Preparation method of compound lactobacillus fermented asparagus yoghurt

Abstract

The invention discloses a preparation method of compound lactobacillus fermented asparagus yoghurt, and belongs to the technical field of food biotechnology and microbial fermentation. The lactobacillus fermented asparagus yoghurt is prepared by taking asparagus primary pulp and fresh cow milk as raw materials, and mixing, fermenting and ripening the raw materials by adopting the lactobacillus paracasei JGSLP08 and the lactobacillus pentosus JGS34, and the obtained compound lactobacillus fermented asparagus yoghurt has excellent antioxidant activity and high-dose lactobacillus viable count; meanwhile, the traditional Chinese medicine composition has a remarkable prevention and treatment effect on inflammatory bowel diseases. The lactobacillus fermented asparagus yoghurt can be widely applied to the field of preventing and / or treating inflammatory bowel diseases, in particular to preparation of food for preventing and / or treating inflammatory bowel diseases.

Description

Technical Field

[0001] This invention relates to the fields of food biotechnology and microbial fermentation technology, and in particular to a method for preparing asparagus yogurt fermented with compound lactic acid bacteria. Background Technology

[0002] Inflammatory bowel disease (IBD) is an immune-mediated chronic inflammatory disease, primarily including Crohn's disease and ulcerative colitis. Its occurrence is closely related to an abnormal immune response to the normal gut microbiota. This dysregulated immune response can trigger persistent intestinal inflammation, leading to damage to the intestinal mucosal barrier. In recent years, the prevalence of IBD in my country has been rising. Symptoms mainly include abdominal pain, diarrhea, and bloody stools, accompanied by fatigue, malnutrition, and fever. Patients with long-term recurrent IBD have a higher risk of secondary infections and colon cancer, seriously endangering their physical and mental health. Currently, clinical treatment of IBD mainly relies on aminosalicylic acid preparations, glucocorticoids, and immunosuppressants. While these drugs can control inflammation to some extent, long-term use often leads to a series of adverse reactions such as immunosuppression, metabolic disorders, and secondary infections, and they also have limited efficacy and a high relapse rate. Therefore, exploring safe, effective, and sustainable alternative treatment options has become an important frontier in the current research field of IBD treatment.

[0003] Asparagus, a plant resource with both medicinal and edible properties, is rich in various bioactive components such as saponins, polysaccharides, flavonoids, and dietary fiber. Its abundant dietary fiber and oligosaccharides can act as potential prebiotics, selectively promoting the proliferation of beneficial bacteria in the gut and indirectly inhibiting inflammatory responses. However, asparagus generally has a wild, unpleasant flavor and a short shelf life due to its vigorous metabolism. Fermenting asparagus with lactic acid bacteria can not only improve its sensory characteristics but may also release or produce new active substances through microbial biotransformation.

[0004] Yogurt, a popular fermented dairy product, is an ideal carrier of lactic acid bacteria. Studies have shown that dietary interventions and probiotic-based therapies are highly effective in alleviating inflammatory bowel disease (IBD), and are more readily accepted by patients and have higher safety and fewer side effects compared to conventional drug treatments and surgery. However, most yogurt products currently on the market have limited functions, primarily focusing on basic gut health regulation, and lack specificity in alleviating IBD and other inflammatory bowel diseases. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing asparagus yogurt fermented with compound lactic acid bacteria, thereby solving the problems existing in the prior art. This invention uses asparagus and milk as raw materials, applying the natural anti-inflammatory activity of asparagus and the probiotic function of specific lactic acid bacteria to yogurt development, and studying its efficacy in alleviating and / or treating inflammatory bowel disease (IBD), aiming to fill the technological gap in alleviating and / or treating IBD, and providing IBD patients with a new and easily acceptable dietary intervention option.

[0006] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is a method for preparing asparagus yogurt fermented with compound lactic acid bacteria, comprising the following steps: (1) Take asparagus pulp, raw milk, whey protein concentrate, white sugar and sodium alginate and mix them thoroughly. Then, homogenize the mixed materials and pasteurize them. (2) Inoculate with 0.05% of the total amount of compound lactic acid bacteria agent and ferment to obtain the compound lactic acid bacteria fermented asparagus yogurt.

[0007] The second technical solution of the present invention is a compound lactic acid bacteria fermented asparagus yogurt prepared by the preparation method.

[0008] Based on the above technical solution, the present invention has the following technical effects: (1) The compound lactic acid bacteria fermented asparagus yogurt provided by the present invention has a delicate and smooth taste, and has a light asparagus flavor and inherent yogurt aroma.

[0009] (2) The flavonoids and polyphenols in the compound lactic acid bacteria fermented asparagus yogurt provided by the present invention are significantly higher than those in single-strain fermented asparagus yogurt and unfermented lactic acid yogurt.

[0010] (3) The compound lactic acid bacteria fermented asparagus yogurt provided by the present invention exhibits excellent antioxidant activity and a high dose of live lactic acid bacteria.

[0011] (4) The compound lactic acid bacteria fermented asparagus yogurt provided by this invention has the effect of preventing and treating inflammatory bowel disease: After DSS-induced model mice ingested asparagus yogurt, the mice's growth performance gradually improved, colon length gradually recovered, TNF-α and IL-6 were significantly reduced, IL-10 was significantly increased, and MPO activity in colon tissue showed varying degrees of reduction. Therefore, this invention provides consumers with a safe, natural, palatable, and easily acceptable functional yogurt, which has broad market prospects and commercial value.

[0012] (5) The present invention discovered a strain of Lactobacillus paracasei JGSLP08 that can alleviate inflammatory bowel disease. After the strain was combined with Lactobacillus pentosus JGS34 to ferment asparagus and fresh milk, the resulting asparagus yogurt had excellent antioxidant function and preventive and therapeutic effects on inflammatory bowel disease. Attached Figure Description

[0013] Figure 1 The effect of asparagus yogurt fermented with compound lactic acid bacteria on the body weight of mice.

[0014] Figure 2 The effect of asparagus yogurt fermented with compound lactic acid bacteria on DIA scores in mice during modeling (A) and drug administration (B).

[0015] Figure 3 The effect of asparagus yogurt fermented with compound lactic acid bacteria on colon length in mice.

[0016] Figure 4 The effect of asparagus yogurt fermented with compound lactic acid bacteria on the TNF-α content in the colon tissue of mice.

[0017] Figure 5 Effects of asparagus yogurt fermented with compound lactic acid bacteria on IL-6 levels in mouse colon tissue.

[0018] Figure 6 Effects of asparagus yogurt fermented with compound lactic acid bacteria on IL-10 content in mouse colon tissue.

[0019] Figure 7 Effects of asparagus yogurt fermented with compound lactic acid bacteria on MPO activity in mouse colon tissue.

[0020] Figure 8 This is a colony morphology diagram of Lactobacillus paracasei JGSLP08.

[0021] Figure 9 Image of Lactobacillus paracasei JGSLP08 after Gram staining.

[0022] Figure 10 The effect of Lactobacillus paracasei JGSLP08 on mouse body weight.

[0023] Figure 11 The effect of Lactobacillus paracasei JGSLP08 on DIA scores in mice during modeling (A) and drug administration (B).

[0024] Figure 12 The effect of Lactobacillus paracasei JGSLP08 on colon length in mice.

[0025] Figure 13The effects of Lactobacillus paracasei JGSLP08 on the levels of TNF-α, IL-6 and IL-10 in mouse colon tissue. Detailed Implementation

[0026] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0027] This invention provides a method for preparing asparagus yogurt fermented with compound lactic acid bacteria, comprising the following steps: (1) Take asparagus pulp, raw milk, whey protein concentrate, white sugar and sodium alginate and mix them thoroughly. Then, homogenize the mixed materials and pasteurize them. (2) Inoculate with 0.05% of the total amount of compound lactic acid bacteria agent and ferment to obtain the compound lactic acid bacteria fermented asparagus yogurt.

[0028] In some specific implementations, the preparation method of the asparagus pulp is as follows: select fresh asparagus, rinse and cut it, pre-cook the cut asparagus for 1-5 minutes, then mix the asparagus and water at a mass ratio of 1:2-1:5 and blend them into a pulp, homogenize at 20-70 MPa for 1-6 minutes at a homogenization temperature of 30-45℃, and then pass it through a 30-mesh sieve to obtain the asparagus pulp.

[0029] In some specific implementations, the length of the cut segment is preferably 10 mm to 20 mm; In some specific implementations, the weight ratio of the asparagus puree, raw milk, whey protein concentrate, white sugar and sodium alginate is (80-150):(800-1000):(10-20):(40-90):(2-10).

[0030] In some specific implementations, the pasteurization conditions are pasteurization at 95°C for 5 minutes.

[0031] In some specific implementations, the colony count of the compound lactic acid bacteria agent is (1-9) × 10⁻⁶. 10 CFU / g, composed of Lactobacillus paracasei JGSLP08 and Lactobacillus pentosacchari JGS34 in a live count ratio of 1:1; The Lactobacillus paracasei JGSLP08 was deposited at the China General Microbiological Culture Collection Center on April 21, 2025, with accession number CGMCC No. 34274. The Pentosacchari lactis JGS34 was deposited on June 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.30985.

[0032] In some specific implementations, the fermentation conditions are as follows: fermentation at 25-38℃ for 8-12 hours, followed by post-ripening at 2-4℃.

[0033] This invention also provides a compound lactic acid bacteria fermented asparagus yogurt prepared by the aforementioned preparation method.

[0034] This invention provides a lactic acid bacteria fermented asparagus yogurt with excellent flavor, smooth and creamy texture, and superior antioxidant activity. Animal experiments have also shown that it has preventive and therapeutic effects on inflammatory bowel disease.

[0035] The Lactobacillus paracasei JGSLR08 has the following properties: (1) Acid resistance: The survival rates at pH 3.0 and pH 2.0 were 76.46% and 71.76%, respectively.

[0036] (2) Bile salt tolerance: The survival rate was as high as 72.99% after being treated in an environment with a bile salt concentration of 0.3% for 4 hours.

[0037] (3) Resistance to gastrointestinal fluid: After 3 hours of treatment with artificial gastric fluid, the survival rate can reach 85.51%, and the survival rate in artificial intestinal fluid can reach 80.82%.

[0038] (4) The scavenging rates of DPPH free radicals and hydroxyl free radicals were 56.82% and 67.59%, respectively.

[0039] (5) It has a strong antibacterial effect against Salmonella cytogenes, Escherichia coli and Staphylococcus aureus.

[0040] (6) Prevention and treatment effects on IBD mouse models: ① Inhibits the shortening of the colon in IBD model mice; ② Increased anti-inflammatory factors in IBD model mice and decreased pro-inflammatory factors in colitis model mice; ③ Reduce oxidative stress damage in IBD model mice; ④ Reduce intestinal mucosal damage in IBD model mice.

[0041] The *Lactobacillus pentosus* involved in this invention and its embodiments is *Lactobacillus pentosus* (… Lactiplanttibacillus pentosus JGS34 was deposited on June 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNO. 30985. *Lactobacillus pentosus* JGS34 has been published in Chinese invention patent application 202411118189.4 (CN 118755635A).

[0042] MRS broth medium (HB0384-1), MRS agar medium (HB0384), and Gram staining kit (HB8278) were all purchased from Haibo Biotechnology Co., Ltd., and bacterial genomic DNA extraction kit (D1600) was purchased from Thermo Fisher Scientific Co., Ltd.

[0043] Preparation of MRS broth medium: Weigh 52.4g of MRS broth medium powder, heat and dissolve it in 1000ml of distilled water, autoclave at 121℃ for 15 minutes, and set aside.

[0044] Preparation of MRS agar medium: Weigh 66.2g of MRS agar medium powder, heat and dissolve it in 1000ml of distilled water, autoclave at 121℃ for 15 minutes, and set aside.

[0045] Example 1 This embodiment provides a method for preparing asparagus yogurt fermented with compound lactic acid bacteria, as detailed below: Asparagus washing → cutting → pre-cooking → grinding → filtering → cooling → asparagus pulp → material preparation → inoculation and fermentation → post-ripening → finished product → inspection and evaluation.

[0046] Preparation of compound lactic acid bacteria inoculum: *Lactobacillus paracasei* JGSLP08 and *Lactobacillus pentosus* JGS34 were separately inoculated into MRS broth medium. After centrifugation at 5000 r / min for 10 min, a 5% skim milk powder aqueous solution (mass fraction 10%) was added to the precipitate. The mixture was then freeze-dried under vacuum for 48 hours. The resulting 1:1 mixture (viable bacteria ratio) yielded the compound lactic acid bacteria inoculum, with a colony count of 1×10⁻⁶. 10 CFU / g. Preparation method of asparagus yogurt: Select fresh asparagus, rinse and cut it. Pre-cook the cut asparagus for 1 minute, then blend it into a puree. The mass ratio of asparagus to water is 1:2-1:5. Homogenize at 50 MPa for 1 minute, then pass through a 30-mesh sieve to obtain asparagus puree. Take 100 parts of cooled asparagus puree, 900 parts of raw milk, 15 parts of whey protein concentrate, 60 parts of white sugar, and 7 parts of sodium alginate, mix them thoroughly, homogenize the mixture, pasteurize at 95℃ for 5 minutes, cool to below 40℃, inoculate with 0.05% of the total amount of compound lactic acid bacteria, ferment at 30℃ for 10 hours, and then place at 4℃ for post-ripening to obtain asparagus yogurt.

[0047] Example 2 This embodiment provides a method for preparing asparagus yogurt fermented with compound lactic acid bacteria. The only difference from Example 1 is that "100 parts asparagus puree, 900 parts raw milk, 15 parts whey protein concentrate, 60 parts white sugar, and 7 parts sodium alginate" are replaced with "80 parts asparagus puree, 1000 parts raw milk, 10 parts whey protein concentrate, 90 parts white sugar, and 2 parts sodium alginate", and "0.05% of the total amount of compound lactic acid bacteria inoculation" is replaced with "0.01% of the total amount of compound lactic acid bacteria inoculation". The rest of the preparation method is the same as in Example 1.

[0048] Example 3 This embodiment provides a method for preparing asparagus yogurt fermented with compound lactic acid bacteria. The difference from Embodiment 1 is that "100 parts asparagus puree, 900 parts raw milk, 15 parts whey protein concentrate, 60 parts white sugar, and 7 parts sodium alginate" are replaced with "150 parts asparagus puree, 800 parts raw milk, 20 parts whey protein concentrate, 40 parts white sugar, and 10 parts sodium alginate," and "0.05% of the total amount of compound lactic acid bacteria inoculation" is replaced with "0.1% of the total amount of compound lactic acid bacteria inoculation." The remaining preparation method is the same as in Embodiment 1.

[0049] Comparative Example 1 This comparative example provides a lactic acid bacteria fermented asparagus yogurt, which differs from Example 1 in that 0.05% of *Lactobacillus paracasei* JGSLP08 bacterial agent (colony count 1×10⁻⁶) was added during inoculation. 10 (CFU / g), the rest of the preparation method is the same as in Example 1.

[0050] Comparative Example 2 This comparative example provides a lactic acid bacteria fermented asparagus yogurt, which differs from Example 1 in that 0.05% of the total amount of Lactobacillus pentosus JGS34 lactic acid bacteria starter (colony count of 1×10⁻⁶) was added during inoculation. 10 (CFU / g), the rest of the preparation method is the same as in Example 1.

[0051] Experimental Example 1 This experiment evaluated the sensory characteristics of Examples 1-3, the unfermented example (Example 1), and Comparative Examples 1-2, mainly including four aspects: appearance, taste, texture, and flavor. The sensory evaluation indicators are as follows: Table 1 Sensory Evaluation Criteria for Asparagus Yogurt

[0052] Table 2 Sensory Evaluation Scores of Asparagus Yogurt

[0053] The sensory evaluation results are shown in Table 2. Compared with Comparative Examples 1-2 and Unfermented Example 1, the asparagus yogurt of Examples 1-3 had higher sensory evaluation scores. It had the pale yellow-green color that asparagus yogurt should have, and no whey separation. The taste was delicate and smooth, with a moderate sweetness and sourness and asparagus flavor. The curd was uniform and firm, with high viscosity and no bubbles. It had the inherent flavor of yogurt, and at the same time had a slight asparagus aroma.

[0054] Experiment Example 2 This experiment investigated the determination of flavonoid and polyphenol content in Examples 1-3, the unfermented Example 1, and Comparative Examples 1-2. The determination methods are as follows: Take 10 g of yogurt sample, add 20 mL of acidic 80% methanol solution (containing 0.16 mol / L HCl), and extract at 25 ℃ with shaking at a frequency of 150 times / min for 2 h. Then centrifuge at 4000 r / min for 10 min, separate and collect the supernatant. The residue is further extracted with 70% acetone with shaking at the same temperature for 2 h. After centrifugation, combine the two extracts and freeze at 18 ℃ for subsequent analysis.

[0055] The flavonoid content was determined using the NaNO2-Al(NO3)3 colorimetric method. 0.5 mL of the extract was placed in a 10 mL stoppered graduated test tube, and 0.3 mL of 5% NaNO2 solution was added. After 6 min, 0.3 mL of 10% Al(NO3)3 solution was added, followed by 4 mL of 4% NaOH solution after another 6 min. The solution was then diluted to the mark with 60% ethanol, and the absorbance was measured at 510 nm. A standard curve was plotted using rutin as a standard to calculate the flavonoid content in the sample.

[0056] Polyphenol content determination: The Folin-Ciocalteu colorimetric method was used. 0.5 mL of the extract was placed in a 10 mL stoppered graduated test tube, 1 mL of Folin-Ciocalteu was added, and the mixture was reacted in the dark for 2 min. Then, 2 mL of 7.5% sodium carbonate solution was added, and the volume was adjusted to 10 mL. The reaction was allowed to proceed for 1 h, and the absorbance was measured at 760 nm. A standard curve was plotted using gallic acid as a standard to calculate the polyphenol content in the sample.

[0057] Table 3 Flavonoid and Polyphenol Content in Asparagus Yogurt

[0058] As shown in Table 3, the content of natural active substances flavonoids and polyphenols in Examples 1-3 is much higher than that in Comparative Examples 1-2 with single-strain fermentation and Example 1 without fermentation.

[0059] Experimental Example 3 This experiment measured the acidity and titratable acidity of samples from Examples 1-3 and the unfermented samples from Examples 1-3. The pH value of the examples was measured using a pH meter, and the titratable acidity was measured using the phenolphthalein indicator method as per GB 5009.239-2023, "National Food Safety Standard - Determination of Acidity in Food". The results are shown in Table 4.

[0060] Table 4. Acidity and Titratable Acidity of Asparagus Yogurt

[0061] As shown in Table 4, the asparagus yogurt prepared in Examples 1-3 showed a significant increase in titratable acidity and a significant decrease in pH after fermentation, giving the asparagus yogurt a unique aroma and taste.

[0062] Experiment Example 4 This experiment measured the water-holding capacity and viscosity of the samples from Examples 1-3 and the unfermented samples from Examples 1-3. The specific measurement methods are as follows: Water-holding capacity determination: The viscosity of the finished yogurt was measured using a Kinexus Pro+ rotational rheometer. The tray was PP35 mm, and the clamp was P35 TiL; the shear rate ranged from 0.01 to 150 s⁻¹. -1 Data was collected from 15 points.

[0063] Viscosity determination: Take approximately 10 g of sample, centrifuge at 6000 g, 10℃ for 30 min, discard the supernatant, invert for 10 min to allow the supernatant to flow out completely, and finally immediately weigh the mass of the precipitate. The calculation formula is as follows: ; In the formula: the mass of the centrifuge tube is denoted as m0; the total mass of the centrifuge tube plus the fermented milk is denoted as m1; and the total mass of the precipitated centrifuge tube is denoted as m2.

[0064] Table 5. Water-holding capacity and viscosity of asparagus yogurt

[0065] The test results are shown in Table 5. Compared with the unfermented Examples 1-3, the viscosity and water-holding capacity of the asparagus yogurt prepared by the present invention are significantly improved, which can effectively prevent whey separation and make the yogurt taste more creamy and smooth.

[0066] Experimental Example 5 This experiment measured the antioxidant capacity of the samples from Examples 1-3 and the unfermented Examples 1-3. Specifically, the DPPH radical scavenging rate and ABTS radical scavenging rate were measured, and the total antioxidant capacity was determined according to the iron reduction / antioxidant capacity (FRAP) method. The results are expressed as FeSO4 equivalents (mmol / L).

[0067] Table 6. Antioxidant capacity of asparagus yogurt

[0068] As shown in Table 6, the asparagus yogurt prepared in Examples 1-3 all showed strong DPPH radical scavenging rate, ABTS radical scavenging rate and total antioxidant capacity, indicating that the asparagus yogurt prepared in this invention has excellent antioxidant activity.

[0069] Experimental Example 6 This experiment tested the viable count of lactic acid bacteria in samples from Examples 1-3 and unfermented Examples 1-3. The specific testing method was based on GB 4789.35-2016 "Food Microbiology Examination: Lactic Acid Bacteria Examination". The test results are shown in Table 7 below.

[0070] Table 7 Total viable count of lactic acid bacteria in asparagus yogurt

[0071] As shown in Table 7, the viable count of lactic acid bacteria in the asparagus yogurt prepared in Examples 1-3 is much higher than the ≥10 specified in GB 7101-2022. 6 The CFU / g indicates that the asparagus yogurt prepared by this invention may contain a sufficient dose of Lactobacillus paracasei JGSLP08, which has positive significance for the treatment and prevention of inflammatory bowel disease.

[0072] Experimental Example 7 This experimental example demonstrates animal experiments conducted on the samples from Examples 1-3.

[0073] Sixty 8-week-old C57BL6 mice were purchased and randomly divided into 6 groups of 60 mice each. The groups were: blank group, model group, positive control group, Example 1 group, Example 2 group, and Example 3 group.

[0074] After one week of acclimatization, except for the control group, all other groups were given 3% DSS solution and allowed free access to drink for 14 consecutive days to establish an animal model of acute IBD inflammation.

[0075] Then, a 7-day intervention period was initiated. According to the group, the mice were administered gavage for 7 consecutive days. The gavage method was as follows: the blank group and the model group were given 200 μL of physiological saline, the positive control group was given 200 μL of 75 mg / kg / d mesalazine aqueous solution, and the groups in Examples 1-3 were administered asparagus yogurt prepared in Examples 1-3 by gavage. The intervention was carried out at a dose of 26 mL per kilogram of mice. The gavage volume of the mice in the intervention groups in Examples 1-3 was converted with reference to the body surface area ratio of 20 g mice to 70 kg adults of 0.0026, which is based on the adult drinking 200 g / d per day.

[0076] During the modeling and intervention period, the weight and living status of mice were observed and recorded daily, and DIA scores were calculated using the same methods as in Example 4. After the experiment, the length of the mouse colon, the colonic inflammatory factors IL-10, IL-6 and TNF-α, and the activity of myeloperoxidase (MPO) in the colonic tissue were measured.

[0077] Experimental results: The present invention tested the efficacy of asparagus yogurt fermented with a mixture of *Lactobacillus paracasei* JGSLP08 and *Lactobacillus pentosus* JGS34 in Examples 1-3 for preventing and treating inflammatory bowel disease. The specific results are as follows: (1) Effects on mouse body weight and DIA score The effects of asparagus yogurt fermented with compound lactic acid bacteria on body weight and DIA score in mice with colitis are as follows: Figure 1 and Figure 2 As shown, the weight of mice in the blank control group gradually increased, and their DIA scores were close to 0. Meanwhile, the mice in the blank control group had good fecal and mental status. The other groups all showed a trend of weight loss and high DIA scores during the modeling period. However, after intervention with the asparagus yogurt of Examples 1-3 of this invention, the mice gradually recovered their weight, and their DIA scores were lower than those of the model group. Therefore, asparagus yogurt fermented with a mixed culture of *Lactobacillus paracasei* JGSLP08 and *Lactobacillus pentosus* JGS34 can, to some extent, improve the symptoms of colitis in mice.

[0078] (2) Effect on colon length in mice DSS-induced IBD causes a shortening of the colon in mice. This invention measured the colon length of each mouse, and the results are as follows: Figure 3 As shown, the colon length in the model group mice was significantly shorter than that in the control group, indicating that DSS successfully induced IBD mice. Furthermore, the colon length in Examples 1-3 showed varying degrees of recovery, demonstrating that the compound lactic acid bacteria asparagus yogurt prepared using *Lactobacillus paracasei* JGSLP08 and *Lactobacillus pentosus* JGS34 can effectively prevent colon shortening in mice.

[0079] (3) Effects on colonic inflammatory factors in mice Upregulation of the secretion of pro-inflammatory factors TNF-α and IL-6 is an important pathway for intestinal mucosal damage, while reduced IL-10 secretion disrupts the homeostasis between pro-inflammatory and anti-inflammatory factors, ultimately inducing colitis.

[0080] Effects of Examples 1-3 on inflammatory factors in mouse colon as follows Figure 4 , Figure 5 and Figure 6As shown, after 7 days of intervention according to Examples 1-3 of the present invention, compared with the model group, mice showed a significant decrease in TNF-α and IL-6, and a significant increase in IL-10. Therefore, the asparagus yogurt fermented by mixed fermentation of *Lactobacillus paracasei* JGSLP08 and *Lactobacillus pentosus* JGS34 provided by the present invention can effectively regulate the expression of pro-inflammatory and anti-inflammatory factors, thereby preventing or treating IBD.

[0081] (4) Effects on mouse MPO Myeloperoxidase (MPO) is a heme protein whose activity directly reflects the infiltration of neutrophils in mouse colon tissue. In a DSS-induced colitis model, intestinal inflammation is a characteristic pathological change. Myeloperoxidase (MPO) activity shows a significant positive correlation with the level of inflammation; therefore, this enzyme activity can serve as a key biochemical indicator for assessing the severity of colitis. Under inflammatory conditions, MPO is released into the extracellular fluid and enters the circulation.

[0082] Depend on Figure 7 It can be seen that the MPO activity of the model group mice was significantly increased, while after 7 days of intervention with asparagus yogurt fermented with compound lactic acid bacteria, the MPO activity of Examples 1-3 showed varying degrees of decrease, indicating that the asparagus yogurt fermented with compound lactic acid bacteria provided by the present invention can effectively alleviate the infiltration of inflammatory cells, thereby alleviating the inflammation level of IBD mice to a certain extent.

[0083] In summary, the asparagus yogurt prepared by the mixed fermentation of *Lactobacillus paracasei* JGSLP08 and *Lactobacillus pentosus* JGS34 of this invention has excellent taste and flavor, superior antioxidant activity, and a high count of viable lactic acid bacteria. Animal experiments show that the asparagus yogurt prepared by this invention can effectively regulate the expression of inflammatory factors and alleviate the infiltration of inflammatory cells, thereby inducing anti-inflammatory responses and improving the colonic environment, achieving the effects of preventing, improving, or treating inflammatory bowel disease.

[0084] Example 4 Relevant properties of Lactobacillus paracasei JGSLP08: 1. Isolation, purification and identification of Lactobacillus paracasei JGSLP08 (1) Isolation and purification of strains Obtaining *Lactobacillus paracasei* JGSLP08 of this invention: A sample of naturally fermented milk yogurt from a farmer's home in Nanchang County, Nanchang City, Jiangxi Province was collected using sterile instruments and transferred to the laboratory within 2 hours for lactic acid bacteria enrichment culture. The sampling date was October 15, 2024. The milk sample was inoculated into sterile MRS broth medium at a 2% inoculation rate in a laminar flow hood and incubated at 37°C for 18 hours. When the MRS broth medium became turbid and a large amount of white bacterial precipitate formed at the bottom, the next step of isolation and purification could be carried out. 1 mL of the MRS broth bacterial suspension was serially diluted 10-fold with sterile physiological saline in a laminar flow hood; 10... -5 ~10 -8 100 μL of diluted bacterial suspension was inoculated into MRS medium, spread, and then the culture dish was inverted and incubated in a 37°C incubator for 48 hours to obtain lactic acid bacteria colonies.

[0085] Single colonies from the above plates were picked and inoculated into fresh MRS medium for streaking isolation. The culture was carried out at 37°C for 48 h, and this process was repeated until morphologically consistent single colonies were obtained. These single colonies were then Gram-stained according to the Gram staining kit instructions. The morphological characteristics of strain JGSLP08, including Gram staining positivity / positivity, presence of capsule, and presence of flagella, were observed under an optical microscope. After Gram staining screening, single colonies of the target bacterium, *Lactobacillus paracasei*, were retained. These single colonies were inoculated into MRS broth medium to obtain a bacterial suspension (10⁻⁶). 9 The culture (CFU / mL) was mixed with an equal volume of 20% glycerol in a culture preservation tube and stored at -80°C.

[0086] (2) Molecular identification of strains DNA extraction and 16S rDNA PCR amplification were performed on suspected *Lactobacillus paracasei* strains. The purified suspected target strain was inoculated into MRS broth medium and cultured at 37°C for 24 h. DNA was then extracted using a bacterial genomic DNA extraction kit. The extracted DNA was amplified by PCR using 1 μL of 10 μM upstream primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1), 1 μL of 10 μM downstream primer 1495R (5'-CTACGGCTACCTTGTTACGA-3', SEQ ID NO.2), 12.5 μL of 2×Taq plus Buffer, and 1 μL of template DNA. The volume was brought to 25 μL with sterile dd H2O. Sterile ultrapure water was used as a negative control instead of template DNA. The amplification conditions were: 94℃ for 5 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, for a total of 29 cycles, with a final extension at 72℃ for 5 min. The target DNA fragment was recovered and then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0087] The sequencing results are shown in SEQ ID NO.3:

[0088] Morphological observation of colonies suspected to be *Lactobacillus paracasei* purified by streak plating revealed that the colonies in the culture medium were milky white, moist, round, smooth, with regular and opaque edges (see...). Figure 8 Gram staining revealed that the strain was Gram-positive, with rod-shaped cells that were non-capsulated and non-flagellated (see...). Figure 9 ).

[0089] Lactobacillus paracasei ( Lacticaseibacillus paracasei JGSLP08 was deposited on April 21, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.34274.

[0090] 2. Determination of the acid resistance, bile salt resistance, and artificial gastrointestinal fluid resistance of Lactobacillus paracasei JGSLP08 (1) Acid resistance Take 1 mL of the overnight bacterial suspension and place it in a 1.5 mL centrifuge tube. Centrifuge at 10000 r / min for 2 min. Then, wash the bacterial cells once with PBS at pH 7.0, followed by resuspending the cells with PBS at pH 2.0 and pH 3.0. Using the original bacterial suspension as a control group, incubate the pH 2.0 and pH 3.0 bacterial suspensions at 35℃ for 3 h, then perform 10-fold serial dilutions, plate the suspensions, and incubate at 37℃ for 24 h. Calculate the bacterial survival rate using the following formula: ; In the formula: N1 is the number of viable bacteria after 3 hours of inoculation; N0 is the number of viable bacteria after 0 hours of inoculation.

[0091] Table 8. Tolerance of Lactobacillus paracasei JGSLP08 to acidic environments.

[0092] The results are shown in Table 8. As can be seen from the data in Table 8, the survival rates of Lactobacillus paracasei JGSLP08 provided by the present invention were 76.46% and 71.76% in pH 3.0 and pH 2.0 environments, respectively, indicating that Lactobacillus paracasei JGSLP08 provided by the present invention has good tolerance to acidic environments.

[0093] (2) Tolerance to bile salts Cultivate bacterial suspensions using 1 mL of MRS medium containing different concentrations of bile salts (0.2%, 0.3%, and 0.5%). Use MRS medium without bile salts as a control group. Incubate the bacterial suspensions in a shaker at 37°C. After 3 h of incubation, perform 10-fold serial dilutions, plate the suspensions, and incubate at 37°C for 24 h. Calculate the survival rate of the strains in each treatment group using the following formula: ; In the formula: N1 is the number of viable bacteria after 3 hours of inoculation; N0 is the number of viable bacteria after 0 hours of inoculation.

[0094] Table 9. Tolerance of Lactobacillus paracasei JGSLP08 to bile salts.

[0095] The results are shown in Table 9. As can be seen from the data in Table 9, the Lactobacillus paracasei JGSLP08 provided by the present invention has a strong tolerance to bile salts. Even at a bile salt concentration of 0.5%, the survival rate of the strain is still as high as 64.84%.

[0096] (3) Tolerance to artificial gastrointestinal fluid Preparation of artificial gastric fluid: Dissolve 0.3 g of pepsin in 100 mL of 0.9% sterile physiological saline. Adjust the pH to 3.0 with 1 mol / L HCl and filter through a 0.22 μm filter membrane.

[0097] Preparation of artificial intestinal fluid: Dissolve 0.2g trypsin and 0.3g ox bile salt in 100mL of 0.9% sterile physiological saline. Adjust the pH to 8.0 with 1mol / L sodium hydroxide and filter through a 0.22μm filter membrane.

[0098] Lactobacillus paracasei JGSLP08 was revived and activated. The overnight culture was centrifuged at 8000 r / min, and the bacterial sludge was washed three times with 0.9% sterile physiological saline. It was then resuspended in 10 mL of artificial gastric fluid and digested at 37℃ for 3 h. Using the original bacterial culture as a control, the treatment and control groups were serially diluted 10-fold. 1 mL of gastric fluid was spread onto MRS agar plates and incubated at 37℃ for 24 h before counting. Another 1 mL of bacterial culture was added to 9 mL of artificial intestinal fluid, and viable bacteria were counted using the same method after 3 h. The formula for calculating the bacterial survival rate is as follows: ; In the formula: N1 is the number of viable bacteria after 3 hours of inoculation; N0 is the number of viable bacteria after 0 hours of inoculation.

[0099] The results showed that after 3 hours of treatment with artificial gastric fluid, Lactobacillus paracasei JGSLP08 still had a survival rate of 85.51% and an intestinal fluid survival rate of 80.82%. Therefore, the strain provided by this invention showed good tolerance in both simulated gastric and intestinal fluids.

[0100] 3. Inhibitory ability of Lactobacillus paracasei JGSLP08 against common pathogenic bacteria The Oxford cup method was used to screen for the inhibitory effect of *Lactobacillus paracasei* on common pathogenic bacteria. *Salmonella cytomegalovirus* (ATCC 14028), *Escherichia coli* (CMCC 44350), and *Staphylococcus aureus* (CMCC 26003) were inoculated into LB broth to prepare a culture medium containing 10... 8 Salmonella cytomegalovirus (CFU / mL), Escherichia coli, and Staphylococcus aureus were incubated. 100 μL of each LB solution was plated onto sterilized LB agar plates. Oxford cups were placed on the LB agar plates, and 200 μL of the same concentration of bacterial suspension was added to each cup. Three replicates were set up, and the plates were incubated at 37°C for 24 h. The diameter of the inhibition zone was recorded. 200 μL of sterilized PBS solution was used as a blank control group.

[0101] Table 10. Diameter of the inhibition zone (mm) of Lactobacillus paracasei JGSLP08

[0102] The results are shown in Table 10. Compared with the blank control group (PBS), the inhibition zone diameter of Lactobacillus paracasei JGSLP08 against Salmonella cytotoxicum, Escherichia coli and Staphylococcus aureus was more than 10 mm, indicating that Lactobacillus paracasei JGSLP08 bacterial solution has a significant inhibitory effect on the three common foodborne pathogens Salmonella cytotoxicum, Escherichia coli and Staphylococcus aureus.

[0103] 4. Test of antioxidant capacity of Lactobacillus paracasei JGSLP08 Preparation of bacterial suspension: Lactobacillus paracasei JGSLP08 was cultured in MRS broth at 37℃ until the concentration reached 10. 9 CFU / mL. Subsequently, the culture medium was removed by centrifugation at 8000 r / min for 5 minutes, and the bacterial cells were collected. The bacterial pellet was resuspended in 2 mL of physiological saline to prepare the test sample. The antioxidant activity of this strain was evaluated using DPPH radical scavenging rate and hydroxyl radical scavenging rate.

[0104] Experimental results showed that the DPPH radical scavenging rate of strain JGSLP08 provided by this invention was 56.82%, and the hydroxyl radical scavenging rate was 67.59%. Combining the results of the DPPH and hydroxyl radical scavenging experiments, the *Lactobacillus paracasei* strain JGSLP08 provided by this invention exhibits strong antioxidant effects.

[0105] 5. Application of Lactobacillus paracasei JGSLP08 in alleviating colitis in mice (1) Animal grouping and modeling Eight-week-old C57BL6 mice were used to establish a DSS colitis model. After acclimatization for 7 days, they were randomly divided into 4 groups of 10 mice each. The specific details of each group are as follows: During the modeling period, the control group (CK) was given 200 μL of sterile saline by gavage; the polysaccharide sodium sulfate group (DSS), the Lactobacillus paracasei intervention group (LP), and the mesalazine control group (MS) were given 200 μL of 3% DSS solution by gavage daily.

[0106] To verify the successful establishment of a mouse colitis model, multiple comprehensive indicators, including body weight, DIA score, and colon length, were used for evaluation after the modeling period and before drug intervention. The model was considered successfully established and could be used to assess the therapeutic effect of subsequent drug administration only when the model group (e.g., the DSS group) showed statistically significant differences (p<0.05) in all the above indicators compared to the normal control group (CK).

[0107] After modeling, the CK group continued to receive 200 μL of sterile saline via gavage daily; the DSS group received 200 μL of sterile saline via gavage daily; and the LP group received 1×10⁻⁶ μL of sterile saline via gavage daily. 9 CFU / mL Lactobacillus paracasei bacterial suspension; MS group received 75 mg / kg / d mesalazine via gavage daily.

[0108] (2) Sample collection Mice were fasted for 12 hours prior to the experiment (with free access to water), and then whole blood samples were collected by enucleation. Immediately after blood collection, the animals were euthanized by cervical dislocation, and the colon tissue was quickly dissected and its length measured. Blood samples were allowed to stand at room temperature for 2-3 hours, then centrifuged at 4°C and 2500 r / min for 10 minutes. The serum supernatant was carefully aspirated, aliquoted, and frozen at -80°C for later use.

[0109] (3) Analysis of mouse body weight, DIA score and colon length Before and after modeling, the mice were monitored for changes in body weight, water and food consumption, and mental status. In addition, the characteristics of mouse feces and occult blood were observed, and the disease activity index (DAI) was calculated according to the scoring criteria shown in Table 11.

[0110] Table 11 DAI Scores

[0111] Experimental results are as follows Figure 10 , Figure 11 , Figure 12 As shown, strain JGSLP08 can effectively alleviate the symptoms of colitis in mice. Figure 10 The results showed that, during the modeling period, except for the CK group, the body weight of all other mice decreased significantly. After the modeling period, the body weight of the mice in the LP group increased more rapidly compared with the DSS group. Figure 11 The results showed that on the day the modeling process ended, DIA scores were performed on the mice. Except for the CK group, all other mice experienced weight loss, soft stools, watery stools, darker or black stools in severe cases, and even bloody stools in some cases. After drug administration, the weight of the LP group mice gradually recovered, and the stool shape and color were close to those of healthy mice. Figure 12 The results showed that the colon length of mice in the DSS group was significantly shorter compared to the CK group. The LP group samples inhibited this shortening of the mouse colon. This indicates that the *Lactobacillus paracasei* provided in this invention can effectively alleviate DSS-induced intestinal damage in mice.

[0112] (4) Measurement of inflammatory factors The inflammatory factors IL-10, IL-6, and TNF-α in mouse colon were measured according to the ELISA kit instructions. The specific steps were as follows: 100 μL of standard and serum were added to a 96-well plate. The wells were sealed with sealing tape and incubated at 37°C in the dark for 90 min, followed by 5 washes. 100 μL of enzyme conjugate diluent was added to the blank wells, and 100 μL of enzyme conjugate working solution was added to the remaining wells. The plates were incubated at 37°C in the dark for 30 min, followed by 5 washes. 100 μL of chromogenic substrate (TMB) was added, and the plates were incubated at 37°C in the dark for 15 min. 100 μL of stop solution was added, and the mixture was thoroughly mixed before measuring the absorbance at 450 nm.

[0113] Figure 13 The results showed that the levels of pro-inflammatory factors TNF-α and IL-6 in the colon of mice in the DSS group were significantly higher than those in other groups, while the expression level of the anti-inflammatory factor IL-10 was significantly lower than that in the other three groups. Compared with the DSS group, the IL-10 concentration in the LP group increased by 27.27%. This indicates that *Lactobacillus paracasei* has a significant anti-inflammatory effect and can effectively alleviate colitis symptoms.

[0114] (5) Determination of malondialdehyde (MDA) and myeloperoxidase (MPO) activity in mouse colon tissue Take an appropriate amount of mouse colon tissue, add 9 times pre-cooled physiological saline to homogenize, centrifuge the homogenate at 3500 r / min for 15 min, collect the supernatant, and detect the MDA content according to the kit instructions. Add 0.1 mL of the above supernatant and 0.1 mL of reagent one to a centrifuge tube, vortex to mix, then add 1.5 mL of reagent two and reagent three, cap the centrifuge tube, vortex to mix, incubate at 95℃ for 40 min, cool, centrifuge at 4000 r / min for 10 min, collect the supernatant and measure its absorbance at 532 nm. Use an equal volume of anhydrous ethanol instead of the test sample as a blank, and use an equal volume of 50% glacial acetic acid instead of reagent three as a control.

[0115] Take an appropriate amount of mouse colon tissue, add 9 times pre-cooled physiological saline to homogenize, centrifuge the homogenate at 3500 r / min for 15 min, collect the supernatant, and detect the MPO content according to the kit instructions. Take an appropriate amount of the above homogenized tissue and mix thoroughly with 0.02 mL of reagent three, then incubate at 37℃ for 15 min. Add 0.2 mL of reagent four and 3 mL of chromogenic reagent, mix thoroughly, and incubate at 37℃ for 30 min. Remove from the water, add 0.05 mL of reagent seven, mix thoroughly, and incubate at 60℃ for 10 min. Immediately after removal, measure the absorbance at 460 nm. Use 3 mL of distilled water instead of 3 mL of chromogenic reagent as a control sample.

[0116] Table 12 MDA content and MPO activity in mouse colon tissue

[0117] The results of MDA content and MPO activity measurements in mouse colon tissue are shown in Table 12. Compared with the CK group, the MDA content and MPO activity in the DSS group were significantly increased. However, after intervention with *Lactobacillus paracasei* JGSLP08 and mesalazine, both MDA content and MPO activity decreased to varying degrees. This indicates that *Lactobacillus paracasei* JGSLP08 provided by this invention can effectively reduce oxidative stress damage and alleviate inflammatory cell infiltration in mice, which has a certain alleviating effect on inflammatory bowel disease.

[0118] (6) Changes in tight junction proteins Immunohistochemical analysis of mouse colon homogenate supernatant was performed using qRT-PCR to detect changes in the mRNA expression of four proteins: tight junction protein-1 (Claudin-1), occluder band protein-1 (ZO-1), and mucin-2 (MUC-2).

[0119] Table 13 Relative expression levels of tight junction proteins in mouse colon tissue

[0120] The results are shown in Table 13. Compared with the CK group, the mRNA expression levels of ZO-1, Claudin-1, and MUC-2 in the colon tissue of the DSS group were significantly reduced, while the expression levels of the LP group were significantly increased to varying degrees compared with the DSS group. This indicates that the Lactobacillus paracasei JGSLP08 provided in this invention can increase the expression level of tight junction proteins, effectively protect the connections between colonic epithelial cells, restore the intestinal barrier function, protect the integrity of the colonic mucosal epithelium, and reduce mucosal damage.

[0121] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing asparagus yogurt fermented with compound lactic acid bacteria, characterized in that, Includes the following steps: (1) Take asparagus pulp, raw milk, whey protein concentrate, white sugar and sodium alginate and mix them thoroughly. Then, homogenize the mixed materials and pasteurize them. (2) Inoculate with 0.01%-0.1% of the total amount of compound lactic acid bacteria agent and ferment to obtain the compound lactic acid bacteria fermented asparagus yogurt.

2. The preparation method according to claim 1, characterized in that, The preparation method of the asparagus pulp is as follows: Select fresh asparagus, rinse and cut into sections, pre-cook the cut asparagus for 1-5 minutes, then mix the asparagus and water at a mass ratio of 1:2-1:5 and blend into a pulp, homogenize at 20-70 MPa for 1-6 minutes at a homogenization temperature of 30-45℃, and then pass through a 30-mesh sieve to obtain the asparagus pulp.

3. The preparation method according to claim 1, characterized in that, The weight ratio of the asparagus puree, raw milk, whey protein concentrate, white sugar and sodium alginate is (80-150):(800-1000):(10-20):(40-90):(2-10).

4. The preparation method according to claim 1, characterized in that, The pasteurization conditions are: pasteurization at 95°C for 5 minutes.

5. The preparation method according to claim 1, characterized in that, The colony count of the compound lactic acid bacteria agent is (1-9) × 10⁻⁶. 10 CFU / g, composed of Lactobacillus paracasei JGSLP08 and Lactobacillus pentosacchari JGS34 in a live count ratio of 1:1; The Lactobacillus paracasei JGSLP08 was deposited at the China General Microbiological Culture Collection Center on April 21, 2025, with accession number CGMCC No. 34274. The Pentosacchari lactis JGS34 was deposited on June 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.30985.

6. The preparation method according to claim 1, characterized in that, The fermentation conditions are as follows: ferment at 25-38℃ for 8-12 hours, and then place at 2-4℃ for post-ripening.

7. The compound lactic acid bacteria fermented asparagus yogurt prepared by the preparation method according to any one of claims 1-6.

Images