A strain of Lactobacillus fermentum HN-8 and its application in preparing fermented coconut milk beverage for lowering blood sugar and blood lipids

Abstract

The present invention relates to the field of microbial technology, and in particular to a strain of Lactobacillus fermentum HN-8 and its application in preparing a fermented coconut milk beverage for reducing blood sugar and blood lipids. The fermented lactobacillus is classified and named as Lactobacillus fermentum HN-8 Limosilactobacillus leaven HN‑8, strain number HN‑8, was deposited in the China Center for Type Culture Collection on September 5, 2024, with a deposit number of CCTCC NO: M 20241917. The fermented lactobacillus HN‑8 has good surface hydrophobicity, self-aggregation ability, acid resistance, bile salt resistance, and has an inhibitory effect on amylase and pancreatic lipase. The fermented coconut milk beverage prepared by the bacteria has a variety of biological activities, which can help lower blood sugar and blood lipids in the body, and has good compatibility and adaptability with lactic acid bacteria, can promote the proliferation of lactic acid bacteria, inhibit the growth of harmful bacteria, and generate volatile compounds at the same time to make the coconut milk flavor mellow, delicate taste, reduce sugar content and increase protein content, and has certain market application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of fermented lactobacillus HN-8 and application thereof in preparing a fermented coconut milk beverage capable of lowering blood sugar and blood lipids. Background Art

[0002] Diabetes is a common metabolic disease characterized by chronic hyperglycemia, and its pathogenesis is related to the patient's insulin resistance. Insulin helps the patient's body synthesize a large amount of triglycerides and very low-density lipoproteins, which accumulate in the liver, causing lipid metabolism disorders and hyperlipidemia; hyperlipidemia has become a potential factor for coronary heart disease, myocardial infarction, atherosclerosis and other diseases. Diabetes and hyperlipidemia are common chronic diseases in modern society. Long-term chemotherapy will affect the patient's liver and kidney function and cause problems such as drug resistance.

[0003] Coconut is a monocotyledonous palm plant with extremely high economic value. It is widely planted in many tropical and subtropical regions. The milky white coconut meat is separated from the shell after processing and made into coconut flakes, coconut flakes, coconut oil and other products according to production needs. Coconut milk is made from fresh and mature coconuts through peeling, shelling, pressing, filtering, homogenization and other procedures. Coconut milk is called santan by people in Malaysia and Indonesia, which shows that its nutritional value is extremely high. Coconut milk is rich in fat and protein, of which 46% of the fat is lauric acid, which is the main source of medium-chain triglycerides and has physiological activities such as lowering cholesterol, inhibiting bacteria and enhancing immunity. Coconut protein contains 18 kinds of amino acids required by the human body, 8 kinds of essential amino acids are complete, and it has certain therapeutic effects in lowering blood lipids, lowering cholesterol and inhibiting hyperlipidemia.

[0004] Lactobacillus fermentum is a Gram-positive bacterium of the genus Lactobacillus. It is a probiotic that is widely found in fermented foods, the intestines of animals and plants, soil and other environments in nature. Lactobacillus fermentum is believed to maintain the balance of intestinal microecology, promote digestion and absorption, and improve immunity. It can ferment lactose and produce lactic acid in dairy fermentation, and has a wide range of applications in the fields of medicine and food health care.

[0005] Therefore, screening a new strain of fermented lactobacillus to ferment coconut milk to increase the economic value of coconut and develop a green hypoglycemic lipid food has important practical significance. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a strain of fermented lactobacillus HN-8 in view of the deficiencies of the prior art.

[0007] The technical problem that the present invention also solves is to provide the application of the fermented lactobacillus in preparing a fermented coconut milk beverage for lowering blood sugar and blood lipids.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0009] A strain of Lactobacillus fermentum, classified and named Lactobacillus fermentum HN-8 Limosilactobacillus fermentum HN-8, strain number is HN-8, was deposited in the China Center for Type Culture Collection (abbreviated as: CCTCC) on September 5, 2024, the preservation number is CCTCC NO: M 20241917, and the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0010] Wherein, the nucleotide sequence of 16S rDNA of the fermented Lactobacillus is shown as SEQ ID No.1.

[0011] SEQ ID No.1:

[0012]

[0013] Wherein, the morphological and physiological identification results of described fermented lactobacillus are as follows:

[0014] Lactobacillus fermentum HN-8 is a type of Gram-positive bacteria, and its cell morphology is rod-shaped under a microscope. It was cultured on MRS solid medium, and the results were as follows Figure 1 As shown in the figure, it can be observed that the colonies of Lactobacillus fermentum HN-8 are medium-sized, raised, moist, have neat edges, and are white and round.

[0015] Furthermore, by measuring the surface hydrophobicity, self-aggregation ability, acid resistance and bile salt resistance of Lactobacillus fermentation HN-8 strain, it was found that the hydrophobicity of Lactobacillus fermentation HN-8 strain was 1.78±0.18%; it had a strong self-aggregation ability; within the pH range of 3.0~5.0, the strain had a certain acid resistance and could maintain good activity during the fermentation process; within the bile salt concentration range of 0~0.5%, Lactobacillus fermentation HN-8 had good bile salt tolerance.

[0016] Furthermore, by measuring the tolerance of Lactobacillus fermentum HN-8 in artificial gastric juice and intestinal juice, it was found that Lactobacillus fermentum HN-8 has a high tolerance to artificial intestinal juice and can have a certain resistance in the gastrointestinal tract, thereby exerting its probiotic function.

[0017] Furthermore, by measuring the amylase inhibition rate and pancreatic lipase inhibition rate of Lactobacillus fermentum HN-8, it was found that the concentration of the bacterial solution of Lactobacillus fermentum HN-8 had a positive correlation with the inhibitory effect on the amylase activity and the pancreatic lipase activity.

[0018] A food leavening agent comprising the fermented lactobacillus is also within the scope of protection of the present invention.

[0019] The application of the fermented lactobacillus in the preparation of fermented coconut milk beverage is also within the scope of protection of the present invention.

[0020] A method for preparing a fermented coconut milk beverage by using the fermented lactobacillus comprises inoculating activated fermented lactobacillus into sterilized coconut milk, allowing the milk to ferment statically, and then post-ripening the milk at low temperature to obtain the fermented coconut milk beverage.

[0021] The activation is specifically carried out by inoculating the fermented lactobacillus into MRS liquid culture medium, culturing at 37°C for 24 hours, applying the fermentation liquid to MRS solid culture medium, culturing at 37°C for 48 hours, picking a single colony, inoculating it again into MRS liquid culture medium, and activating it at 37°C for 18 hours.

[0022] Specifically, the MRS solid culture medium formula is as follows: 20.0 g glucose, 10.0 g peptone, 10.0 g beef extract, 5.0 g yeast extract, 2.0 g triammonium citrate, 2.62 g dipotassium hydrogen phosphate, 5.0 g anhydrous sodium acetate, 0.58 g magnesium sulfate, 0.198 g ammonium sulfate, 1.0 mL Tween 80, 15.0 g agar, add 1000 mL distilled water, adjust the pH to 6.0 after fully dissolving, and sterilize at 121° C. for 20 min after aliquoting.

[0023] Wherein, the inoculation amount is 2-5% v / v; when the fermented lactobacillus is inoculated into the sterilized coconut milk, the concentration of the fermented lactobacillus is 10 6 ~10 9 CFU / mL.

[0024] Preferably, the inoculation amount is 3% v / v; when the fermented lactobacillus is inoculated into the sterilized coconut milk, the concentration of the fermented lactobacillus is 10 8 CFU / mL.

[0025] The coconut milk is obtained by mixing coconut meat and sterile water, filtering and sterilizing; the solid-liquid ratio of the coconut meat to the sterile water is 1:2-4, preferably 1:2.

[0026] Specifically, the coconut milk is old coconut milk.

[0027] Specifically, the sterilization is performed by pasteurization, and the pasteurization conditions are: sterilization temperature 65° C., and sterilization time 30 min.

[0028] In some embodiments of the present invention, the coconut meat is white solid endosperm obtained by shelling old high-quality coconuts from Wenchang, Hainan, which are free of mildew, disease and insect pests.

[0029] The static fermentation is carried out under the following conditions: 2 to 12 h at 37°C; and the low-temperature ripening is carried out under the following conditions: 8 to 12 h at 4°C.

[0030] Preferably, the static fermentation is carried out under the following conditions: static fermentation for 12 h at 37°C; and the low-temperature ripening is carried out under the following conditions: ripening for 12 h at 4°C.

[0031] The fermented coconut milk beverage prepared by the method for preparing a fermented coconut milk beverage using the fermented lactobacillus is also within the scope of protection of the present invention.

[0032] Specifically, the fermented coconut milk beverage helps to lower blood sugar and blood lipids in the body and maintain healthy blood sugar and blood lipid levels.

[0033] Beneficial effects:

[0034] (1) After separation and purification, the present invention screened a new strain of Lactobacillus fermentum Limosilactobacillus fermentum HN-8. This strain has good surface hydrophobicity, self-aggregation ability, acid resistance, and bile salt resistance; it also has high tolerance to artificial intestinal fluid and can have a certain resistance in the gastrointestinal tract, thereby exerting its probiotic function; it also has an inhibitory effect on amylase and pancreatic lipase.

[0035] (2) The fermented coconut milk beverage prepared by the fermented lactobacillus obtained by the present invention has multiple biological activities, good compatibility and adaptability with lactic acid bacteria, and can promote the proliferation of lactic acid bacteria. Coconut milk is fermented by microorganisms to produce new organic acids to inhibit the growth of harmful bacteria, and at the same time, volatile compounds are generated to make coconut milk mellow and delicious, with a delicate taste, reduced sugar content and significantly increased protein content.

[0036] (3) The present invention proves through the study of hypoglycemic and hypolipidemic effects that fermented lactobacillus can work synergistically with coconut milk. The prepared fermented coconut milk beverage has significant advantages in inhibiting the activity of amylase and pancreatic lipase, providing a new solution for the development of foods that help lower blood sugar and blood lipids in the body and maintain healthy blood sugar and blood lipid levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The present invention will be further described in detail below with reference to the accompanying drawings, and the above and / or other advantages of the present invention will become more clear.

[0038] Figure 1 This is the colony morphology of Lactobacillus fermentum HN-8.

[0039] Figure 2 The self-aggregation of Lactobacillus fermentatus HN-8 changes over time. Note: Different lowercase letters indicate significant differences in the number of live bacteria between different states ( P <0.05).

[0040] Figure 3 is the acid resistance of fermented Lactobacillus HN-8. Note: Different lowercase letters indicate significant differences in the number of live bacteria between strains in different states ( P <0.05).

[0041] Figure 4 is the bile salt tolerance of Lactobacillus fermentum HN-8. Note: Different lowercase letters indicate significant differences in the number of live bacteria between strains in different states ( P <0.05).

[0042] Figure 5The inhibitory effect of fermented Lactobacillus HN-8 on amylase and pancreatic lipase. Note: Different uppercase / lowercase letters indicate significant differences in the number of live bacteria between different states. P <0.05).

[0043] Figure 6 The pH changes of coconut milk fermented by Lactobacillus HN-8 over time.

[0044] Figure 7 Titrate the change of acidity over time for coconut milk fermented by Lactobacillus HN-8.

[0045] Figure 8 The change of the inhibition rate of coconut milk amylase by fermented Lactobacillus HN-8 over time. Note: Different lowercase letters indicate significant differences in the number of lyophilized live bacteria between strains in different states ( P <0.05).

[0046] Fig. 9 The change of pancreatic lipase inhibition rate of coconut milk fermented by Lactobacillus fermentum HN-8 over time. Note: Different lowercase letters indicate significant differences in the number of lyophilized live bacteria between strains in different states ( P <0.05).

[0047] A strain of Lactobacillus fermentum, classified and named Lactobacillus fermentum HN-8 Limosilactobacillus fermentum HN-8, strain number is HN-8, was deposited in the China Center for Type Culture Collection (abbreviated as: CCTCC) on September 5, 2024, the preservation number is CCTCC NO: M 20241917, and the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. DETAILED DESCRIPTION

[0048] The present invention will be further described in detail below in conjunction with specific implementations, and the above and / or other advantages of the present invention will become more clear.

[0049] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0050] Example 1: Cultivation of Lactobacillus fermentum HN-8

[0051] 1. Preparation of MRS medium

[0052] The specific components of MRS medium are as follows: glucose 20.0 g / L, peptone 10.0 g / L, beef extract 10.0 g / L, yeast extract powder 5.0 g / L, triammonium citrate 2.0 g / L, dipotassium hydrogen phosphate 2.62 g / L, anhydrous sodium acetate 5.0 g / L, magnesium sulfate 0.58 g / L, ammonium sulfate 0.198 g / L, Tween 80 1 mL / L, agar powder 15.0 g / L.

[0053] Preparation method: Dissolve the above ingredients in 1 L of distilled water. After complete dissolution, adjust the pH to 6.0, then divide into portions and sterilize at 121°C for 20 min.

[0054] 2. Isolation and purification of strain HN-8

[0055] The fermented beverage Boza obtained from Yining City, Xinjiang in June 2008 was inoculated into MRS liquid culture medium and cultured at 37°C for 24 h. Then 0.1 L of the fermentation liquid was evenly spread on an MRS solid plate using an L-shaped coating rod and cultured at 37°C for 48 h. A single colony was picked and isolated, purified and preserved by the streak method.

[0056] (1) Slant preservation: Use a disposable inoculation loop to dip a small amount of purified strain and inoculate it in a zigzag streak method on the slant MRS solid culture medium. After streaking, burn the mouth of the test tube and place it in a 37°C environment for static culture. After the strain grows to maturity, store it in a 4°C refrigerator.

[0057] (2) Glycerol tube freezing: Mix the purified bacterial solution with 80% glycerol in a 1:1 ratio and store it evenly in a cryogenic tube. Then freeze it in a -80℃ refrigerator.

[0058] 3. Identification of Lactobacillus fermentum HN-8

[0059] (1) Morphological and physiological identification

[0060] Lactobacillus fermentum HN-8 is a type of Gram-positive bacteria, and its cell morphology is rod-shaped under a microscope. It was cultured on MRS solid medium, and the results were as follows Figure 1 As shown in the figure, it can be observed that the colonies of Lactobacillus fermentum HN-8 are medium-sized, raised, moist, have neat edges, and are white and round.

[0061] (2) Molecular biological identification

[0062] The genomic DNA of strain HN-8 was extracted using a genome extraction kit, and the 16S rDNA gene fragment of the strain was amplified by PCR using universal primers 27F (SEQ ID No.2: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID No.3: 5'-TACGGTTACCTTGTTACGACTT-3') (the 16S rDNA sequence is shown in SEQ ID No.1). The PCR product was sent to Qingke Biotech for sequencing, and the sequencing results were compared in the Genbank database of NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and it was found that strain HN-8 was similar to Lactobacillus fermentum. Limosilactobacillus fermentum The homology of is the highest, which is 99.93%. Therefore, it is identified that the present invention has obtained a strain of fermented lactobacillus through separation and purification. Limosilactobacillus fermentum HN-8 。

[0063] (3) Strain preservation

[0064] The fermented Lactobacillus HN-8 was sent to the China Center for Type Culture Collection (CCTCC) for preservation on September 5, 2024, and was classified and named as Lactobacillus fermented HN-8 Limosilactobacillus fermentum HN-8, the strain number is HN-8, the preservation number is CCTCC NO: M 20241917, and the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0065] Example 2: Determination of surface hydrophobicity, self-aggregation ability, acid resistance and bile resistance of Lactobacillus fermentum HN-8 strain

[0066] The frozen Lactobacillus fermentum HN-8 was inoculated into MRS medium and cultured at 37 ℃ for 24 h. Then, it was activated for the second time with an inoculum size of 3% v / v. After culturing for 18 h, the Lactobacillus fermentum HN-8 cells were collected by centrifugation and diluted to an appropriate dilution by adding PBS buffer. The surface hydrophobicity, self-aggregation ability, acid resistance and bile resistance of the strain were determined.

[0067] 1. The process of surface hydrophobicity determination:

[0068] Take the fermented Lactobacillus HN-8 bacterial suspension in a 50 mL centrifuge tube, centrifuge at 5000 rpm and 4 °C for 5 min, discard the supernatant, mix it with PBS buffer, repeat the washing three times, and then resuspend the bacterial slurry with PBS buffer to adjust the OD 600 To 0.6 ± 0.02, record as A0. Add 1 mL of xylene to a 10 mL centrifuge tube, then add 3 mL of OD 600The bacterial suspension was vortexed and placed at room temperature for 20 min. After 20 min, the aqueous phase was aspirated and the OD was measured. 600 The absorbance value at is recorded as A1.

[0069] The calculation formula of strain surface hydrophobicity is as follows: strain hydrophobicity (%) = (1-A1 / A0) × 100.

[0070] The surface hydrophobicity of a strain refers to the ability of the strain to leave the aqueous phase and move toward other organic phases, which is related to the nonspecific adhesion ability of the strain. After measurement, it was found that the hydrophobicity of Lactobacillus fermentum HN-8 was 1.78±0.18%, which is far less than 10%), indicating that the surface hydrophobicity is very weak.

[0071] 2. The process of self-aggregation ability determination:

[0072] Take the fermented Lactobacillus HN-8 bacterial suspension in a centrifuge tube, centrifuge at 5000 rpm, 4 ℃, and then resuspend the bacterial slurry with PBS buffer to adjust the OD 600 The suspension was kept at room temperature, and then the upper bacterial suspension was taken at 2 h, 4 h and 24 h to measure the OD 600 The absorbance at is recorded as B1.

[0073] The calculation formula of the strain self-aggregation ability is as follows: self-aggregation (%) = (1-B1 / B0) × 100.

[0074] Strain self-aggregation refers to the ability of bacteria to combine together to form cell aggregates, which is related to the formation of biofilms and helps the strains colonize in intestinal epithelial cells. Figure 2 It can be seen that with the increase of time, the self-aggregation ability of the strain showed a significant change, and the longer the placement time, the more significant the improvement of the self-aggregation ability of the strain. After 24 hours, the self-aggregation ability reached 51.42%, indicating that fermented Lactobacillus HN-8 has the potential to adhere to intestinal cells and colonize the intestine.

[0075] 3. The process of acid resistance determination:

[0076] Take 1 mL of the culture medium of Lactobacillus fermentum HN-8 strain with good growth, collect the bacteria by centrifugation, resuspend them with the corresponding MRS liquid culture medium with pH of 3.0, 4.0 and 5.0, and culture the bacterial suspension at 37°C. Samples were taken at 0 h and 3 h, and the bacterial suspension was gradiently diluted with physiological saline, and spread on MRS solid agar plates for colony counting.

[0077] The formula for calculating the strain survival rate is as follows: strain survival rate (%) = Nt / N0×100, where Nt is the number of colonies at 3 h or 6 h, and N0 is the number of colonies at 0 h.

[0078] The tolerance of Lactobacillus fermentum to acid Figure 3 As shown in the figure, under the condition of pH 4.0-5.0, the survival rate of the strain did not change significantly. When the pH was 3.0, the survival rate of the strain dropped significantly to 427.04%, but the survival rate was much greater than 100%, which showed that within the pH range of 3.0-5.0, the strain had a certain acid resistance and could maintain good activity during the fermentation process.

[0079] 4. The process of bile salt tolerance determination:

[0080] Take 1 mL of the culture medium of Lactobacillus fermentum HN-8 strain with good growth, collect the bacteria by centrifugation, and resuspend them in the corresponding MRS liquid culture medium with bile salt content of 0.0%, 0.3%, 0.4% and 0.5% (w / v), respectively. The bacterial suspensions were cultured at 37 ℃, and samples were taken at 0 h and 3 h. The samples were gradient diluted with PBS buffer and spread on MRS solid agar plates for colony counting.

[0081] The formula for calculating the strain survival rate is as follows: strain survival rate (%) = Nt / N0×100, where Nt is the number of colonies at 3 h and N0 is the number of colonies at 0 h.

[0082] Bile salts in human intestinal fluid can affect cell permeability and even cause bacterial death. Therefore, bacterial strains need to have a certain degree of bile salt tolerance in order to colonize in the body and exert their probiotic functions. Figure 4 It can be seen that with the increase of bile salt concentration, the survival rate of the strain continued to decrease. When the bile salt concentration was 0.3%, the survival rate of the strain decreased significantly from the initial 377.97% to 143.77%. When the bile salt concentration was further reduced, the survival rate also decreased significantly but was still greater than 100%. This indicates that in the range of bile salt concentration of 0~0.5%, Lactobacillus fermentum HN-8 has good bile salt tolerance.

[0083] Example 3: Determination of tolerance of Lactobacillus fermentum HN-8 in artificial gastric juice and intestinal juice

[0084] In order to simulate the digestive effect of artificial gastric juice and intestinal juice on Lactobacillus fermentum HN-8, this example measured the survival rate of the strain in artificial gastric juice and intestinal juice. The specific process is as follows:

[0085] Gastric buffer: NaCl 2.758 g, KCl 0.5144 g, KH2PO4 0.225 g, NaHCO3 2.1 g, MgCl2 (H2O) 60.203 g, (NH4)2CO3 0.0786 g, 1 L deionized water. Before the test, weigh 15 mg of gastric mucin and 6.25 mg of pepsin and dissolve them in 10 mL of gastric buffer respectively, mix them and combine them, adjust the pH of the buffer to 1.6, and place them in a 37°C water bath for 20 min to stimulate enzyme activity, and use them as simulated gastric fluid for later use.

[0086] Preparation of intestinal buffer: KCl 0.507 g, KH2PO4 0.1089 g, NaHCO3 7.1409 g, NaCl 2.2441 g, MgCl2 (H2O) 60.0671 g, 1 L deionized water. Before the experiment, 8.17 mg bile salt and 5.62 mg pancreatin were dissolved in 10 mL intestinal buffer solution, mixed and combined, the pH of the buffer was adjusted to 7.0, and the solution was incubated at 37°C for 20 min to stimulate enzyme activity, and used as simulated intestinal fluid.

[0087] The culture fluid of Lactobacillus fermentum HN-8 strain with good growth condition was taken, centrifuged, the bacteria were collected, resuspended with simulated gastric fluid and simulated intestinal fluid, and cultured on a shaker at 37℃ and 150 rpm. The simulated gastric fluid was sampled at 0 h and 3 h, and the simulated intestinal fluid was sampled at 0 h, 3 h, and 6 h. The sampled fluids were gradiently diluted with PBS buffer and then spread on MRS solid agar plates for colony counting.

[0088] The survival rate is calculated as follows: Survival rate (%) = (N t / N0)×100; where Nt is the number of colonies at 3 h or 6 h, and N0 is the number of colonies at 0 h.

[0089] Table 1 Tolerance of Lactobacillus fermentum HN-8 to artificial gastrointestinal fluid

[0090]

[0091] The results are shown in Table 1: Food stays in the stomach for about 3 hours for digestion. The survival rate of Lactobacillus fermentum HN-8 in simulated gastric juice for 3 hours is 68.72%, while the survival rate of Lactobacillus fermentum HN-8 after 3 hours and 6 hours of culture in simulated intestinal juice is 164.57% and 135.64%. Compared with the survival rate in gastric juice, the survival rate of the strain in intestinal juice is significantly increased, both greater than 100%. This shows that Lactobacillus fermentum HN-8 has a high tolerance to artificial intestinal juice and can have a certain resistance in the gastrointestinal tract, thereby exerting its probiotic function.

[0092] Example 4: Determination of amylase inhibition rate and pancreatic lipase inhibition rate of Lactobacillus fermentum HN-8

[0093] 1. Determination of amylase inhibition

[0094] Take the culture medium of Lactobacillus fermentum HN-8 strain with good growth status, collect the cells by centrifugation, and adjust the cell concentration to 1×10 6 ~1×10 9 CFU / mL. Add 250 μL of bacterial sample and 250 μL of 1 U / mL α-amylase to a 10 mL ep tube, react at 25 °C for 10 min, then add 250 μL of 1% soluble starch solution, mix well and react at 25 °C for 10 min, then add 500 μL of DNS reagent and heat in a boiling water bath for 5 min. After the solution cools, add 5 mL of PBS buffer to dilute, measure the absorbance at 540 nm, record it as C0, use PBS buffer instead of enzyme solution 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 calculation formula for amylase inhibition rate is as follows:

[0095] Amylase inhibition rate (%) = )×100.

[0096] 2. Determination of pancreatic lipase inhibition

[0097] Take the culture medium of Lactobacillus fermentum HN-8 strain with good growth status, collect the cells by centrifugation, and adjust the cell concentration to 1×10 6 ~1×10 9 CFU / mL, the inhibitory activity of bacterial samples on pancreatic lipase was determined by colorimetry. 60 μL of bacterial sample solution was taken into a 2 mL centrifuge tube, and 150 μL Tris-Hcl (0.05M, pH 8.0) and 60 μL pancreatic lipase (40 U / mL) were added to the centrifuge tube in sequence, and reacted in a tabletop constant temperature blast drying oven at 37°C for 10 min, and then 300 μL PNPB solution (18% v / v) was added and placed in a drying oven for 15 min. After the reaction was completed, centrifuged at 5000 rpm for 5 min, and the supernatant was taken to measure the sample absorbance (OD) at 405 nm, recorded as D1, Tris-Hcl buffer was used instead of enzyme solution as the control, recorded as D2, Tris-Hcl buffer was used instead of sample solution as the sample blank, recorded as D3, and Tris-Hcl buffer was used instead of sample solution and enzyme solution as the control blank, recorded as D4, and the determination was repeated 3 times. The calculation formula for the inhibition rate of pancreatic lipase is as follows:

[0098] Pancreatic lipase inhibition rate (%) = ( .

[0099] Figure 5 The results showed that the concentration of Lactobacillus fermentum HN-8 had a positive correlation with the inhibitory effect on amylase activity and pancreatic lipase activity. The inhibitory effect of Lactobacillus fermentum HN-8 on amylase gradually increased with the increase of the concentration of the culture solution. 6 ~10 9 Within the CFU / mL range, the inhibitory effect of Lactobacillus fermentum HN-8 on amylase activity increased from 8.84% to 10.90%, and the inhibitory effect of the strain on pancreatic lipase activity also showed similar results, increasing from 6.84% to 13.42%. This may be due to the binding of the bacteria to the active site of pancreatic lipase, thereby inhibiting the activity of pancreatic lipase.

[0100] Example 5: Preparation of fermented coconut milk beverage with hypoglycemic and lipid-lowering effects

[0101] S1. Strain activation: fermented Lactobacillus HN-8 was inoculated into MRS liquid medium and cultured at 37°C for 24 h. The fermented liquid was then spread onto MRS solid medium and cultured at 37°C for 48 h. A single colony was picked and inoculated into MRS liquid medium again for activation. The activation conditions were: cultured at 37°C for 18 h.

[0102] S2. Raw material pretreatment: Select old coconuts grown in Wenchang, Hainan without mildew, disease or insect pests, break the shells to obtain the solid endosperm (coconut meat), homogenize the chopped coconut meat with sterile water in a solid-liquid ratio of 1:2, and filter through four layers of gauze to remove impurities to obtain old coconut milk.

[0103] S3, sterilization: the old coconut milk filtered and impurities removed in S2 is packaged into sterilized glass bottles for pasteurization at a temperature of 65°C for 30 min. After sterilization, it is cooled to room temperature.

[0104] S4, strain fermentation: take the activated fermented Lactobacillus HN-8 and 10 8 The bacterial solution with a concentration of CFU / mL was inoculated into the sterilized old coconut milk at an inoculum size of 3% v / v, mixed evenly, and fermented at 37 °C for 2-12 h.

[0105] S5, post-ripening: the old coconut milk after fermentation in S4 is placed in a 4°C refrigerator for post-ripening for 12 h to obtain a fermented coconut milk beverage.

[0106] The pH value and titratable acidity after fermentation were measured. Figure 6-7 As shown. Figure 6 It can be seen that the pH of fermented coconut milk gradually decreased with the increase of fermentation time, began to stabilize at 6 h, and was 3.94 after 12 h. Figure 7It can be seen that the titratable acidity of coconut milk increases with the increase of fermentation time, and the change is obvious after 8 h. After 12 h, the titratable acidity reaches 52 。 T.

[0107] Comparative Example 1: Preparation of coconut milk beverage with hypoglycemic and lipid-lowering effects

[0108] S1. Raw material pretreatment: Select old coconuts grown in Wenchang, Hainan without mildew, disease or insect pests, break the shells to obtain the solid endosperm (coconut meat), homogenize the chopped coconut meat with sterile water in a solid-liquid ratio of 1:2, and filter through four layers of gauze to remove impurities to obtain old coconut milk.

[0109] S2. Sterilization: pasteurize the old coconut milk filtered and impurity-removed in S1 at a temperature of 65°C for 30 min, and then cool to room temperature.

[0110] S3. Place the sterilized old coconut milk in S2 in a 4°C refrigerator for 12 h to obtain a coconut milk beverage.

[0111] Implementation effect:

[0112] 1. Sensory evaluation

[0113] The fermented coconut milk beverages fermented for 8 h, 10 h, and 12 h in Example 5 and the coconut milk beverage in Comparative Example 1 were subjected to sensory evaluation. The prepared coconut milk beverages were placed in 50 mL transparent plastic cups and evaluated by 10 trained sensory tasters. The products were evaluated and scored (whole values) in terms of color, smell, texture state, and taste according to the evaluation criteria in Table 2, and the average value was taken for each item. The results showed that the sensory evaluation results of the fermented coconut milk beverages fermented for 10 h and 12 h had higher scores.

[0114] Table 2 Sensory scoring table

[0115]

[0116] Table 3 Sensory evaluation results of different groups

[0117]

[0118] 2. In vitro hypoglycemic and hypolipidemic effects

[0119] Taking the amylase inhibition and pancreatic lipase inhibition as evaluation indicators, the in vitro hypoglycemic and hypolipidemic effects of the fermented coconut milk beverages prepared in Example 5 for 8 h, 10 h, and 12 h were compared with those prepared in Comparative Example 1 (see Figure 8 , Fig. 9). The results showed that the fermented coconut milk beverage fermented for 12 hours had better amylase inhibition and pancreatic lipase inhibition than other groups, which indicated that the fermented coconut milk beverage fermented for 12 hours had higher in vitro hypoglycemic and hypolipidemic activity. The fermented coconut milk beverage fermented for 12 hours was selected and its protein content, amino acid content, total sugar content, reducing sugar content and total phenol content were determined. The specific parameters are shown in Table 4. As can be seen from Table 4, the protein content in the fermented coconut milk beverage fermented for 12 hours was increased by 80.48% compared with the protein content in Comparative Example 1; while the amino acid content, total sugar content, reducing sugar content and total phenol content in the fermented coconut milk beverage fermented for 12 hours were reduced by 64.93%, 41.39%, 26.53% and 28.34% respectively compared with the contents of each component in Comparative Example 1. It shows that the old coconut milk can utilize the sugars, phenols and amino acids in the coconut milk and synthesize protein after the fermentation of Lactobacillus HN-8.

[0120] Table 4 Determination results of protein content, amino acid content, total sugar content, reducing sugar content and total phenol content

[0121]

[0122] The present invention provides a strain of fermented lactobacillus HN-8 and a method and method for its application in preparing fermented coconut milk beverage for reducing blood sugar and blood lipids. There are many methods and approaches for realizing the technical scheme. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in the technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention. All components not clearly defined in this embodiment can be realized by existing technology.

Claims

1. A strain of Lactobacillus fermentum HN-8, classified and named Lactobacillus fermentum Limosilactobacillus fermentum , strain number is HN-8, and it was deposited in the China Center for Type Culture Collection on September 5, 2024, with the deposit number CCTCC NO: M20241917.

2. Use of the fermented lactobacillus according to claim 1 in inhibiting amylase and / or pancreatic lipase.

3. Application of the fermented lactobacillus according to claim 1 in preparing fermented coconut milk beverage.

4. A food leavening agent, characterized in that: It contains the fermented lactobacillus according to claim 1.

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