Compound bacterial agent for reducing blood sugar and blood fat, application of compound bacterial agent and method for preparing fermented coconut milk beverage from compound bacterial agent

Abstract

The invention discloses a compound bacterial agent for reducing blood sugar and blood fat, application of the compound bacterial agent and a method for preparing a fermented coconut milk beverage from the compound bacterial agent, belongs to the technical field of microorganisms, and particularly relates to the compound bacterial agent for reducing blood sugar and blood fat, the compound bacterial agent comprises fermented lactobacillus mucus AL-8 and lactobacillus paracasei DX-5, and the compound bacterial agent is prepared from the fermented lactobacillus mucus AL-8, the lactobacillus paracasei DX-5, the lactobacillus paracasei DX-5, the lactobacillus paracasei DX-5 and the lactobacillus paracasei DX-5 and the lactobacillus paracasei DX-5 and the lactobacillus paracasei DX-5 and the lactobacillus paracasei DX-5 and the lactobacillus paracasei DX-5. The preservation number of the lactobacillus mucus AL-8 is CCTCC (China Center For Type Culture Collection) NO: M20252735; the preservation number of the lactobacillus paracasei DX-5 is CCTCC (China Center For Type Culture Collection) NO: M20252736. According to the invention, the fermented coconut milk beverage is prepared by utilizing the inhibition effect of a blood sugar and blood fat reducing compound bacterial agent consisting of fermented lactobacillus mucus AL-8 and casei paracasei DX-5 on alpha-amylase and pancreatic lipase.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a blood glucose and lipid-lowering compound bacterial agent and application thereof and a method for preparing fermented coconut milk beverage. BACKGROUND

[0002] With the development of economy and the change of lifestyle, the incidence of diseases related to abnormal glucose and lipid metabolism, such as diabetes, obesity, non-alcoholic fatty liver and atherosclerosis, continues to rise worldwide, and has become a serious public health problem. In addition to drug treatment, dietary intervention to regulate the body's metabolism is an important prevention and management strategy. In recent years, the use of probiotics to target the regulation of glucose and lipid metabolism has become a research hotspot for the development of functional foods to improve glucose and lipid metabolism.

[0003] Hainan is a tropical region in China, with rich tropical agricultural resources, especially a developed coconut industry. Coconut milk, as a natural beverage raw material with rich nutrients, has a unique flavor and nutritional value. At the same time, Hainan Island has a rich variety of fermented foods, including distiller's grains vinegar, fish acid, meat acid, pickled fruits and vegetables, etc., which are good sources of lactic acid bacteria resources. Lactic acid bacteria fermentation technology has been widely used in food processing, and through the selection of high-quality lactic acid bacteria for fermentation of food materials, not only the nutritional value of the product can be increased, but also a variety of bioactive substances can be produced. At present, there are a variety of probiotic fermented dairy products on the market, such as yogurt, fermented milk beverages, etc., which mainly contain traditional probiotics such as lactobacillus and bifidobacterium, and are generally claimed to regulate intestinal flora balance and promote digestion and absorption. However, most of the strains used in existing products have a wide range of functions, and there is a lack of special probiotic combinations that are precisely selected and synergistically combined for key aspects of glucose and lipid metabolism, such as inhibition of fat absorption, promotion of glucose utilization, improvement of insulin sensitivity, regulation of bile acid metabolism, etc.

[0004] On the other hand, consumers' demand for plant-based health foods is growing. Coconut milk, as a nutrient-rich plant-based raw material, is rich in medium-chain fatty acids, which have unique metabolic characteristics, but traditional coconut milk products have a single form. However, existing coconut milk fermentation technology mostly follows traditional yogurt starters such as Streptococcus thermophilus and Lactobacillus bulgaricus, and the main purpose is to produce acid coagulation, form texture and basic flavor. The growth adaptability of these strains in coconut milk matrix, the ability to produce adhesion, and the specific regulation function of glucose and lipid metabolism have not been fully researched and optimized. In addition, coconut milk has a high fat content, and how to optimize its fatty acid composition through the fermentation of specific probiotics and produce active substances with metabolic regulation function is a problem that existing technology has not systematically solved.

[0005] Therefore, there is an urgent need in this field to develop a specialized combination of probiotics that have undergone rigorous screening and scientific formulation. This combination can not only grow and ferment stably and with high activity in coconut milk substrate, producing a pleasant flavor and suitable texture, but also, through the synergistic effect between strains, specifically produce or enhance the function of regulating host glucose and lipid metabolism, such as improving insulin sensitivity, lowering blood lipids, and reducing fat accumulation. This would provide the market with a new type of fermented food that combines plant-based nutrition with precise metabolic regulation. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a hypoglycemic and lipid-lowering compound microbial agent and its application, as well as a method for preparing fermented coconut milk beverages. The method utilizes the inhibitory effect of the hypoglycemic and lipid-lowering compound microbial agent, composed of *Lactobacillus fumaricus* AL-8 and *Lactobacillus paracasei* DX-5, on α-amylase and pancreatic lipase to prepare fermented coconut milk beverages. After fermentation with the compound microbial agent, the coconut milk produces new organic acids that inhibit the growth of harmful bacteria. Simultaneously, volatile compounds are generated, resulting in a mellow and fragrant coconut milk flavor, a delicate taste, reduced sugar content, and significantly increased protein content.

[0007] To achieve the above objectives, the present invention provides a compound bacterial agent for lowering blood sugar and blood lipids, wherein the compound bacterial agent includes *Lactobacillus fermentum* (… Limosilactobacillus fermentum AL-8 and Lactobacillus paracasei ( Lacticaseibacillus paracasei DX-5; The fermenting Lactobacillus mucinus AL-8 was deposited at the China Center for Type Culture Collection on December 1, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO:M20252735. The *Lactobacillus paracasei* DX-5 was deposited on December 1, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M20252736.

[0008] Preferably, the volume ratio of *Lactobacillus fermentans* AL-8 and *Lactobacillus paracasei* DX-5 in the compound bacterial agent is 1:1.

[0009] Preferably, the effective viable count of *Lactobacillus fermentum* AL-8 in the compound bacterial agent is 10. 8 CFU / mL, the effective viable count of *Lactobacillus paracasei* DX-5 in the compound bacterial agent is 10. 8 CFU / mL.

[0010] The present invention also provides the application of the compound microbial agent in the preparation of fermented coconut milk beverages.

[0011] The present invention also provides a method for preparing fermented coconut milk beverage using the compound microbial agent, comprising the following steps: 1) Crack the coconut shell, extract the coconut meat and coconut water, homogenize them, filter with gauze, and sterilize to obtain coconut milk; 2) Inoculate the compound microbial agent into coconut milk and ferment to obtain fermented coconut milk beverage.

[0012] Preferably, the coconut mentioned in step 1) is a black-shelled coconut.

[0013] Preferably, the ratio of coconut meat to coconut water in step 1) is 1g: 1~3mL.

[0014] Preferably, the sterilization in step 1) is pasteurization, the pasteurization temperature is 65°C, and the pasteurization time is 30 min.

[0015] Preferably, the inoculation amount of the compound microbial agent in step 2) is 2~4% v / v.

[0016] Preferably, the fermentation temperature in step 2) is 37°C and the fermentation time is 2~12h.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects: (1) After isolation and purification, this invention screened a new strain of *Lactobacillus fermentatus* AL-8 and *Lactobacillus paracasei* DX-5. *Lactobacillus fermentatus* AL-8 has good surface hydrophobicity, self-aggregation ability, acid resistance, and bile salt resistance. *Lactobacillus paracasei* DX-5 can impart better aroma and flavor to coconut milk during fermentation. Both *Lactobacillus fermentatus* AL-8 and *Lactobacillus paracasei* DX-5 have high tolerance to artificial intestinal fluid and can exhibit certain resistance in the gastrointestinal tract, thereby exerting their probiotic function. Both *Lactobacillus fermentatus* AL-8 and *Lactobacillus paracasei* DX-5 also have inhibitory effects on α-amylase and pancreatic lipase, and have hypoglycemic and hypolipidemic effects.

[0018] (2) The fermented coconut milk beverage prepared using the compound microbial agent composed of Lactobacillus fermentum AL-8 and Lactobacillus paracasei DX-5 obtained by the present invention has a variety of biological activities and good compatibility and adaptability with lactic acid bacteria, which can promote the proliferation of lactic acid bacteria. After the coconut milk is fermented by the compound microbial agent of the present invention, new organic acids are produced to inhibit the growth of harmful bacteria. At the same time, volatile compounds are generated, which makes the coconut milk flavor mellow and fragrant, with a delicate taste, reduces sugar content and significantly increases protein content. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a colony morphology diagram of Lactobacillus fermentans AL-8. Figure 2 This is a colony morphology diagram of Lactobacillus paracasei DX-5. Figure 3 The graph shows the change in self-aggregation of Lactobacillus fermentum AL-8 and Lactobacillus paracasei DX-5 over time. In the graph, AL-8 represents Lactobacillus fermentum AL-8 and DX-5 represents Lactobacillus paracasei DX-5. Different uppercase / lowercase letters indicate significant differences in the viable number of strains in different states (P<0.05). Figure 4 The graph shows the change in hydrophobicity of Lactobacillus fermentum AL-8 and Lactobacillus paracasei DX-5 over time. In the graph, AL-8 represents Lactobacillus fermentum AL-8 and DX-5 represents Lactobacillus paracasei DX-5. Different uppercase / lowercase letters indicate significant differences in the viable count of strains in different states (P<0.05). Figure 5 The acid resistance of Lactobacillus fermentum AL-8 and Lactobacillus paracasei DX-5 is shown in the figure. AL-8 represents Lactobacillus fermentum AL-8 and DX-5 represents Lactobacillus paracasei DX-5. Different uppercase / lowercase letters indicate that there are significant differences in the number of viable bacteria between different states of the strain (P<0.05). Figure 6 To evaluate the bile salt tolerance of Lactobacillus fermentum AL-8 and Lactobacillus paracasei DX-5, AL-8 represents Lactobacillus fermentum AL-8 and DX-5 represents Lactobacillus paracasei DX-5. Different uppercase / lowercase letters indicate significant differences in viable cell counts between strains in different states (P<0.05). Figure 7 The pH changes of coconut milk beverages fermented with Lactobacillus mucilaginosus AL-8 and Lactobacillus paracasei DX-5; Figure 8 The titratable acidity changes of coconut milk beverages fermented with Lactobacillus mucilaginosus AL-8 and Lactobacillus paracasei DX-5; Figure 9The graph shows the pancreatic lipase inhibition rate of fermented coconut milk beverage in Example 2 and fermented coconut milk beverage in Comparative Example 2. In the graph, AL-8+DX-5 represents fermented coconut milk beverage in Example 2, and Z-15+S-NB represents fermented coconut milk beverage in Comparative Example 2. Different uppercase / lowercase letters indicate that there are significant differences in the number of viable bacteria of the strains in different states (P<0.05). Figure 10 The graph shows the α-amylase inhibition rate of fermented coconut milk beverage in Example 2 and fermented coconut milk beverage in Comparative Example 2. In the graph, AL-8+DX-5 represents fermented coconut milk beverage in Example 2, and Z-15+S-NB represents fermented coconut milk beverage in Comparative Example 2. Different uppercase / lowercase letters indicate that there are significant differences in the number of viable bacteria in different states (P<0.05).

[0021] Preservation Certificate Lactobacillus fermentans AL-8, classified and named Lactobacillus fermentans AL-8 Limosilactobacillus fermentum AL-8, this strain is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on December 1, 2025, with accession number CCTCC NO:M 20252735.

[0022] Lactobacillus paracasei DX-5, classified as Lactobacillus paracasei DX-5 Lacticaseibacillus paracasei DX-5, this strain is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on December 1, 2025, with accession number CCTCC NO:M 20252736. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] The materials used in this invention were sourced as follows: the genome extraction kit was purchased from Sangon Biotech (Shanghai) Co., Ltd.; PBS buffer was purchased from Beijing Solarbio Science & Technology Co., Ltd.; PNPB solution was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; Lactobacillus fermentum Z-15, preservation number CCTCC NO: M 2024024; and Lactobacillus paracasei S-NB, preservation number CCTCC NO: M 2021461.

[0029] The MRS liquid culture medium used in this invention consists of: 20.0g glucose, 10.0g peptone, 10.0g beef extract, 5.0g yeast extract, 2.0g triammonium citrate, 2.62g dipotassium hydrogen phosphate, 5.0g anhydrous sodium acetate, 0.58g magnesium sulfate, 0.198g ammonium sulfate, and 1mL Tween 80. The above components are dissolved in 1L distilled water. After complete dissolution, the pH is adjusted to 6.0, and then dispensed and sterilized at 121℃ for 20min.

[0030] The MRS solid culture medium containing calcium carbonate used in this invention consists of: 20.0g glucose, 10.0g peptone, 10.0g beef extract, 5.0g yeast extract, 2.0g triammonium citrate, 2.62g dipotassium hydrogen phosphate, 5.0g anhydrous sodium acetate, 0.58g magnesium sulfate, 0.198g ammonium sulfate, 1.0mL Tween 80, 15.0g agar, and 5g calcium carbonate. 1L of distilled water is added, and after complete dissolution, the pH is adjusted to 6.0. The medium is then dispensed and sterilized at 121°C for 20 minutes.

[0031] The MRS solid culture medium used in this invention consists of: 20.0g glucose, 10.0g peptone, 10.0g beef extract, 5.0g yeast extract, 2.0g triammonium citrate, 2.62g dipotassium hydrogen phosphate, 5.0g anhydrous sodium acetate, 0.58g magnesium sulfate, 0.198g ammonium sulfate, 1.0mL Tween 80, and 15.0g agar. 1L of distilled water is added, and after complete dissolution, the pH is adjusted to 6.0. The mixture is then dispensed and sterilized at 121°C for 20 minutes.

[0032] Example 1 I. Isolation and purification of strains AL-8 and DX-5.

[0033] Fermented radishes and fish acid obtained from Nanbin Agricultural Market, Yazhou District, Sanya City, Hainan Province in June 2024 were inoculated into MRS solid medium containing calcium carbonate and cultured at 37°C for 48 hours. Single colonies with calcium carbonate clear zones were picked, isolated and purified by streak plating, and then preserved.

[0034] Slant culture preservation: Use a disposable inoculation loop to pick up a small amount of purified strain and inoculate it in the MRS slant solid medium using a zigzag streak method. After streaking, flame the mouth of the test tube and place it in a static culture at 37°C. After the strain has matured, store it in a refrigerator at 4°C.

[0035] Glycerol tube cryopreservation: The purified bacterial solution was mixed with 80% glycerol at a volume ratio of 1:1 and stored in cryovials, and then frozen in a freezer at -80°C.

[0036] II. Identification of Lactobacillus fermentans AL-8 and Lactobacillus paracasei DX-5.

[0037] 1. Morphological and physiological identification.

[0038] *Lactobacillus fermentans* AL-8 is a Gram-positive bacterium that appears as rod-shaped cells under a microscope. Its culture on MRS solid medium yielded the following results: Figure 1 As shown in the figure, it can be observed that the colonies of Lactobacillus fermentans AL-8 are round with white raised bumps and a colony diameter of 0.6 mm.

[0039] *Lactobacillus paracasei* DX-5 is a Gram-positive bacterium that appears as rod-shaped cells under a microscope. Its culture on MRS solid medium yielded the following results: Figure 2 As shown in the figure, it can be observed that the colonies of Lactobacillus paracasei DX-5 are medium to small, raised, moist, with neat edges, white, round, and glossy.

[0040] 2. Molecular biological identification.

[0041] Genomic DNA was extracted from strains AL-8 and DX-5 using a genomic DNA extraction kit. Using these as templates, the 16S rDNA gene fragments of the strains were amplified by PCR using universal primer pairs 27F (SEQ ID NO.3: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID NO.4: 5'-TACGGTTACCTTGTTACGACTT-3') (the 16S rDNA sequences are shown in SEQ ID NO.1 and SEQ ID NO.2). The PCR products were sent to Blast Biotech for sequencing, and the sequencing results were compared with those in the NCBI Genbank database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). It was found that strain AL-8 is similar to *Lactobacillus fermentum* (…). Limosilactobacillus fermentum The highest homology was found in strain DX-5, at 99.93%; strain DX-5 showed the highest homology with *Lactobacillus paracasei* (…). Lacticaseibacillus paracasei The homology was highest, at 100%. Therefore, the present invention, through isolation and purification, yielded one strain of Lactobacillus fermentans AL-8 and one strain of Lactobacillus paracasei DX-5.

[0042] The nucleotide sequence of the 16S rDNA of Lactobacillus fermentans AL-8 is shown in SEQ ID NO.1.

[0043] The nucleotide sequence of the 16S rDNA of Lactobacillus paracasei DX-5 is shown in SEQ ID NO.2.

[0044] III. Determination of the self-aggregation ability, surface hydrophobicity, acid resistance, and bile salt resistance of fermenting *Lactobacillus mucinus* AL-8 and *Lactobacillus paracasei* DX-5 strains.

[0045] Frozen Lactobacillus fermentans AL-8 and Lactobacillus paracasei DX-5 were inoculated into MRS liquid medium and cultured at 37°C for 24 h. After secondary activation, the bacteria were inoculated at 3% v / v in MRS liquid medium and cultured at 37°C for 18 h. After centrifugation, the bacterial cells were collected and diluted with PBS buffer. The surface hydrophobicity, self-aggregation ability, acid resistance and bile salt resistance of the strains were then determined.

[0046] 1. Determination of self-aggregation ability.

[0047] Bacterial cultures of two strains, *Lactobacillus mucinus* AL-8 and *Lactobacillus paracasei* DX-5, were collected in centrifuge tubes and centrifuged at 5000 rpm for 5 min. The bacterial culture was then resuspended in PBS buffer and the OD was adjusted. 600 The concentration was reduced to 0.60 ± 0.02, and recorded as A0. The suspension was allowed to stand, and then the supernatant was collected at 1 hour, 3 hours, and 5 hours after standing to determine the OD value.600 The absorbance at that point is denoted as A1.

[0048] The formula for calculating the self-aggregation ability of the strain is as follows: Self-aggregation (%) = (1-A1 / A0)×100%.

[0049] Strain self-aggregation refers to the ability of bacterial cells to bind together and form cell aggregates, which is associated with biofilm formation and helps the strain colonize intestinal epithelial cells. Figure 3 It can be seen that the self-aggregation ability of both Lactobacillus fermentum AL-8 and Lactobacillus paracasei DX-5 changed significantly with time, and the longer the storage time, the more significant the increase in the self-aggregation ability of the two strains, proving that these two strains have the potential to adhere to intestinal cells and colonize the intestine.

[0050] 2. Surface hydrophobicity determination.

[0051] Two bacterial cultures, *Lactobacillus mucinus* AL-8 and *Lactobacillus paracasei* DX-5, were respectively placed in 15 mL centrifuge tubes and centrifuged at 5000 rpm for 5 min. The supernatant was discarded, and the cultures were mixed with PBS buffer by pipetting. The bacterial sludge was washed three times, and then resuspended in PBS buffer and the OD was adjusted. 600 The result was adjusted to 0.6 ± 0.02, and recorded as B0. 1 mL of xylene was added to a 10 mL centrifuge tube, followed by 3 mL of OD. 600 The bacterial suspension was 0.6 ± 0.02 mg / L. After vortexing and mixing, it was allowed to stand for 1 h, 3 h, and 5 h. The aqueous phase was then collected and the OD was measured. 600 The absorbance value at that point is denoted as B1.

[0052] The formula for calculating the surface hydrophobicity of a strain is as follows: Strain hydrophobicity (%) = (1 - B1 / B0) × 100%.

[0053] Strain surface hydrophobicity refers to the ability of a strain to detach from the aqueous phase and move towards other organic phases, and is related to the strain's non-specific adhesion ability. Figure 4 It can be seen that the surface hydrophobicity of both Lactobacillus fermentum AL-8 and Lactobacillus paracasei DX-5 changed significantly with time, and the longer the storage time, the more significant the increase in surface hydrophobicity of the two strains.

[0054] 3. Acid resistance test.

[0055] Take 1 mL of culture medium of Lactobacillus fermentum AL-8 and Lactobacillus paracasei DX-5 with good growth, centrifuge to collect the cells, resuspend them in the corresponding MRS liquid culture medium at pH 2.5, 3.0 and 3.5 respectively, and incubate the bacterial suspension at 37℃. Take samples at 0h and 3h, serially dilute the bacterial suspension with physiological saline, spread them on MRS solid agar plates and count the colonies.

[0056] The formula for calculating strain survival rate is as follows: Strain survival rate (%) = N t / N0×100%, where N t N is the colony count after 3 hours, and N0 is the colony count after 0 hours.

[0057] Tolerance to acid, such as Figure 5 As shown, the survival rates of *Lactobacillus fermentum* AL-8 and *Lactobacillus paracasei* DX-5 were highest at pH 3.5. At pH 3.0, the survival rates of both strains decreased, and at pH 2.5, the survival rate of *Lactobacillus paracasei* DX-5 was below 50%. This indicates that *Lactobacillus fermentum* AL-8 and *Lactobacillus paracasei* DX-5 possess a certain degree of acid resistance within the pH range of 3.0–3.5 and can maintain good activity after entering the human body.

[0058] 4. Test of bile salt tolerance.

[0059] Take 1 mL of culture medium of Lactobacillus fermentum AL-8 and Lactobacillus paracasei DX-5 with good growth, centrifuge to collect the cells, and resuspend them in the corresponding MRS liquid medium with bile salt contents of 0.1%, 0.2% and 0.3% (w / v). Incubate the bacterial suspensions at 37℃, and take samples at 0h and 3h. Dilute the samples serially with PBS buffer, spread them on MRS solid medium and count the colonies.

[0060] The formula for calculating strain survival rate is as follows: Strain survival rate (%) = N t / N0×100%, where N t N is the colony count after 3 hours, and N0 is the colony count after 0 hours.

[0061] Bile salts in human intestinal fluid can affect cell permeability and even lead to bacterial death. Therefore, bacterial strains need a certain degree of bile salt tolerance to colonize the body and exert their probiotic functions. Figure 6 It can be seen that with the increase of bile salt concentration, the survival rate of both *Lactobacillus fermentum* AL-8 and *Lactobacillus paracasei* DX-5 strains continuously decreased. At a bile salt concentration of 0.3% (w / v), the survival rate of both strains was significantly lower than at the beginning. Within the bile salt concentration range of 0-0.2% (w / v), both strains exhibited good bile salt tolerance.

[0062] IV. Determination of the tolerance of fermenting Lactobacillus mucinus AL-8 and Lactobacillus paracasei DX-5 in artificial gastric and intestinal fluids.

[0063] To simulate the digestive effects of artificial gastric and intestinal fluids on the bacterial strain, the survival rate of the strain in artificial gastric and intestinal fluids was determined. The specific procedure is as follows: Gastric buffer solution: NaCl 2.758g, KCl 0.5144g, KH2PO4 0.225g, NaHCO3 2.1g, MgCl2(H2O)6 0.203g, (NH4)2CO3 0.0786g, 1L deionized water. Weigh 15mg of gastric mucin and 6.25mg of pepsin and dissolve them separately in 10mL of gastric buffer solution. After mixing, combine the solutions and adjust the pH of the buffer solution to 3.0. Incubate at 37℃ for 20min to activate enzyme activity. This solution is then used as a simulated gastric fluid.

[0064] Preparation of intestinal buffer solution: KCl 0.507g, KH2PO4 0.1089g, NaHCO3 7.1409g, NaCl 2.2441g, MgCl2(H2O)6 0.0671g, 1L deionized water. Dissolve 8.17mg bile salts and 5.62mg secretin separately in 10mL of intestinal buffer solution, mix well, combine, adjust the pH of the buffer solution to 7.0, and incubate at 37℃ for 20min to activate enzyme activity. This is used as a simulated intestinal fluid for later use.

[0065] Cultures of *Lactobacillus fermentans* AL-8 and *Lactobacillus paracasei* DX-5 strains with good growth were collected by centrifugation, and the cells were resuspended in simulated gastric and intestinal fluids. The cells were then incubated at 37°C for 3 hours. Samples of simulated gastric and intestinal fluids were taken at 0 and 3 hours. The samples were serially diluted with physiological saline and then spread onto MRS solid medium for colony counting.

[0066] The survival rate calculation formula is as follows: Strain survival rate (%) = (N) / (N) t / N0)×100%; where N t N is the colony count after 3 hours, and N0 is the colony count after 0 hours.

[0067] Table 1 Results of strain tolerance to artificial gastrointestinal fluid

[0068] The results are shown in Table 1. The food remained in the stomach for digestion for approximately 3 hours. The survival rates of *Lactobacillus fermentum* AL-8 and *Lactobacillus paracasei* DX-5 after 3 hours of incubation in simulated gastric fluid were 65.74% and 60.07%, respectively. However, after 3 hours of incubation in simulated intestinal fluid, the survival rates of *Lactobacillus fermentum* AL-8 and *Lactobacillus paracasei* DX-5 were 101.96% and 101.10%, respectively. Compared to their survival rates in gastric fluid, the survival rates of *Lactobacillus fermentum* AL-8 and *Lactobacillus paracasei* DX-5 were significantly higher in intestinal fluid, both approaching 100%. This indicates that *Lactobacillus fermentum* AL-8 and *Lactobacillus paracasei* DX-5 exhibit good tolerance to artificial intestinal fluid and possess a certain degree of resilience in the gastrointestinal tract, thus exerting their probiotic functions.

[0069] Example 2 Frozen glycerol-based Lactobacillus fermentans AL-8 and Lactobacillus paracasei DX-5 cultures were inoculated separately into MRS liquid medium and cultured at 37°C for 24 hours. After a second activation, the cultures were inoculated into fresh MRS liquid medium and cultured at 37°C for 18 hours. Then, the cultures were inoculated again into MRS liquid medium and activated at 37°C for 12 hours to obtain the activated Lactobacillus fermentans AL-8 solution (with an effective viable count of 10-1). 8 CFU / mL) and Lactobacillus paracasei DX-5 activation solution (effective viable count of 10 CFU / mL) 8 The compound bacterial agent was prepared by mixing Lactobacillus AL-8 activation solution and Lactobacillus paracasei DX-5 activation solution at a mass / volume ratio of 1:1.

[0070] 1) Select local high-quality black-shelled coconuts from Wenchang, Hainan, that are free from mold, rot, and pests. Crack the shells to extract the solid endosperm (coconut meat). Mix the chopped coconut meat with coconut water at a ratio of 1g:2mL. Filter the mixture through gauze to remove impurities and obtain coconut milk. Dispense the coconut milk into sterilized glass bottles and pasteurize at 65℃ for 30 minutes. After sterilization, cool to room temperature.

[0071] 2) The total inoculation amount is 3% v / v. The compound bacterial agent is inoculated into the pasteurized coconut milk at room temperature. After mixing evenly, it is allowed to ferment at 37°C for 2-14 hours. After fermentation, the fermented coconut milk beverage is obtained and stored at 4°C.

[0072] The pH value and titratable acidity were measured during the static fermentation process, and the results are as follows: Figure 7 , Figure 8 As shown. From Figure 7 It can be seen that the pH value gradually decreases with increasing fermentation time, begins to stabilize after 8 hours, and reaches 4.49 after 12 hours; from Figure 8It can be seen that the titratable acidity increases continuously with the increase of fermentation time, and the change is obvious after 6 hours, reaching 48°T after 12 hours.

[0073] Determination of α-amylase inhibition rate: 200 μL of the supernatant of the compound bacterial agent (prepared by centrifuging the compound bacterial agent at 5000 r / min for 5 min) was mixed with 200 μL of α-amylase enzyme solution (1 U / mL), and incubated at 25℃ for 10 min. Then, 200 μL of 1% (w / v) starch solution was added, and the mixture was incubated at 25℃ for 10 min. Subsequently, 400 μL of DNS was added to the sample, and the mixture was boiled in a water bath for 5 min. After the sample cooled to room temperature, 4 mL of distilled water was added for dilution, and the absorbance was measured at 540 nm. The control group used PBS buffer instead of the sample, and the blank group used PBS buffer instead of the enzyme solution. The α-amylase inhibition rate was calculated using the following formula: α-amylase inhibition rate (%) = (1 - (A... 样品 -A 空白 ) / A 对照 ) × 100%.

[0074] Determination of pancreatic lipase inhibition rate: 60 μL of the supernatant of the compound bacterial agent (prepared by centrifuging the compound bacterial agent at 5000 r / min for 5 min) was placed in a 10 mL centrifuge tube. The inhibitory activity of the sample on pancreatic lipase was determined by colorimetry. 150 μL of Tris-HCl (0.05 M, pH 8.0) and pancreatic lipase (40 U / mL) were added sequentially to the sample. After reacting at 37℃ for 10 min, 300 μL of PNPB solution (18%, v / v) was added, and the mixture was reacted at 37℃ for 15 min. After the reaction was complete, the sample was centrifuged at 5000 rpm for 5 min, and the absorbance of the supernatant was measured at 405 nm. The pancreatic lipase inhibition rate was calculated using the following formula: Pancreatic lipase inhibition rate (%) = (1 - (A3 - A4) / (A1 - A2)) × 100%, where A1 is the blank group, A2 is the blank control group, A3 is the sample group, and A4 is the sample control group. The specific composition of the blank group, blank control group, sample group, and sample control group is shown in Table 2 below.

[0075] Table 2. Components for determining pancreatic lipase inhibition rate

[0076] The compound bacterial agent inhibited α-amylase by 30.31% and pancreatic lipase by 38.79%.

[0077] Comparative Example 1 1) Select local high-grade black-shelled coconuts from Wenchang, Hainan, that are free from mold, rot, and pests. Crack the shells to extract the solid endosperm (coconut meat). Mix the chopped coconut meat with coconut water at a ratio of 1g:2mL. Filter the mixture through gauze to remove impurities and obtain coconut milk. Dispense the coconut milk into sterilized glass bottles and pasteurize at 65℃ for 30 minutes. After sterilization, cool to room temperature to obtain aged coconut milk, and refrigerate at 4℃.

[0078] Comparative Example 2 Frozen glycerol-based Lactobacillus fermentation culture Z-15 and Lactobacillus paracasei S-NB were inoculated separately into MRS liquid medium and cultured at 37°C for 24 hours. After a second activation, the culture was inoculated into fresh MRS liquid medium and cultured at 37°C for 18 hours. It was then inoculated again into MRS liquid medium and activated at 37°C for 12 hours to obtain the activated Lactobacillus fermentation culture Z-15 (effective viable count of 10⁻⁶). 8 CFU / mL) and Lactobacillus paracasei S-NB activation solution (effective viable count of 10). 8 The activated liquid of Lactobacillus fumarate Z-15 and the activated liquid of Lactobacillus paracasei S-NB were mixed at a mass / volume ratio of 1:1 to obtain compound bacterial agent II.

[0079] 1) Select local high-quality black-shelled coconuts from Wenchang, Hainan, that are free from mold, rot, and pests. Crack the shells to extract the solid endosperm (coconut meat). Mix the chopped coconut meat with coconut water at a ratio of 1g:2mL. Filter the mixture through gauze to remove impurities and obtain coconut milk. Dispense the coconut milk into sterilized glass bottles and pasteurize at 65℃ for 30 minutes. After sterilization, cool to room temperature.

[0080] 2) With a total inoculation amount of 3% v / v, inoculate the compound bacterial agent II into the pasteurized coconut milk at room temperature, mix well, and let it ferment at 37℃ for 2~14h. After fermentation, you will get fermented coconut milk beverage II, which can be refrigerated at 4℃.

[0081] Experimental Example 1 I. Determination of α-amylase inhibition rate and pancreatic lipase inhibition rate after fermentation of coconut milk with compound microbial agents.

[0082] 1. Determination of amylase inhibition.

[0083] Take coconut milk from different fermentation times in Example 2, dilute it 50 times, add 250 μL to a 10 mL centrifuge tube, then add 250 μL of 1 U / mL α-amylase, react at 25℃ for 10 min, then add 250 μL of 1% (w / w) soluble starch solution, mix well, react at 25℃ for 10 min, then add 500 μL of DNS reagent, heat in a boiling water bath for 5 min. After the solution cools, dilute with 5 mL of PBS buffer, measure the absorbance at 540 nm, and record it as C0. Use PBS buffer instead of enzyme as a control, record it as C1, and use PBS buffer instead of bacterial sample and enzyme solution as a blank, record it as C2. The formula for calculating the amylase inhibition rate is as follows: Amylase inhibition rate (%) = [1-(C0-C1) / C2]×100%.

[0084] The results are as follows Figure 9 As shown, with the extension of fermentation time, the inhibitory effect of fermented coconut milk on α-amylase activity showed a trend of first increasing and then decreasing, reaching the highest value at 12h, with the inhibitory effect on α-amylase activity increasing from 21.15% to 39.41%.

[0085] 2. Determination of pancreatic lipase inhibition.

[0086] Coconut milk from different fermentation times in Example 2 was diluted 50 times. The inhibitory activity against pancreatic lipase was determined using a colorimetric method. 60 μL of the diluted sample was placed in a 1.5 mL centrifuge tube. 150 μL of Tris-HCl (0.05 M, pH 8.0) and 60 μL of pancreatic lipase (40 U / mL) were added sequentially to the centrifuge tube. The tube was then incubated at 37°C for 10 min. Next, 300 μL of PNPB solution (18% v / v) was added, and the tube was incubated at 37°C for 15 min. After the reaction was complete, the tube was centrifuged at 5000 rpm for 5 min. The supernatant was measured at 405 nm, and the absorbance (OD) was recorded as D1. Tris-HCl buffer was used as a control instead of the enzyme solution, recorded as D2. Tris-HCl buffer was used as a blank instead of the sample solution, recorded as D3. Tris-HCl buffer was used as a control blank instead of both the sample solution and the enzyme solution, recorded as D4. The determination was performed in triplicate. The formula for calculating the pancreatic lipase inhibition rate is as follows: Pancreatic lipase inhibition rate (%) = [1-(D1-D2) / (D3-D4)]×100%.

[0087] The results are as follows Figure 10 As shown, with the extension of fermentation time, the inhibitory effect of fermented coconut milk on pancreatic lipase activity showed a trend of first increasing and then decreasing, reaching the highest value at 12h, with the inhibitory effect on pancreatic lipase activity increasing from 22.14% at 6h to 42.10%.

[0088] II. Sensory evaluation.

[0089] Table 3 Sensory Rating Table

[0090] Sensory evaluations were conducted on the fermented coconut milk beverages of Example 2 (fermented for 8h, 10h, and 12h) and the old coconut milk of Comparative Example 1. The products were placed in 50mL transparent glass cups and evaluated by 13 trained sensory evaluators. The products were evaluated and scored (integer values) in terms of color, aroma, texture, and taste according to the evaluation criteria in Table 3. The average value of each item was taken.

[0091] Table 4 Sensory Evaluation Results

[0092] The results are shown in Table 4. The sensory evaluation scores of the fermented coconut milk beverages fermented for 10h and 12h in Example 2 of the present invention are relatively high.

[0093] III. Inhibition of α-amylase and pancreatic lipase.

[0094] Using α-amylase inhibition and pancreatic lipase inhibition as evaluation indicators, the fermented coconut milk beverage fermented for 6-14 hours in Example 2 was compared with the old coconut milk prepared in Comparative Example 1.

[0095] Table 5. Results of protein content, reducing sugar content, fat content, and bacterial count.

[0096] Fermented coconut milk beverage prepared in Example 2 and fermented for 12 hours were compared with old coconut milk prepared in Comparative Example 1. Their protein content, amino acid content, total sugar content, fat content, and viable bacteria count were measured. Specific parameters are shown in Table 5. The protein content of the fermented coconut milk beverage prepared in Example 2 was 36.38% higher than that of Comparative Example 1; while the total sugar content and fat content were 51.82% and 5.01% lower than those of Comparative Example 1, respectively. This indicates that the fermentation process using the compound microbial agent of this invention can utilize the sugars, amino acids, and fats in coconut milk to synthesize proteins.

[0097] like Figure 9 and Figure 10 As shown, the fermented coconut milk beverage of Example 2, fermented for 12 hours, exhibited superior α-amylase and pancreatic lipase inhibition compared to other groups. Comparative Example 2, also fermented for 12 hours, showed higher α-amylase and pancreatic lipase inhibition rates, but its performance was slightly inferior to that of Comparative Example 2.

[0098] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A compound bacterial agent for lowering blood sugar and blood lipids, characterized in that, The compound microbial agent includes *Lactobacillus fermentum* (… Limosilactobacillus fermentum AL-8 and Lactobacillus paracasei ( Lacticaseibacillus paracasei DX-5; The fermenting *Lactobacillus mucinus* AL-8 was classified and named *Lactobacillus mucinus* AL-8. Limosilactobacillus fermentum AL-8 was deposited at the China Center for Type Culture Collection on December 1, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M20252735. The *Lactobacillus paracasei* DX-5 is classified and named *Lactobacillus paracasei* DX-5. Lacticaseibacillus paracasei DX-5 was deposited on December 1, 2025 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO: M20252736.

2. The compound microbial agent according to claim 1, characterized in that, The volume ratio of *Lactobacillus mucinus* AL-8 and *Lactobacillus paracasei* DX-5 in the compound microbial agent is 1:

1.

3. The compound microbial agent according to claim 1, characterized in that, The effective viable count of *Lactobacillus fermentum* AL-8 in the compound bacterial agent is 10. 8 CFU / mL, the effective viable count of *Lactobacillus paracasei* DX-5 in the compound bacterial agent is 10. 8 CFU / mL.

4. The use of the compound microbial agent as described in any one of claims 1 to 3 in the preparation of fermented coconut milk beverages.

5. A method for preparing fermented coconut milk beverage using the compound microbial agent as described in any one of claims 1 to 3, characterized in that, Includes the following steps: 1) Crack the coconut shell, extract the coconut meat and coconut water, homogenize them, filter with gauze, and sterilize to obtain coconut milk; 2) Inoculate the compound microbial agent into coconut milk and ferment to obtain fermented coconut milk beverage.

6. The method for preparing fermented coconut milk beverage according to claim 5, characterized in that, The coconut mentioned in step 1) is the black-shelled coconut king.

7. The method for preparing fermented coconut milk beverage according to claim 5, characterized in that, The ratio of coconut meat to coconut water mentioned in step 1) is 1g: 1~3mL.

8. The method for preparing fermented coconut milk beverage according to claim 5, characterized in that, The sterilization described in step 1) is pasteurization, with a pasteurization temperature of 65°C and a pasteurization time of 30 minutes.

9. The method for preparing fermented coconut milk beverage according to claim 5, characterized in that, The inoculation amount of the compound bacterial agent mentioned in step 2) is 2~4% v / v.

10. The method for preparing fermented coconut milk beverage according to claim 5, characterized in that, The fermentation temperature in step 2) is 37°C, and the fermentation time is 2~12h.

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