Lactobacillus fermentum with hypoglycemic function and application thereof

Abstract

The invention discloses lactobacillus fermentum with a hypoglycemic function and application of the lactobacillus fermentum. According to the invention, lactobacillus fermentum with high hypoglycemic ability is screened from Yunnan Baoshan freshly brewed yoghurt samples, the strain is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20241504. In the fermentation process of the strain, alpha-glucosidase and alpha-amylase can be inhibited, and the inhibition rates are 43.91% and 59.44% respectively. The bacterial colony morphology of the lactobacillus fermentum E003 is milky white, semitransparent, relatively moist and smooth, the edge is neat, and obvious bulges are formed. When being used for preparing the anti-sugar fermented milk, the fermented milk can also inhibit alpha-glucosidase and alpha-amylase, and meanwhile, the fermented milk also has various characteristic nutritional ingredients; the lactobacillus fermentum E003 fermentation liquor also has relatively strong bacteriostatic ability, antioxidant activity and GABA high-yield ability.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a fermented lactobacillus with blood sugar lowering function and application thereof. Background Art

[0002] High blood sugar can lead to diabetes, a chronic disease that seriously affects the quality of life of humans and can easily lead to a series of complications such as kidney disease, cardiovascular disease, blindness and disability. Patients need to take medication for life to control blood sugar. Although there are many drugs for the treatment of diabetes, they also have side effects.

[0003] Lactic acid bacteria have been recognized as beneficial bacteria for the human body, with multiple functionalities. The research on the hypoglycemic effect of lactic acid bacteria and the development of health products has gradually become a hot topic. Lactic acid bacteria have mild effects, stable properties, obvious and long-lasting effects, and have opened up a new direction for the development of long-acting hypoglycemic products. Therefore, lactic acid bacteria hypoglycemic has great research value and application potential. However, there are few studies on the hypoglycemic effect of lactic acid bacteria dairy products in China, and the material basis and mechanism of its hypoglycemic effect have not yet been clarified. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a fermented lactobacillus and an application thereof. The fermented lactobacillus can inhibit α-glucosidase and α-amylase and has a good blood sugar lowering effect. In addition, the fermentation broth also has antioxidant function, antibacterial function and high γ-aminobutyric acid production function.

[0005] In order to solve the above technical problems, the technical solution of the present invention is as follows:

[0006] The invention provides a fermented lactobacillus with a blood sugar lowering function. The fermented lactobacillus (Limosilactobacillus fermentum) is named as fermented lactobacillus E003, with a preservation number of CCTCC NO: M20241504, a preservation unit: China Center for Type Culture Collection, a preservation date of July 8, 2024, and a preservation address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, on the campus of Wuhan University.

[0007] The present invention also provides a fermentation liquid obtained by fermenting the lactobacillus fermentum.

[0008] The present invention also provides the use of the fermented lactobacillus or the fermented liquid in preparing blood sugar lowering, anti-oxidation or antibacterial drugs.

[0009] The present invention also provides application of the fermented lactobacillus in preparing blood sugar-lowering fermented milk.

[0010] In the application of preparing fermented milk, preferably, the inoculation amount of Lactobacillus fermentum E003 is 1.0×10 8CFU / mL-3.0×10 8 CFU / mL.

[0011] In the application of preparing fermented milk, preferably, the fermented Lactobacillus E003 is used in combination with a commercial yogurt starter; preferably, the usage amount of the commercial yogurt starter is 0.01-0.03% (m / m); the fermentation time is 6h-10h, and the fermentation temperature is 36.5℃-39.5℃.

[0012] The characteristics of the present invention are as follows: the present invention screens out a fermented lactobacillus with high blood sugar lowering ability from a sample of freshly brewed yogurt in Baoshan, Yunnan. The strain can inhibit alpha-glucosidase and alpha-amylase during fermentation, and the inhibition rates are 43.91% and 59.44% respectively. The colony morphology of the fermented lactobacillus E003 is milky white, translucent, relatively moist, smooth, with neat edges and obvious protrusions. It is used to prepare sugar-resistant fermented milk, and the fermented milk can also inhibit alpha-glucosidase and alpha-amylase. At the same time, the fermented milk also has a variety of characteristic nutrients; the fermented lactobacillus E003 fermentation liquid also has strong antibacterial ability, antioxidant activity and high GABA production ability.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] (1) The present invention isolates and screens a lactic acid bacterium with high blood sugar lowering ability from a freshly brewed yogurt sample. The lactic acid bacterium is identified by morphological identification and whole genome sequencing. It is classified as Limosilactobacillusfermentum and is a fermentation lactobacillus. The lactic acid bacterium is named Limosilactobacillusfermentum E003, which can effectively inhibit α-glucosidase and α-amylase.

[0015] (2) The fermented milk with good flavor and texture, high viable count and high inhibition rate of α-glucosidase and α-amylase can be produced by using Lactobacillus fermentum E003.

[0016] (3) The fermentation broth of Lactobacillus fermentum E003 also has strong antibacterial ability, antioxidant activity and high GABA production capacity. DETAILED DESCRIPTION

[0017] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific embodiments. The description in this section is only exemplary and explanatory and should not have any limiting effect on the protection scope of the present invention. The comparative lactic acid bacteria applied in the following embodiments: Lactobacillus plantarum B103 is derived from yak milk and is identified as Lactobacillus plantarum by 16s rDNA gene sequence analysis; Lactobacillus plantarum C039 is derived from American cockroaches and is identified as Lactobacillus plantarum by 16s rDNA gene sequence analysis; Lactobacillus fermentum D051 is derived from milk and is identified as Lactobacillus fermentum by 16s rDNA gene sequence analysis; Lactobacillus fermentum D021 is derived from pickled vegetables and is identified as Lactobacillus fermentum by 16s rDNA gene sequence analysis.

[0018] Example 1 Isolation and purification of bacterial strains

[0019] Take 10g of freshly brewed yogurt sample collected from Baoshan City, Yunnan Province, add it to 100mL of sterile saline and shake it evenly. After the sample is diluted gradiently, 100 microliters are applied to MRS solid medium containing 1% calcium carbonate and cultured at 37°C for 48h. Select the colonies that form calcium-dissolving circles on the solid medium, and repeatedly purify them on the MRS solid medium until the colony morphology of the strain is consistent. Inoculate the purified lactic acid bacteria into MRS slant medium and store it at 4°C. Mix the strain with 20% glycerol and insert it into a cryopreservation tube. Mix evenly and store it at -80°C.

[0020] (1) Morphological characteristics of bacterial species

[0021] After culturing Lactobacillus fermentum E003 in MRS agar medium for 24 hours, the colony morphology was milky white, translucent, relatively moist, smooth, with neat edges and obvious protrusions. Under an optical microscope, the cells of the strain Lactobacillus fermentum E003 were rod-shaped, non-spore-forming, and Gram-positive.

[0022] (2) Molecular genetic identification of bacterial species

[0023] Whole genome sequencing

[0024] The DNA extraction kit of TGUIDE S96 magnetic bead technology was used to extract the DNA of the screening strain. The Qubit method was used to determine its content, the Nanodrop method was used to determine its DNA integrity, and the agarose electrophoresis method was used to determine its DNA integrity. After the sample test was qualified, the library was constructed, and the sequencing test was carried out on the machine after the library quality inspection was qualified. Through PacBio third-generation sequencing technology, the genome size of Lactobacillus fermentum E003 was 2,096,565bp and the GC content was 51.52%.

[0025] Finally, the strain screened was determined to be Lactobacillus fermentum through morphology and whole genome sequencing, and was named Lactobacillus fermentum E003, and preserved in the China Center for Type Culture Collection with the preservation number: CCTCC NO: M 20241504. Example 2 Determination of the inhibition rate of Lactobacillus fermentum E003 on α-glucosidase and α-amylase

[0026] (1) Growth curve of Lactobacillus fermentum E003

[0027] The fermented lactobacillus E003 seed liquid activated for three generations in MRS liquid medium was inoculated into MRS liquid medium at an inoculum size (V / V) of 1%, and 200 μL of the seed liquid was placed in a 96-well plate, and the absorbance at 600 nm was measured, and the absorbance was measured every 2 hours for a total of 24 hours. Each time point was repeated 3 times, and the average value was taken with time as the horizontal coordinate and the absorbance as the vertical coordinate to draw a growth curve.

[0028] As the fermentation time increases, the OD600nm value of Lactobacillus fermentum E003 shows an upward trend, and the growth conforms to the S-shaped curve. 0-2h is in the lag phase of growth, and the growth rate of the strain is relatively slow; 2-14h is in the logarithmic phase of growth, and the growth rate of the strain increases sharply; 14-16h is in the stable phase of growth, and the OD600nm value of the strain is basically stable; 16-24h is in the decay phase of growth, and the OD600 nm value of the bacteria decreases slightly.

[0029] (2) Determination of α-glucosidase inhibition rate of Lactobacillus fermentum E003

[0030] The fermented lactobacillus seed liquid activated for three generations in MRS liquid medium was inoculated into the MRS liquid medium at an inoculum size (V / V) of 1%, and cultured at 37°C for 24h, and centrifuged at 8000r / min for 15min to obtain the lactobacillus fermentation supernatant. In 96-well plates, 50 μL sample solutions were added respectively, and then 100 μL of α-glucosidase (0.1U / mL) was added, and then 50 μL PNPG (5mmol / L) was added after 37°C for 10min, and then 37°C for 20min was cultivated, and finally 1mL of Na2CO3 (0.1mol / L) was added to terminate the reaction, and absorbance was measured at 405nm wavelength. And the inhibition rate was calculated according to the formula, and the formula is as follows:

[0031]

[0032] Where:

[0033] A – OD405 value of the solution containing α-glucosidase but without sample;

[0034] B——OD405 value of the solution without α-glucosidase and the sample to be tested;

[0035] C——OD405 value of α-glucosidase solution and sample to be tested;

[0036] D——Does not contain α-glucosidase solution but contains OD405 value of the sample to be tested.

[0037] α-glucosidase is located in the brush border of small intestinal epithelial cells. It is a key enzyme that catalyzes the hydrolysis of oligosaccharides and increases blood sugar levels after meals. Its enhanced activity can easily lead to increased blood sugar levels in diabetic patients. Inhibiting the activity of α-glucosidase is an effective way to treat type 2 diabetes. The α-glucosidase inhibition rate of Lactobacillus fermentum E003 is the highest, with an inhibition rate of 43.91%.

[0038] (3) Determination of α-amylase inhibition rate of Lactobacillus fermentum E003

[0039] The fermented lactobacillus seed liquid activated for three generations in MRS liquid medium was inoculated into MRS liquid medium at an inoculum volume of 1% (V / V), cultured at 37°C for 24h, and centrifuged at 8000r / min for 15min to obtain the lactic acid bacteria fermentation supernatant. 125μL lactic acid bacteria supernatant and 1mg / mL α-amylase solution were added to each solution and mixed in equal volumes, incubated in a 37°C constant temperature water bath for 10min, then the reaction solution was added to 250μL 1.5% soluble starch solution at 37°C, reacted at 37°C for 15min, then 500μL DNS solution was added, reacted in a boiling water bath for 5min, then cooled rapidly to room temperature, diluted 20 times, and allowed to stand for 30min, and the absorbance was measured at 540nm. PBS solution (0.1mol / L, pH=6.8) was used as a blank control for α-amylase solution and the sample to be tested. The inhibition rate was calculated according to the formula, which is as follows:

[0040]

[0041] Where:

[0042] A: Sample set, containing sample solution and α-amylase solution

[0043] B: Sample blank group, containing sample solution but not α-amylase solution

[0044] C: control group, containing no sample solution and α-amylase solution

[0045] D: blank group, without sample solution and α-amylase solution.

[0046] α-Amylase is an important starch hydrolyzing enzyme that can cut the glycosidic bonds inside starch to produce dextrin, oligosaccharides and glucose, etc. It can promote the hydrolysis and digestion of carbohydrates in food, promote the intake of sugar, and increase the levels of blood sugar and blood lipids. After the human body eats, α-glucosidase and α-amylase synergistically promote the increase of blood sugar, which in turn causes symptoms of hyperglycemia. Therefore, the α-amylase inhibitory activity of lactic acid bacteria is also an important indicator for evaluating its anti-sugar ability. The α-amylase inhibition rate of fermented Lactobacillus E003 is the highest, reaching 59.44%. Therefore, fermented Lactobacillus E003 or its fermented liquid can be used to lower blood sugar.

[0047] Example 3 Sugar-resistant fermented milk prepared by Lactobacillus fermentum E003

[0048] (1) Preparation of sugar-resistant fermented milk

[0049] The activated Lactobacillus fermentum E003 strain was inoculated into MRS broth at a 1% inoculation rate and cultured (37°C, 24h) to the third generation. The Lactobacillus fermentum E003 seed solution was diluted with 1-3% (1.0×10 8 CFU / mL-3.0×10 8 CFU / mL) (V / V) and commercial direct-inoculation starter cultures (Lactobacillus bulgaricus and Streptococcus thermophilus were purchased from Royal DSM of the Netherlands, with a ratio of 1:1) were inoculated into bovine milk at a rate of 0.01%, and fermented at a constant temperature. The fermented milk was placed in a refrigerator at 4°C for 12 h, and then sensory evaluation was performed.

[0050] Process Optimization of Fermented Milk

[0051] (1) Single factor experiment of fermentation process

[0052] ①Effect of inoculation amount of Lactobacillus fermentum E003 on the sugar resistance of fermented milk

[0053] The inoculation amount was set at 1, 1.5, 2, 2.5, and 3%, the fermentation temperature was 37°C, and the fermentation time was 10 h to study the effect of inoculation amount on the sugar resistance of fermented milk.

[0054] When the fermentation temperature was 37°C, the fermentation time was 10 h, and the inoculation amount of strain E003 was 1%, 1.5%, 2%, 2.5%, and 3%, the sugar resistance of fermented milk showed a trend of first increasing and then decreasing. When the inoculation amount was 2.5%, the sugar resistance was the highest. Therefore, 2%, 2.5%, and 3% were selected as the subsequent response surface optimization levels.

[0055] ②The influence of fermentation temperature on sugar resistance

[0056] The fermentation temperature was set at 33, 35, 37, 39, and 41°C, the inoculation amount was 2%, and the fermentation time was 10 h to study the effect of fermentation temperature on the sugar resistance of fermented milk.

[0057] When the fermentation time was 10 h, the CE inoculation rate was 2%, and the fermentation temperature was 33°C, 35°C, 37°C, 39°C, and 41°C, the sugar resistance of the fermented milk increased first and then decreased. When the fermentation temperature was 39°C, the sugar resistance was the highest. Therefore, 37°C, 39°C, and 41°C were selected as the subsequent response surface optimization levels.

[0058] ③The influence of fermentation time on sugar resistance

[0059] The fermentation time was set to 6, 8, 10, 12, and 14 h, the inoculation amount was 2%, and the fermentation temperature was 37 ° C to study the effect of fermentation time on the sugar resistance of fermented milk. The experimental results showed that the sugar resistance was the highest when the fermentation time was 10 h, so 8 h, 10 h, and 12 h were selected as the subsequent response surface optimization levels.

[0060] ④ Sensory evaluation of fermented milk

[0061] Ten laboratory members who have a certain understanding of the sensory characteristics of fermented milk conducted a sensory evaluation of the finished fermented milk. In order to avoid the influence of cool temperature on the taste, the samples were placed at room temperature for 15 minutes during the sensory evaluation; the color and texture of the fermented milk were observed, and then the odor and taste of the fermented milk were smelled. The sensory scoring table is shown in the table, using a 100-point scoring method, and finally calculating the score of the fermented milk. The scoring criteria are detailed in Table 1.

[0062] Table 1 Sensory rating table

[0063]

[0064] (2) Optimization of process conditions for sugar-resistant fermented milk using response surface analysis

[0065] On the basis of the single factor experiment of sugar-resistant fermented milk, the BoxBehnken in the design expert software was used to conduct a 3-factor 3-level response surface design with the dual response values ​​of sugar-resistant ability and sensory score. The three factors of inoculation amount, fermentation temperature and fermentation time were selected to optimize the optimal fermentation process of sugar-resistant fermented milk.

[0066] According to the results of the single-factor experiment, the inoculation amount of bacteria (% (A), fermentation temperature ℃ (B), and fermentation time h (C) were used as the investigation factors, and the α-glucosidase inhibition rate and sensory score double response value were used. The Box-Behnken Design central composite test was used to conduct a three-factor three-level test to obtain the optimal fermentation process conditions for fermented milk. The variance analysis is shown in Table 2.

[0067] Table 2 Response surface experimental design and experimental results

[0068]

[0069]

[0070] ①Model establishment and significance test

[0071] The Design-Expert8.0.6 software was used to perform multiple regression fitting on Table 2, and the quadratic equation model of the inhibition rate of α-glucosidase of fermented milk (Y1), sensory score (Y2) and strain ratio v / v (A), fermentation temperature ℃ (B), and fermentation time h (C) was obtained as follows:

[0072] Y1=44.54-1.33A-1.55B+0.45C+0.23AB-0.12AC+1.11BC-2.28A 2 -3.68B 2 -5.57C 2

[0073] Y2=87.32-2.55A-0.65B-3.18C+0.85AB+0.90AC-4.15BC-4.84A 2 -1.64B 2 -10.13C 2

[0074] The results of the variance analysis of the regression model: Both models are significant, and the lack of fit terms are not significant. R1 2 =0.9727, R2 2 =0.9904, R 2 Adj1＝ 0.9375, R 2 Adj2 =0.9780, which also shows that the fit between the actual measured values ​​and the predicted values ​​during the experiment is relatively good, and the credibility and authenticity are both very high, so the model can be used to optimize the process of fermented milk.

[0075] ②Verification of regression model and determination of optimal conditions

[0076] After analysis by the design software, the optimal process conditions for fermented milk were obtained, which were: inoculation volume 2.35%, fermentation temperature 38.55℃, fermentation time 9.89h. Under these conditions, the inhibition rate of α-glucosidase and sensory score were 44.882% and 87.8796 points. In order to verify the accuracy of the optimal process conditions, and taking into account the actual situation, the process conditions were adjusted to: inoculation volume 2.5%, fermentation temperature 39℃, fermentation time 10h. Under these process conditions, the verification test was carried out, and the fermented milk α-glucosidase inhibition rate was 45.33%, and the sensory score was 86.3 points, which was close to the theoretical value analyzed by the response surface method, proving that this model is more reliable and can be used in experiments.

[0077] Example 4 Quality Analysis of Sugar-Resistant Fermented Milk by Lactobacillus fermentum E003

[0078] (1) Determination of physical and chemical indicators of fermented milk

[0079] The protein content was determined by the Kjeldahl method according to the national standard GB 5009.5-2016; the fat content was determined by the alkaline hydrolysis method according to the national standard GB 5009.6-2016; the acidity was determined according to the national standard GB 5009.239-2016; the viscosity was determined by a viscometer (spindle No. 3, speed 30 rpm, time 30 s); and the water holding capacity was determined according to the method of S Guo.

[0080] The physical and chemical indicators of fermented milk are shown in Table 3. As can be seen from Table 3, the protein, fat and acidity values ​​of fermented milk all meet the national standards. Acidity is the main factor affecting the shelf life and taste of fermented milk. The pH value of fermented milk (4.44) is conducive to maintaining the stability of the taste of fermented milk and effectively extending the market shelf life of fermented milk. Water holding capacity can affect the viscosity and hardness of fermented milk products, causing changes in the quality of fermented milk. The water holding capacity of fermented milk is 72.64±5.85%, which meets the texture requirements of fermented milk. Viscosity is an important indicator for evaluating the texture and taste of fermented milk. The viscosity of fermented milk is (4656.00±1287.51)MPa·s, reaching the viscosity value range of fermented milk reported in research.

[0081] Table 3 Physicochemical properties of fermented milk

[0082]

[0083] (2) Determination of microbial indicators of fermented milk

[0084] Determination of microbial indicators: the total colony count was determined with reference to the relevant methods of GB 4789.2-2016; the mold and yeast were determined with reference to the first method of GB / T 4789.15-2016; the lactic acid bacteria count was determined with reference to the relevant methods of GB 4789.35-2016; the coliform group, Salmonella and Staphylococcus aureus were determined with reference to GB / T 4789.3-2016, GB / T 4789.4-2016 and GB / T 4789.10-2016 respectively.

[0085] As shown in Table 4, the microbial indicators and lactic acid bacteria count of fermented milk are in line with GB19302-2010 "National Food Safety Standard Fermented Milk", which ensures product quality and is beneficial to product storage.

[0086] Table 4 Microbial indicators of fermented milk and number of lactic acid bacteria

[0087]

[0088]

[0089] Example 5 Study on the sugar-resistant fermented milk components prepared by Lactobacillus fermentum E003

[0090] Metabolite profile of fermented milk

[0091] Metabolites in fermented milk were studied by LC-MS / MS. As shown in Table 5, compared with fermented milk prepared by commercial starter and Lactobacillus fermentum E003 (CEBS-FM), 36 differential metabolites were obtained, mainly including amino acids and peptides, organic acids, fatty acids, aromatic compounds and their derivatives, which indicated that the fermentation process of the screened strains was different from that of commercial starter Lactobacillus bulgaricus and Streptococcus thermophilus, and these differences involved multiple fermentation processes such as strain hydrolysis, synthesis and growth and reproduction.

[0092] Table 5 Differential metabolites between CEBS-FM and BS-FM

[0093]

[0094]

[0095]

[0096]

[0097] Example 6 Fermentation of Lactobacillus E003 to produce other beneficial functional factors

[0098] Determination of AI-2 production ability of Lactobacillus fermentum E003

[0099] Vibrio harveyi (V.harveyi) BB170 (ATCC BAA-1117) was used as an indicator bacteria, which was purchased from Guangdong Microbiological Culture Collection Center (GDMCC). V.harveyi BB170 was streaked on Marine 17 agar medium and cultured at 30°C for 24 hours. It was inoculated into an autogenous bioassay (AB) liquid medium and cultured at 30°C for 12 hours. The fermentation broth of different lactic acid bacteria (Lactobacillus plantarum B103, Lactobacillus fermentum E003 and Lactobacillus fermentum D051) cultured for 24 hours and the fermentation broth of V.harveyi BB170 was centrifuged at 4000rpm for 30 minutes, and the resulting supernatant was filtered through a 0.2μm filter. At the same time, V. harveyi BB170 that had been grown overnight was diluted with AB medium at a ratio of 1:100 (v:v), and then mixed with the medium group (sterile MRS broth), different lactic acid bacteria groups, and AB liquid medium group (negative control) at a ratio of 1:50 (v:v). Then, 200 μL of each mixed sample was placed in a 96-well cell culture plate, each sample was placed in one well, and the fluorescence intensity of each sample was detected using the bioluminescence mode of a full-wavelength multifunctional microscope. The AI-2 content of each experimental group was calculated when the fluorescence value of the negative control was the lowest within 1-7h. The AI-2 content is the ratio of the fluorescence value of the experimental group to the fluorescence value of the negative control group, which reached the lowest within 1-7h. The ratio of the fluorescence value of the experimental group to the fluorescence value of the culture medium. It is well known that the quorum sensing system is a mechanism for information exchange between microorganisms. As a signal molecule, AI-2 can exchange information between species / within species, and has a certain regulatory effect on specific physiological functions and gene expression of microorganisms.

[0100] The amount of AI-2 produced by fermented Lactobacillus E003 was higher than that of other lactic acid bacteria, indicating that it can promote the communication between microorganisms and utilize the prebiotic function.

[0101] Determination of GABA production capacity of Lactobacillus fermentum E003

[0102] The determination was performed according to the instructions of the γ-aminobutyric acid (GABA) content kit of Suzhou Grace Biotechnology Co., Ltd. The fermented Lactobacillus E003 seed liquid activated for three generations in MRS liquid medium was inoculated into the MRS liquid medium at a 1% inoculation volume (V / V), cultured at 37°C for 24 hours, centrifuged at 8000r / min for 15 minutes, and the supernatant was taken and the GABA content was determined according to the instructions. The GABA content calculation formula is: GABA (μgml –1 )=1960.8×(ΔA+0.0003)×D

[0103] Where: ΔA = absorbance (sample) - absorbance (CK); D is the dilution ratio.

[0104] As shown in Table 6, fermented Lactobacillus E003 can produce GABA with a content of 119.27 μg ml –1 A, higher than that of Lactobacillus fermentum D051.

[0105] Table 6 GABA content of strains

[0106] Strain name <![CDATA[GABA content (μg / ml –1 )]]> Lactobacillus fermentum E003 119.27±7.69 Lactobacillus fermentum D051 64.91±5.84

[0107] Determination of antibacterial ability of fermentation broth of Lactobacillus fermentum E003

[0108] The fermented Lactobacillus E003 seed liquid activated three generations in MRS liquid medium was inoculated into MRS liquid medium at a 1% inoculation rate (V / V), cultured at 37°C for 24 hours, centrifuged at 8000r / min for 15 minutes, and the supernatant was taken for antibacterial ability determination. Staphylococcus aureus and Escherichia coli were used as indicator bacteria, and 100 μL of indicator bacteria liquid (10 6 ~10 7 CFU / mL) was spread on LB agar plates. A hole was made on the plate with an Oxford cup (10 mm deep, 8 mm wide), 100 μL of lactic acid bacteria fermentation supernatant was added into the hole, and the size of the inhibition zone was observed and measured after culturing at 37°C for 24 h.

[0109] The fermented lactobacillus E003 showed an obvious inhibition zone on the Staphylococcus aureus plate, with an inhibition zone diameter of 16.9±0.7 mm, but no inhibition zone appeared on the Escherichia coli plate, indicating that the fermented lactobacillus E003 had a high inhibition ability against Staphylococcus aureus, but had no inhibition effect on Escherichia coli.

[0110] Determination of Antioxidant Capacity of Lactobacillus fermentum E003 Fermentation Broth

[0111] (1) Determination of DPPH free radical scavenging ability

[0112] The lactic acid bacteria E003 seed liquid activated for three generations in MRS liquid medium was inoculated into the medium MRS liquid medium at a 1% inoculation rate (V / V), cultured at 37°C for 24 hours, centrifuged at 8000r / min for 15 minutes, and the supernatant was taken for DPPH free radical scavenging ability determination. 2mL of fermentation supernatant was mixed with 2mL of 0.2mmol / L DPPH-anhydrous ethanol solution, reacted in the dark for 30 minutes, and the absorbance was measured at 517nm. The DPPH free radical scavenging ability was calculated according to the formula, which is as follows:

[0113]

[0114] Where:

[0115] A1 represents the absorbance of sample + DPPH-anhydrous ethanol solution;

[0116] A2 represents the absorbance of sample + anhydrous ethanol solution;

[0117] A0 represents the absorbance of distilled water + DPPH-anhydrous ethanol solution.

[0118] The DPPH free radical scavenging rate of Lactobacillus fermentum E003 was 79.10%, which was higher than that of other strains, indicating that the strain had a higher antioxidant capacity.

[0119] (2) Determination of ABTS free radical scavenging ability

[0120] Dissolve ABTS in anhydrous ethanol to prepare a 7mM ABTS stock solution. At the same time, prepare a 5mM potassium persulfate solution, mix the two in a volume ratio of 1:1, and react in the dark for 12 hours to obtain an ABTS working solution. Dilute the ABTS working solution 20 times with PBS (pH = 7.4) before use. Inoculate the lactic acid bacteria seed solution activated for three generations in MRS liquid culture medium into the MRS liquid culture medium at a 1% inoculation rate (V / V), culture at 37°C for 24 hours, and centrifuge at 8000r / min for 15 minutes. Take 0.5ml of lactic acid bacteria fermentation supernatant and add it to 4.5mL of diluted ABTS working solution. After shaking, react in the dark for 6 minutes, centrifuge at 12000rpm for 2 minutes, and take the supernatant to measure the absorbance of the supernatant at a wavelength of 734nm. Calculate the scavenging ability according to the formula, as shown below:

[0121] ABTS free radical scavenging rate (%) = [1-(A i -A j ) / A0]

[0122] Where:

[0123] A i It represents the absorbance of ABTS working solution + lactic acid bacteria fermentation supernatant;

[0124] A j It represents the absorbance of lactic acid bacteria fermentation supernatant + anhydrous ethanol solution;

[0125] A0 represents the absorbance of ABTS working solution + anhydrous ethanol solution.

[0126] The ABTA free radical scavenging rate of Lactobacillus fermentum E003 was 89.52%, which was higher than that of other strains, indicating that the strain had a higher antioxidant capacity.

[0127] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A fermented lactobacillus having blood sugar lowering function, characterized in that: The fermented lactobacillus (Limosilactobacillusfermentum) is named as fermented lactobacillus E003, with a preservation number of CCTCC NO: M20241504, a preservation unit: China Center for Type Culture Collection, and a preservation address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University.

2. The fermented liquid obtained by fermenting Lactobacillus fermentum according to claim 1.

3. Application of the fermented lactobacillus according to claim 1 or the fermented liquid according to claim 2 in preparing hypoglycemic or anti-oxidant or antibacterial drugs.

4. Use of the fermented lactobacillus according to claim 1 in preparing hypoglycemic fermented milk.

5. The use of the fermented lactobacillus according to claim 1 in preparing hypoglycemic fermented milk, characterized in that: The inoculation amount of Lactobacillus fermentum E003 is 1.0×10 8 CFU / mL-3.0×10 8 CFU / mL.

6. The use of the fermented lactobacillus according to claim 1 in preparing hypoglycemic fermented milk, characterized in that: The fermented lactobacillus E003 is used in combination with a commercial yogurt starter.

7. The use of the fermented lactobacillus according to claim 6 in preparing hypoglycemic fermented milk, characterized in that: The usage amount of the commercial yogurt starter is 0.01-0.03% (m / m); the fermentation time is 6h-10h, and the fermentation temperature is 36.5℃-39.5℃.

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