional food processing and microbial fermentation preparation technology, and in particular to a method for preparing probiotic carrot juice and postbiotics that improves constipation and intestinal flora and enhances antioxidant capacity. Background Technology

[0002] Functional constipation is a common gastrointestinal dysfunction with a continuously rising incidence in modern society, seriously affecting patients' quality of life. The condition is characterized by decreased bowel movement frequency, hard stools, and difficulty in defecation. Its development is closely related to gut microbiota dysbiosis, often forming a vicious cycle: gut microbiota imbalance leads to weakened intestinal motility and abnormal water absorption, further exacerbating constipation; while constipation further disrupts the microbiota balance.

[0003] The harm of gut microbiota dysbiosis extends beyond the gut itself; the systemic oxidative stress it triggers is a core factor exacerbating various health problems. When beneficial gut bacteria decline while harmful bacteria proliferate, it leads to an imbalance between the gut's oxidative and antioxidant systems, producing excessive reactive oxygen species (ROS). These ROS not only damage the intestinal mucosal barrier but can also enter the circulatory system, attacking major tissues and organs, accelerating cell damage and aging, thereby weakening the body's overall antioxidant defense capabilities. Therefore, restoring a healthy gut microbiota is crucial for fundamentally enhancing the body's endogenous antioxidant levels. Currently, utilizing probiotics to regulate the gut microbiota has become an important strategy for nutritional intervention in sub-health conditions. Probiotics promote gut health through inhibiting pathogens, enhancing intestinal barrier function, and regulating immunity. However, traditional live bacteria preparations (such as common Lactobacillus and Bifidobacterium products) face many challenges in practical applications: First, the survival rate of live bacteria is low during processing, storage, and passage through the human gastrointestinal tract, making it difficult to ensure sufficient live bacteria reach the intestinal site of action; second, insufficient host adaptability and gut ecological targeting of the strains limit the stability of their colonization and efficacy. These factors all limit the stability and reliability of the intervention effect of live bacteria preparations.

[0004] To overcome the aforementioned limitations, the concept of postbiotics emerged. Postbiotics refer to preparations of non-living microorganisms and / or their components that are beneficial to the health of the host. Compared with live bacteria, postbiotics have advantages such as high stability, good safety, and a clear mechanism of action, providing new ideas for the development of functional foods.

[0005] Carrot juice, as a natural fruit and vegetable ingredient, is rich in dietary fiber (such as cellulose, hemicellulose, and pectin), oligosaccharides, and various vitamins, making it an ideal prebiotic matrix. Its fiber components not only physically promote intestinal peristalsis but also selectively promote the proliferation of beneficial gut bacteria (such as Bifidobacteria and Lactobacillus). However, the regulatory effect of simply drinking carrot juice is relatively limited. While there have been attempts to ferment carrot juice using probiotics, current technologies primarily focus on utilizing live bacteria or their primary metabolites. Research on deep fermentation using specific strain combinations and further utilizing "fermentation-inactivation" technology to prepare post-fermentative products with synergistic effects for a systematic solution to gut health remains insufficient.

[0006] Therefore, there is an urgent need in this field to develop a stable and efficient new product and its preparation method that can overcome the defects of live bacteria preparations, fully utilize the potential of carrot prebiotics, and effectively improve constipation, regulate intestinal flora, and enhance antioxidant capacity through multiple synergistic pathways. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing probiotic carrot juice and postbiotics that improve gut health and have antioxidant properties. The probiotic carrot juice and postbiotics prepared by this invention can improve constipation, regulate gut microbiota, and enhance antioxidant capacity, thereby improving gut health. Furthermore, the preparation method is stable and efficient.

[0008] The technical solution of the present invention is: a probiotic fermented carrot juice that improves intestinal health and has antioxidant properties. The carrot juice is made by fermenting carrot raw materials with a compound probiotic agent containing Lactobacillus plantarum YZX21, Lactobacillus casei YRL577, Lactobacillus paracasei X11 and Lactobacillus rhamnosus LR-C111. Among them, the classification name of Lactobacillus plantarum YZX21 is Lactobacillus plantarum ( Lactobacillus plantarum The accession number is CCTCC NO: M 2020829; The Lactobacillus casei YRL577 is classified as Lactobacillus casei ( Lactobacillus casei The accession number is CCTCC NO: M 2020824; The Lactobacillus paracasei X11 is classified as Lactobacillus paracasei ( Lactobacillus paracasei The accession number is CCTCC NO: M 2020826; The Lactobacillus rhamnosus LR-C111 is classified and named Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus (The accession number is CCTCC NO: M 20251647).

[0009] In the aforementioned probiotic fermented carrot juice that improves gut health and provides antioxidant benefits, the live bacteria count ratio of Lactobacillus plantarum YZX21, Lactobacillus casei YRL577, Lactobacillus paracasei X11, and Lactobacillus rhamnosus LR-C111 in the compound probiotic agent is any one of them accounting for 15%-35% of the total live bacteria count.

[0010] The aforementioned probiotic fermented carrot juice that improves gut health and provides antioxidant benefits has a live bacteria ratio of 1:1:1:1 for Lactobacillus plantarum YZX21, Lactobacillus casei YRL577, Lactobacillus paracasei X11, and Lactobacillus rhamnosus LR-C111.

[0011] The application of a probiotic-fermented carrot juice as described above in the preparation of functional foods or health products for improving constipation, regulating intestinal flora and / or enhancing antioxidant capacity.

[0012] The aforementioned method for preparing probiotic-fermented carrot juice that improves gut health and provides antioxidant benefits involves washing, peeling, crushing, and pulping carrots to make carrot puree. The carrot puree is then subjected to high-temperature flash sterilization, followed by vacuum concentration. The concentrated carrot puree is then inoculated with the aforementioned compound probiotic agent at a final concentration of 10. 2 -10 9 CFU / mL, fermented at 30-40℃ for 9-15 hours to obtain probiotic fermented carrot juice.

[0013] An epigenetic product, wherein the epigenetic product is obtained by high-temperature sterilization of probiotic fermented carrot juice as described in claim 5.

[0014] The aforementioned postbiotic products contain bacterial cell lysis components, organic acids and short-chain fatty acids produced by probiotic metabolism, and dietary fiber modified by enzymatic hydrolysis.

[0015] The aforementioned post-biotic product is sterilized at a temperature of 105±3℃.

[0016] The aforementioned epigenetic products are in the form of ready-to-drink liquid beverages or solid powders.

[0017] The aforementioned postbiotic products are used in the preparation of functional foods or health products for improving constipation, regulating gut microbiota, and / or enhancing antioxidant capacity.

[0018] Compared with existing technologies, this invention uses a specific compound probiotic agent composed of Lactobacillus plantarum YZX21, Lactobacillus casei YRL577, Lactobacillus paracasei X11, and Lactobacillus rhamnosus LR-C111 to ferment carrot juice, followed by a post-fermentation sterilization process to prepare a functional product rich in bacterial cell lysis components, metabolically active substances, and modified dietary fiber. This overcomes the shortcomings of traditional live bacteria preparations, such as low survival rate and poor stability. It can synergistically regulate the intestinal flora structure, increase fecal water content, shorten the time to first defecation, and accelerate gastrointestinal transit. It also has good antioxidant capacity, ultimately effectively relieving constipation and promoting intestinal health. At the same time, this product can be processed into various forms such as ready-to-drink liquid or solid powder. Attached Figure Description

[0019] Figure 1 The antioxidant capacity of the compound fermented carrot juice was demonstrated; Figure 2 This displays a stacked bar chart showing the relative abundance of species; Figure 3 This demonstrates the commonalities and unique characteristics of gut microbiota taxa in different experimental groups; Figure 4 (a) is a box plot of the Chao1 index, and (b) is a box plot of the number of observed species. Figure 5 (a) is the Shannon-Wiener index. Figure 5 (b) is a box plot of the Simpson index; Figure 6 (b) represents the Pielou uniformity index. Figure 6 (a) Box plot of Goods coverage index; Figure 7 The principal coordinate analysis (PCoA) plot of the Bray-Curtis distance matrix is ​​shown; Figure 8 Bar chart of relative abundance of bacterial communities at the phylum level in each group; Figure 9 Statistical differences in Bacteroidetes among the groups; Figure 10 Statistical differences among the Bacteroidetes groups; Figure 11 The relative abundance of bacterial communities at the genus level in each group; Figure 12 Statistical differences in Lactobacillus genus among different groups; Figure 13 The statistical differences in the ratio of bacterial cells to colonies among the groups; Figure 14 A phylogenetic tree showing the microbial community structure in different treatment groups; Figure 15 LDA score bar charts of microbial community structure in different treatment groups Figure 16 The fecal water content of mice in each group was shown; Figure 17 The time of the first black stool passage was displayed for each group; Figure 18 The gastrointestinal transit rate of each group was shown; Figure 19 This is a cell morphology diagram of Lactobacillus plantarum YZX21. Figure 20 Image showing the cell morphology of Lactobacillus casei YRL577; Figure 21 This is a cell morphology diagram of Lactobacillus paracasei X11. Figure 22 This is a morphological diagram of Lactobacillus rhamnosus LR-C111. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0021] In the following embodiments, the present invention provides a compound probiotic agent comprising Lactobacillus plantarum YZX21, Lactobacillus casei YRL577, Lactobacillus paracasei X11 and Lactobacillus rhamnosus LR-C111.

[0022] Among them, the classification name of Lactobacillus plantarum YZX21 is Lactobacillus plantarum ( Lactobacillus plantarum The accession number is CCTCC NO: M 2020829, the depositary institution is the China Center for Type Culture Collection, the deposit address is Wuhan University, Luojia Mountain, Wuchang, Wuhan, Hunan Province, China, the deposit date is December 17, 2020, and the status is live. The Lactobacillus casei YRL577 is classified as Lactobacillus casei ( Lactobacillus casei The accession number is CCTCC NO: M 2020824, the depositary institution is the China Center for Type Culture Collection, the deposit address is Wuhan University, Luojia Mountain, Wuchang, Wuhan, Hunan Province, China, the deposit date is December 17, 2020, and the status is live; The Lactobacillus paracasei X11 is classified as Lactobacillus paracasei ( Lactobacillus paracasei The accession number is CCTCC NO: M 2020826; the depositary institution is the China Center for Type Culture Collection, the deposit address is Wuhan University, Luojia Mountain, Wuchang, Wuhan, Hunan Province, China, the deposit date is December 17, 2020, and the status is live. The Lactobacillus rhamnosus LR-C111 is classified and named Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosusThe accession number is CCTCC NO: M 20251647. The depositary institution is the China Center for Type Culture Collection. The deposit address is Wuhan University, Luojia Mountain, Wuchang District, Wuhan City, Hunan Province, China. The deposit date is July 21, 2025. The status is live. The identification method for probiotics is as follows: 16S rDNA sequencing is used for identification. The specific steps include bacterial genomic DNA extraction, 16S rDNA specific primer PCR amplification, amplification product purification, DNA sequencing and sequence alignment.

[0023] Primer sequence: Universal primers were used. 27F: AGAGTTTGATCMTGGCTCAG; 1492R: GGTTACCTTGTTACGACTT.

[0024] Experimental results: The 16S rDNA sequence of Lactobacillus plantarum YZX21 is as follows:

[0025] The strain was identified as Lactobacillus (Lactobacillus), presumed to be Lactobacillus plantarum (Lactobacillus plantarum), hence the name Lactobacillus plantarum ( Lactobacillus plantarum YZX21, its cell morphology is shown in the figure below. Figure 19 As shown.

[0026] The 16S rDNA sequence of Lactobacillus casei YRL577 is as follows:

[0027] The strain was identified as Lactobacillus (Lactobacillus), presumed to be Lactobacillus casei (Lactobacillus casei), hence the name Lactobacillus casei ( Lactobacillus casei YRL577, its cell morphology is shown in the figure below. Figure 20 As shown.

[0028] The 16S rDNA sequence of Lactobacillus paracasei X11 is as follows:

[0029] The strain was identified as Lactobacillus (Lactobacillus), presumed to be Lactobacillus paracasei (Lactobacillus paracasei), hence the name Lactobacillus paracasei. Lactobacillus paracasei X11, its cell morphology diagram is as follows Figure 21 As shown.

[0030] The 16S rDNA sequence of *Lactobacillus rhamnosus* LR-C111 is as follows:

[0031] The strain was identified as Lacticaseibacillus (Lactobacillus), presumed to be Lacticaseibacillus rhamnosus (Lactobacillus rhamnosus), hence the name Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus LR-C111, its cell morphology is shown in the figure. Figure 22 As shown.

[0032] Example 1: Preparation of fermented carrot juice with compound probiotics, including the following steps: Step 1.1 Raw material processing: Select fresh, unrotten carrots, wash them thoroughly, peel them with steam, then mechanically crush and pulp them to make a uniform carrot pulp, which is then temporarily stored in a storage tank.

[0033] Step 1.2, Sterilization of the pulp: To thoroughly inactivate contaminating bacteria and provide a sterile fermentation substrate, the carrot pulp is sterilized at 105±3℃ and a flow rate of 3-4 m³ / h. 3 The system uses a high-temperature instantaneous sterilization system to treat the bacteria and completely kill them.

[0034] Step 1.3, Vacuum Concentration: The sterilized carrot pulp is vacuum concentrated to appropriately increase the solid content and form a high-concentration fermentation substrate, providing a suitable nutrient environment for subsequent probiotic fermentation.

[0035] Step 1.4, Fermentation Inoculation and Cultivation: Four probiotic strains—Lactobacillus plantarum YZX21, Lactobacillus casei YRL577, Lactobacillus paracasei X11, and Lactobacillus rhamnosus LR-C111—were activated and cultured in a ratio of 1.5:2.5:3.5:2.5. The activated strains were then inoculated into concentrated and cooled carrot substrate to a final inoculation concentration of 10. 5 The concentration of CFU / mL was then kept at a constant temperature of 37℃ for 14 hours to complete the fermentation process and enrich the probiotic metabolites, thus obtaining probiotic fermented carrot juice.

[0036] Example 2: Preparation of fermented carrot juice with compound probiotics, including the following steps: Step 1.1 Raw material processing: Select fresh, unrotten carrots, wash them thoroughly, peel them with steam, then mechanically crush and pulp them to make a uniform carrot pulp, which is then temporarily stored in a storage tank.

[0037] Step 1.2, Sterilization of the pulp: To thoroughly inactivate contaminating bacteria and provide a sterile fermentation substrate, the carrot pulp is sterilized at 105±3℃ and a flow rate of 3-4m³ / h. 3 The system uses a high-temperature instantaneous sterilization system to treat the bacteria and completely kill them.

[0038] Step 1.3, Vacuum Concentration: The sterilized carrot pulp is vacuum concentrated to appropriately increase the solid content and form a high-concentration fermentation substrate, providing a suitable nutrient environment for subsequent probiotic fermentation.

[0039] Step 1.4, Fermentation Inoculation and Cultivation: Four probiotic strains—Lactobacillus plantarum YZX21, Lactobacillus casei YRL577, Lactobacillus paracasei X11, and Lactobacillus rhamnosus LR-C111—were activated and cultured in a ratio of 2.5:2.5:1.5:3.5. The activated strains were then inoculated into concentrated and cooled carrot substrate to a final inoculation concentration of 10. 8 The concentration of CFU / mL was then kept at a constant temperature of 37℃ for 10 hours to complete the fermentation process and enrich the probiotic metabolites, thus obtaining probiotic fermented carrot juice.

[0040] Example 3: Preparation of compound probiotic fermented carrot juice and postbiotic products, including the following steps: Step 1.1 Raw material processing: Select fresh, unrotten carrots, wash them thoroughly, peel them with steam, then mechanically crush and pulp them to make a uniform carrot pulp, which is then temporarily stored in a storage tank.

[0041] Step 1.2, Sterilization of the pulp: To thoroughly inactivate contaminating bacteria and provide a sterile fermentation substrate, the carrot pulp is sterilized at 105±3℃ and a flow rate of 3-4 m³ / h. 3 The system uses a high-temperature instantaneous sterilization system to treat the bacteria and completely kill them.

[0042] Step 1.3, Vacuum Concentration: The sterilized carrot pulp is vacuum concentrated to appropriately increase the solid content and form a high-concentration fermentation substrate, providing a suitable nutrient environment for subsequent probiotic fermentation.

[0043] Step 1.4, Fermentation Inoculation and Cultivation: Four probiotic strains—Lactobacillus plantarum YZX21, Lactobacillus casei YRL577, Lactobacillus paracasei X11, and Lactobacillus rhamnosus LR-C111—were activated and cultured in a 1:1:1:1 ratio. The activated strains were then inoculated into concentrated and cooled carrot substrate to a final inoculation concentration of 10. 7 CFU / mL, then kept at a constant temperature of 37℃ for 12 h to complete the fermentation process to enrich the probiotic metabolites and obtain probiotic fermented carrot juice. Step 1.5, Sterilization of fermentation broth: After fermentation, in order to obtain post-biotic components, the probiotic fermented carrot juice is sterilized at 105±3℃ to completely inactivate all fermentation cells and potential microorganisms, while retaining their metabolites.

[0044] Step 1.6, Magnetic Separation and Precision Filtration: The sterilized fermented juice first passes through a magnetic separator with a magnetic field strength of not less than 6000 Gauss to effectively remove any metal impurities that may be mixed in. Then it undergoes precision filtration to obtain post-fermentation juice with a delicate taste and stable clarity.

[0045] Step 1.7, Subsequent Processing: ① Liquid Product Line: The filtered and clarified post-biotic juice is directly bottled in a closed and clean environment, and immediately labeled and packaged to produce ready-to-drink post-biotic liquid beverages.

[0046] ② Solid product line: The filtered post-biotic juice is processed into powder using boiling granulation technology, and then metered and packaged to produce solid beverage products.

[0047] Step 1.8, Filling and Packaging: The processed liquid products or solid powders are finally filled and packaged in a clean environment.

[0048] Control example: The preparation of carrot juice fermented by the control strain includes the following steps: Step 2.1, Raw material pretreatment: The raw material pretreatment method for carrot juice is the same as steps 1.1-1.3 in Example 3.

[0049] Step 2.2, Fermentation Inoculation and Culture: Two probiotic strains, *Lactobacillus rhamnosus* GG (ATCC 53103) and *Bifidobacterium lactis* BB12 (DSM 15954), were activated and cultured (the ratio of the two strains was specified). The activated strains were inoculated into concentrated and cooled carrot substrate, with a final inoculation concentration of 10. 7 CFU / mL, and then incubated at a constant temperature of 37℃ for 12 hours to complete the fermentation process and enrich probiotic metabolites.

[0050] Step 2.3 and subsequent operations are the same as steps 1.5-1.8 in Example 3.

[0051] Experiment Example 4: Experiment on the antioxidant properties of fermented carrots with compound probiotics 1.1 Determination of DPPH scavenging ability Take 0.5 mL of the compound probiotic fermented carrot juice from Experiment 3 (the same applies below), add 0.5 mL of DPPH solution (prepared with anhydrous ethanol to a final concentration of 0.2 mmol / L), mix well, and incubate at 25℃ in the dark for 30 min. Centrifuge at 10000 r / min for 10 min, collect the supernatant, and measure the absorbance of the sample at a wavelength of 517 nm. Measure three times in parallel. The control group sample is prepared by replacing the compound probiotic fermented carrot juice with an equal volume of distilled water.

[0052] Clearance rate (%) = (Am control - A sample) / A control × 100.

[0053] Determination of reducing activity Take 0.25 mL of fermented carrot juice with compound probiotics, add 0.25 mL of 0.2 mol / L (pH = 6.6) PBS solution and 0.25 mL of 1% (m / v) potassium ferricyanide solution, incubate at 50℃ for 20 min, then rapidly cool to room temperature with ice water. Add 0.25 mL of 10% trichloroacetic acid (TCA), mix thoroughly, centrifuge at 10000 r / min for 10 min to remove protein and other precipitates, take 0.25 mL of the supernatant, add 0.05 mL of 0.1% (m / v) ferric chloride, shake thoroughly to mix, let stand for 10 min, and measure the absorbance at 700 nm. The results use L-cysteine ​​as a standard to represent the reducing power.

[0054] Determination of the ability to scavenge hydroxyl radicals (·OH) The ability of the test sample to scavenge hydroxyl radicals (·OH) by catalyzing the formation of ·OH from hydrogen peroxide by ferrous ions was used to determine its ability to scavenge ·OH. 0.25 mL of 0.435 mmol / L Brilliant Green, 0.5 mL of 0.5 mmol / L FeSO4, 0.4 mL of 3.0% (w / v) hydrogen peroxide, and 1 mL of fermented carrot juice with compound probiotics were mixed and incubated in a water bath at 37°C for 30 min. The mixture was then centrifuged at 10000 rpm for 10 min, and the supernatant was collected. The absorbance was measured at 525 nm. The blank group used distilled water instead of the sample but containing hydrogen peroxide, while the control group used distilled water instead of hydrogen peroxide and fermented carrot juice with compound probiotics.

[0055] Hydroxyl radical scavenging rate (%) = (Sample A - Control A) / (Blank A - Control A) x 100.

[0056] Sample group: Contains compound probiotic fermented carrot juice and hydrogen peroxide; Control group: Distilled water was used instead of the sample but contained hydrogen peroxide. Blank group: Distilled water was used instead of hydrogen peroxide and fermented carrot juice with compound probiotics was used. 1.4. Scavenging superoxide anions (·O) 2- ) ability measurement 1 mL of 150 mmol / L Tris-HCl (pH=8.2) was mixed with 1 mL of 3 mmol / L diethylenetriaminepentaacetic acid, 1 mL of 1.2 mmol / L pyrogallol, and 0.5 mL of fermented carrot juice with compound probiotics, for a total reaction volume of 3.5 mL. The mixture was incubated in a water bath at 25°C for 10 min, and the absorbance was measured at 325 nm.

[0057] Superoxide anion scavenging rate (%) = [1 - (A3 - A2) / (A1)] x 100.

[0058] A1 contains no pyrogallol; A2 contains fermented carrot juice with compound probiotics and does not contain pyrogallol; A3 contains fermented carrot juice with compound probiotics and pyrogallol. like Figure 1 As shown, the antioxidant properties of fermented carrot juice with compound probiotics were tested. The DPPH free radical scavenging rate of the fermented carrot juice with compound probiotics was (67.878±0.954)%, the reducing power was (71.483±0.608)%, the hydroxyl free radical scavenging rate was (80.782±1.537)%, and the superoxide anion scavenging rate was (81.065±0.869)%. Therefore, it can be considered that the fermented carrot juice with compound probiotics has good antioxidant properties.

[0059] Experiment Example 5: Effects of compound probiotic fermented carrot juice on the intestinal flora of mice 1.1. Intervention experiment of probiotic fermented carrot juice on mice Male BALB / c mice without specific pathogens were housed at a temperature of 22±2℃ and a relative humidity of 50%±10%. The light / dark cycle was 12h, and the mice were allowed to acclimatize to the environment in the animal room for one week before use. The BALB / c mice were randomly divided into four groups of eight mice each. Table 1 lists the composition of each group and the daily intragastric dosage.

[0060] Table 1

[0061] 1.2 16S rRNA gene sequencing On the last day of gavage treatment, fecal samples were collected from individually housed mice and immediately cryopreserved at −80°C. Genomic DNA was extracted using the Fast DNA SPIN kit (Santa Analog Devices, Inc., USA). Using this DNA as a template, the 16S rRNA gene was amplified using Q5 high-fidelity DNA polymerase. The amplification process employed forward primer 338F and reverse primer 806R, targeting the V3-V4 variable region of the 16S rRNA gene, respectively. After purification and quantification, the PCR amplicon was sequenced by Shanghai Renren Biotechnology Co., Ltd. (Shanghai, China).

[0062] The detection of α-diversity (number of ASY-NOTUs) in mouse fecal microbiota based on 16S rRNA gene MiSeq sequencing Figure 2 The diagram shows stacked bar charts of relative abundance of species. The group types include control groups 1-4, model groups 1-4, fermented carrot juice groups 1-3, and positive drug groups 1-4. The numbers under each group represent parallel samples. Figure 3 This study demonstrates the commonalities and unique characteristics of gut microbiota taxa in different experimental groups. Figure 2 and Figure 3The results showed that, compared with the control group (CON), the model group (MOD, 4577 ASY-NOTUs) exhibited significant dysbiosis (p<0.01), characterized by abnormally increased species abundance, reflecting a pathological state of intestinal microecological imbalance. After intervention with fermented carrot juice, the diversity of the fermented carrot juice group (FCJ, 8004 ASY-NOTUs) was significantly increased by 75% compared with the model group (p<0.001), confirming that probiotic-fermented carrot juice can restore the complexity of the microbial community. However, its value was much higher than that of the CON group, suggesting that further evaluation of the stability of the microbial structure is needed. The diversity of the positive control group (ADC, 976 ASY-NOTUs) was significantly lower than that of the model group and the sample group, indicating that although conventional drugs inhibit the proliferation of pathogenic bacteria, they excessively reduce the diversity of symbiotic bacteria and fail to fully restore the microecological balance.

[0063] By systematically comparing the diversity of different groups of microbial communities Figures 4-6 The box plot of the α-diversity index is shown, in which Figure 4 (a) is the Chao1 index. Figure 4 (b) represents the number of observed species. Figure 5 (a) is the Shannon-Wiener index. Figure 5 (b) is the Simpson index. Figure 6 (a) is the Faith phylogenetic diversity index. Figure 6 (b) represents the Pielou uniformity index. Figure 6 (a) is the Goods coverage index. Figure 6 (a) It can be seen that the Goods coverage index of the model group (MOD) was significantly reduced (p=0.036), indicating insufficient microbial coverage and sequencing bias, consistent with the characteristics of ecological imbalance. After treatment with fermented carrot juice, the α-diversity index (including the Shannon-Wiener index, p=0.031) of the treatment group was significantly restored, and its dilution curve approached a saturation plateau (OTU increment <5% when sequencing depth >4000), confirming that the treatment effectively reconstructed the microbial community structure. Notably, the model group maintained a steep upward slope (OTU increment >15%) when sequencing depth >5000, suggesting the presence of a large number of low-abundance, highly heterogeneous species in its microbial community, which is consistent with the fragmentation phenomenon of microbial communities under disease conditions.

[0064] Figure 7 This shows the principal coordinate analysis (PCoA) plot of the Bray-Curtis distance matrix. Figure 7The results showed significant intergroup differentiation in the gut microbiota structure (PCo1 explained 14.8% of the variation): the model group samples were concentrated in the negative PCo1 range (-0.15 to -0.25), significantly deviating from the control group (p<0.001, permutation multivariate analysis of variance), confirming that microbial imbalance led to niche shift; while the fermented carrot juice group approached the positive range (0.05-0.20) where the control group was located, and its sample distribution overlapped with the model group by less than 15%, indicating that the probiotic could effectively reshape the microbial community structure; it is worth noting that the centroid distance between the fermented carrot juice group and the control group in the PCo1-PCo2 plane was shortened by 68% compared with the model group (based on Procter-Stokes analysis), proving that its restoration effect is not only reflected in α diversity, but also realizes the reconstruction of community functional state at the β diversity level.

[0065] Figure 8 The bar chart shows the relative abundance of bacterial communities at the phylum level for each group. The group types include control group 1-4, model group 1-4, fermented carrot juice group 1-4, and positive drug group 1-4. The numbers under each group represent parallel samples. Figure 9 The differences in the Bacteroidetes phylum among the groups were statistically analyzed. Figure 10 The differences in the Bacteroidetes order among the groups were statistically analyzed. Figure 11 The relative abundance of bacterial communities at the genus level in each group. Figure 12 Statistical differences in Lactobacillus genus among different groups; Figure 13 This represents the statistical differences in the cell-to-colony ratio among the groups. Figures 6-13 It can be seen that by comparing the relative abundance of microbial communities in different treatment groups, the regulatory effect of fermented carrot juice on the gut microbial community structure was revealed. The results showed that in the model group, Firmicutes (… Firmicutes The relative abundance of Bacteroidetes was significantly reduced, while that of other Bacteroidetes ( Bacteroidetes ) and Proteobacteria ( Proteobacteria The relative abundance of Lactobacillus (Lactobacillus) increased significantly, indicating that the model group treatment had a significant impact on the gut microbiota structure. However, the fermented carrot juice group showed a recovery in the relative abundance of Firmicutes, and the Bac-Firm ratio was close to that of the control group, suggesting that fermented carrot juice may help restore the balance of the gut microbiota. In addition, the fermented carrot juice treatment group showed an increase in the relative abundance of Lactobacillus (Lactobacillus). Lactobacillus The relative abundance of ) was significantly higher than that of other groups, further indicating that fermented carrot juice has a positive impact on the gut microbiota.

[0066] Figure 14 A phylogenetic tree showing the microbial community structure in different treatment groups was presented. Figure 15 LDA score bar charts showing the microbial community structure in different treatment groups. Figure 14The phylogenetic tree revealed the diversity and relative abundance of microbial classification. It was found that the model group caused abnormal enrichment of some bacterial communities, while after intervention with fermented carrot juice, the bacterial communities recovered to the enrichment pattern of the control group, and unique enriched groups appeared. Figure 15 The LDA score bar chart quantified the significant differences in different microbial communities among the treatment groups, indicating significant differences in microbial community composition among the treatment groups. The control group had high scores for groups such as Trichophytonceae, which are the characteristic dominant bacteria of the control group. The model group had unique high-scoring bacteria, reflecting the "disorder markers" of the constipation model. The fermented carrot juice group had exclusive high-scoring bacteria, indicating that probiotic intervention specifically promoted the enrichment of beneficial bacteria. The markers in the positive drug group differed significantly from those in other groups, reflecting different microbial regulation patterns compared to other interventions. Therefore, the fermented carrot juice of this invention can reshape the intestinal microbiota by promoting beneficial bacteria and inhibiting disordered bacteria, and the regulatory pattern is significantly different from that of the control group and the positive drug group.

[0067] Experiment Example 6: The effect of compound probiotic fermented carrot juice on relieving constipation 1.1 Intervention Experiment of Carrot Juice Fermented with Compound Probiotics Male BALB / c mice without specific pathogens were housed at a temperature of 22±2℃ and a relative humidity of 50%±10%. The light / dark cycle was 12 h, and the mice were allowed to acclimatize to the environment in the animal room for one week before use. The BALB / c mice were randomly divided into 5 groups of 8 mice each. Table 2 lists the composition of each group and the daily intragastric dosage.

[0068] Table 2

[0069] 1.2 Measurement of constipation-related indicators 1.2.1 Measurement of overall conditions Mice were weighed every 4 days to determine changes, and their food and water intake was measured. Water consumption was recorded for each group. Food utilization rate was calculated using the following formula: ; 1.2.2 Determination of water content in feces On day 17, the water content of mouse feces was measured. After the intragastric administration, four mice from each group were randomly selected and transferred to a clean, individual cage. Two hours later, feces were collected in test tubes. The wet weight of the feces was measured immediately after excretion, and then the feces were thoroughly dried in an oven and weighed again to obtain the dry weight. The criteria for assessing water content were as follows: ; 1.2.3 Time of first black stool passage On day 17, the time of the first black feces was measured. Four mice were randomly selected from each group. The mice were injected intragastricly with Indian ink (0.2 mL / 10 g body weight), and then each mouse was placed individually in a clean cage. Absorbent paper was placed at the bottom of the cage, and the time of the first black feces excreted by each mouse was recorded.

[0070] Gastrointestinal transit rate measurement On day 18, gastrointestinal transit rate was measured. Four mice in each group were sacrificed 20 minutes after receiving the ink (0.2 mL / 10 g body weight) intragastrically. The peritoneum of the mice was dissected, and the gastrointestinal transit rate was calculated using the following method: .

[0071] The results are as follows: Figure 16 The fecal water content of mice in each group was displayed. Data showed that the fecal water content in the model group was significantly reduced, proving the successful replication of the constipation model. A key finding was the comparison of the two fermentation products: the fecal water content in the post-fermentation group recovered to the highest level, showing no statistical difference from the blank control group. This effect was not only significantly better than the positive control group but also significantly better than the control strain group. This result strongly suggests that the specific strain combination used for fermentation is a key determinant of the production of highly effective moisturizing post-fermentation. The three lactic acid bacteria selected in this invention may produce richer bioactive substances (such as specific types of short-chain fatty acids) that regulate intestinal water and salt metabolism during fermentation, with efficacy exceeding that of the traditional combination of strains LGG and BB12.

[0072] Figure 17 The time to first melena was shown for each group. Assessment of intestinal motility further confirmed the importance of strain specificity. The metabiotic group demonstrated a superior effect in shortening the time to first melena, with no significant difference compared to the blank control group and a significantly shorter time than the control strain group. This indicates that the metabiotics produced by mixed-culture fermentation contain more potent pro-motility components. These components may act directly on the enteric nervous system or smooth muscle, alleviating the sluggish intestinal motility caused by constipation at a faster rate, with a higher efficacy than metabiotics produced by LGG and BB12 fermentation.

[0073] Figure 18The gastrointestinal transit rates of each group were presented. Regarding the gastrointestinal transit rate, which comprehensively reflects the overall intestinal motility function, both the metabiotic group and the control strain group demonstrated a strong ability to completely reverse the transit retardation caused by the constipation model. The effects of both groups were comparable and significantly superior to the positive control group. This indicates that the metabiotics produced by both fermentation methods can efficiently restore intestinal motility. Combining the first two indicators, we can conclude that although the two metabiotics are comparable in their ability to "push" the intestinal contents forward, the mixed-culture fermentation product exhibits a more comprehensive and superior overall effect in improving stool characteristics (moisturization) and accelerating intestinal defecation (speeding up), highlighting its significant value as a potential functional agent.

[0074] In summary, this invention utilizes a specific compound probiotic agent composed of Lactobacillus plantarum YZX21, Lactobacillus casei YRL577, and Lactobacillus paracasei X11 to ferment carrot juice, followed by a post-fermentation sterilization process. This process yields a functional product rich in bacterial cell lysis components, metabolically active substances, and modified dietary fiber, overcoming the shortcomings of traditional live bacteria preparations such as low survival rate and poor stability. It synergistically regulates the intestinal flora structure, increases fecal water content, shortens the time to first defecation, and accelerates gastrointestinal transit, ultimately effectively relieving constipation and promoting intestinal health. Furthermore, this product can be processed into various forms, including ready-to-drink liquids and solid powders.

Claims

1. A probiotic fermented carrot juice that improves gut health and provides antioxidant benefits, characterized by: The carrot juice is made by fermenting carrot raw materials with a compound probiotic agent containing Lactobacillus plantarum YZX21, Lactobacillus casei YRL577, Lactobacillus paracasei X11 and Lactobacillus rhamnosus LR-C111. Among them, the classification name of Lactobacillus plantarum YZX21 is Lactobacillus plantarum ( Lactobacillus plantarum The accession number is CCTCC NO: M 2020829; The Lactobacillus casei YRL577 is classified as Lactobacillus casei ( Lactobacillus casei The accession number is CCTCC NO: M 2020824; The Lactobacillus paracasei X11 is classified as Lactobacillus paracasei ( Lactobacillus paracasei The accession number is CCTCC NO: M 2020826; The Lactobacillus rhamnosus LR-C111 is classified and named Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus (The accession number is CCTCC NO: M 20251647).

2. The probiotic fermented carrot juice for improving gut health and providing antioxidant benefits according to claim 1, characterized in that: In the compound probiotic agent, the live bacteria count ratio of Lactobacillus plantarum YZX21, Lactobacillus casei YRL577, Lactobacillus paracasei X11 and Lactobacillus rhamnosus LR-C111 is any one of them accounting for 15%-35% of the total live bacteria count.

3. The probiotic fermented carrot juice for improving gut health and providing antioxidant benefits according to claim 2, characterized in that: The viable count ratio of *Lactobacillus plantarum* YZX21, *Lactobacillus casei* YRL577, *Lactobacillus paracasei* X11, and *Lactobacillus rhamnosus* LR-C111 is 1:1:1:

1.

4. The use of probiotic fermented carrot juice as described in any one of claims 1-3 in the preparation of functional foods or health products for improving constipation, regulating intestinal flora and / or anti-oxidation.

5. The method for preparing probiotic fermented carrot juice that improves gut health and has antioxidant properties according to any one of claims 1-3, characterized in that: Carrots are washed, peeled, crushed, and pulped to make carrot puree. The carrot puree is then subjected to high-temperature flash sterilization, followed by vacuum concentration. The concentrated carrot puree is then inoculated with the aforementioned compound probiotic agent at a final concentration of 10. 2 -10 9 CFU / mL, fermented at 30-40℃ for 9-15 hours to obtain probiotic fermented carrot juice.

6. An epigenetic product, characterized in that, The postbiotic product is obtained by high-temperature sterilization of probiotic fermented carrot juice as described in claim 5.

7. The post-natal product according to claim 6, characterized in that, The postbiotic product contains bacterial cell lysis components, organic acids and short-chain fatty acids produced by probiotic metabolism, and dietary fiber modified by enzymatic hydrolysis.

8. The post-natal product according to claim 6, characterized in that, The high-temperature sterilization conditions are 105±3℃.

9. The post-natal product according to claim 6, characterized in that, The post-biotic product is in the form of an instant liquid beverage or a solid powder.

10. The use of the postbiotic product as described in claim 6 in the preparation of functional foods or health products for improving constipation, regulating gut microbiota and / or providing antioxidant effects.

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