CN113528383B - Hypoglycemic lactobacillus ZJUIDS09 and application thereof - Google Patents
Hypoglycemic lactobacillus ZJUIDS09 and application thereof Download PDFInfo
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- CN113528383B CN113528383B CN202110774556.6A CN202110774556A CN113528383B CN 113528383 B CN113528383 B CN 113528383B CN 202110774556 A CN202110774556 A CN 202110774556A CN 113528383 B CN113528383 B CN 113528383B
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- GNBVPFITFYNRCN-UHFFFAOYSA-M sodium thioglycolate Chemical compound [Na+].[O-]C(=O)CS GNBVPFITFYNRCN-UHFFFAOYSA-M 0.000 description 1
- 229940046307 sodium thioglycolate Drugs 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 229960005404 sulfamethoxazole Drugs 0.000 description 1
- JLKIGFTWXXRPMT-UHFFFAOYSA-N sulphamethoxazole Chemical compound O1C(C)=CC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 JLKIGFTWXXRPMT-UHFFFAOYSA-N 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 235000019640 taste Nutrition 0.000 description 1
- 229960003080 taurine Drugs 0.000 description 1
- AWDRATDZQPNJFN-VAYUFCLWSA-N taurodeoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS(O)(=O)=O)C)[C@@]2(C)[C@@H](O)C1 AWDRATDZQPNJFN-VAYUFCLWSA-N 0.000 description 1
- 235000019587 texture Nutrition 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
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- 229940088594 vitamin Drugs 0.000 description 1
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- 235000013343 vitamin Nutrition 0.000 description 1
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- 238000004260 weight control Methods 0.000 description 1
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Abstract
The invention relates to the technical field of food microorganisms, and discloses lactobacillus johnsonii ZJUIDS09 with the preservation number of CGMCC NO. 22608. The invention also discloses the application of the lactobacillus johnsonii ZJUIDS09 in preparing products with the function of reducing blood sugar.
Description
Technical Field
The invention relates to the technical field of food microorganisms, in particular to lactobacillus johnsonii ZJUIDS09 with a blood sugar function and application thereof.
Background
Diabetes has now become the most abundant metabolic disease in the Chinese and world population. High serum blood glucose levels are considered to be a major factor in metabolic diseases such as diabetes. Therefore, lowering serum glucose levels is directly related to the health of the human body. So far, a great number of experiments prove that the partial taking of the lactic acid bacteria and the related products thereof has the effects of reducing the blood sugar content of serum of a human body, reducing the incidence rate of diabetes or relieving symptoms. Lactic acid bacteria are important probiotics in human intestinal tracts, and the quantity and the composition of the lactic acid bacteria play a vital role in maintaining the microecological balance of hosts and improving the functions of immune systems.
Lactobacillus is closely related to human life, and is one of beneficial microorganisms widely applied to the fields of food fermentation, industrial lactic acid fermentation and medical care. Lactobacillus johnsonii is a species of Lactobacillus, and it can be added to food to improve the taste, texture and flavor of the food. The lactobacillus johnsonii can also be planted in a human body to play a probiotic role, such as the aspects of regulating intestinal flora, inhibiting the growth of intestinal pathogenic bacteria, reducing serum and blood sugar, enhancing the immunity of the organism, improving lactose digestion, resisting tumors, resisting oxidation and the like.
CN102026554B lactobacillus paracasei and weight control teaches the use of lactobacillus paracasei ST11 for the preparation of a composition for the treatment or prevention of overweight, obesity and/or associated metabolic diseases selected from diabetes, cirrhosis, metabolic syndrome, cardiovascular diseases and combinations thereof.
CN1380902B entitled "microorganism for treating or preventing obesity and diabetes and pharmaceutical composition containing the same" teaches a pharmaceutical composition for treating or preventing obesity or diabetes, which is selected from Acetobacter (Acetobacter) KCTC 0773BP and Lactobacillus (Lactobacillus) KCTC 0774 BP.
CN112075638A food compositions and pharmaceutical compositions comprising a blood glucose lowering lactic acid bacterial strain, said compositions comprising an isolated lactic acid bacterial strain selected from Lactobacillus reuteri (Lactobacillus reuteri) GL-104 strain CCTCC NO: m209138, Lactobacillus salivarius subsp.salicinus AP-32 strain CCTCC NO: m2011127, Lactobacillus acidophilus (Lactobacillus acidophilus) TYCA06 strain CGMCC No.15210, and Lactobacillus johnsonii (Lactobacillus johnsonii) MH-68 strain CCTCC NO: one or a combination of M2011128.
Disclosure of Invention
The invention aims to solve the technical problem of providing a human-derived blood sugar-reducing lactobacillus johnsonii ZJUIDS09 and application thereof.
In order to solve the technical problems, the invention provides Lactobacillus johnsonii (Lactobacillus johnsonii) ZJUIDS09 with the preservation number of CGMCC NO. 22608.
The improvement of lactobacillus johnsonii zjuid 09 as the invention: the 16S rDNA full sequence of Lactobacillus johnsonii ZJUIDS09 is SEQ ID No: 1 is shown.
The invention also provides the application of the lactobacillus johnsonii ZJUIDS09 in preparing products (such as medicines) with the function of reducing blood sugar.
As an improvement of the application of the invention: the product with blood sugar lowering function contains bacterial powder preparation (viable bacteria preparation) of Lactobacillus johnsonii ZJUIDS 09.
In the bacterial powder preparation (viable bacterial preparation) of Lactobacillus johnsonii ZJUIDS09, the viable bacterial count of Lactobacillus johnsonii ZJUIDS09 is 1.0 × 1011～5×1011CFU/g。
The strain ZJUIDS09 of the invention has the preservation name: lactobacillus johnsonii; the preservation unit: china general microbiological culture Collection center, preservation Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, on Beijing, with a deposit number: CGMCC NO.22608, preservation time 2021 year 05 month 26 days.
The invention screens a strain of Lactobacillus johnsonii ZJUIDS09(Lactobacillus johnsonii ZJUIDS09) from feces of healthy breast-fed infants, and identifies the strain by combining an API 50CH identification reagent strip (French Merrier) and 16S rDNA sequencing and the like through the morphological, physiological and culture characteristics of bacteria.
The colony morphology characteristics of the Lactobacillus johnsonii ZJUIDS09(Lactobacillus johnsonii ZJUIDS09) provided by the invention are as follows: obvious colonies are formed on an MRS agar culture medium, and the size of the colonies is 0.3-1.5 mm. The colony is round, the edge is neat, white, and the surface is moist and smooth. The morphological characteristics of the thallus are as follows: gram staining is positive, no spore is produced, and the bacillus circulans is single, paired or short chain.
The DPP-IV inhibition rate in the blood sugar reducing capability of the Lactobacillus johnsonii ZJUIDS09(Lactobacillus johnsonii ZJUIDS09) provided by the invention is 30.6-38.9%, and the alpha-glucosidase inhibition rate is 23.6-40.1%. It also has the following capabilities:
1. has stronger bile salt hydrolase activity;
2. can resist acid and bile salt, and has certain proliferation ability; sensitivity to common antibiotics; has antibacterial activity; namely, it can resist the gastrointestinal environment, has no antibiotic resistance, and can inhibit pathogenic bacteria harmful in the intestine.
In conclusion, the present invention selects Lactobacillus johnsonii ZJUIDS09 having a strong hypoglycemic effect from probiotics isolated from feces of healthy breast-fed infants. The strain has strong bile salt hydrolase activity, has obvious advantages in acid resistance and bile salt resistance compared with other lactic acid bacteria, is suitable for gastrointestinal environment and has proliferation capacity. Has no antibiotic resistance and has antibacterial activity. The lactobacillus johnsonii ZJUIDS09 can be widely used for developing products with related probiotic functions for reducing blood sugar, such as fermented fruits and vegetables with the function of reducing blood sugar and fermented sour meat with the function of reducing blood sugar; yogurt with hypoglycemic function, silage with hypoglycemic function, pet milk powder and the like.
Drawings
FIG. 1 is a colony morphology of Lactobacillus johnsonii ZJUIDS 09.
FIG. 2 is a gram-stained bacterial morphology of Lactobacillus johnsonii ZJUIDS 09.
FIG. 3 is an electrophoretic identification chart of 16S rDNA of Lactobacillus johnsonii ZJUIDS 09;
note that M is 5000, 3000, 2000, 1000, 750, 500, 250, 100bp from top to bottom.
FIG. 4 is an example of a plate of antibacterial activity of Lactobacillus johnsonii ZJUIDS 09;
from left to right, the inhibition zones for salmonella enteritidis, staphylococcus aureus, escherichia coli and listeria monocytogenes are respectively.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1, screening and characterization of lactobacillus johnsonii ZJUIDS 09:
1. screening of lactic acid bacteria
1.1 sample sources
The bacterial strains used in the invention are all separated from feces samples of breast-fed healthy infants (3-6 months). 30 parts of fecal samples are collected, wherein all the sampling objects do not take any fermented food and probiotic preparation containing live bacteria, gastrointestinal tract diseases such as diarrhea and the like, and any antibiotics medicines in at least one month before being sampled.
1.2 isolation and purification of the Strain
Approximately 5g of fresh fecal samples were collected using sterile tubes and immediately sent to the laboratory for strain isolation. Putting 1g of sample into 9mL of MRS liquid culture medium, carrying out vortex mixing, and then carrying out enrichment culture at 37 ℃ for 48 h; then sucking 1mL of enrichment liquid in a super clean bench, performing tenfold gradient dilution by using sterile physiological saline, and selecting 10-6、10-7、10-8And (3) three dilution gradients, wherein 100 mu L of bacterial liquid of each gradient is taken and smeared on an MRS agar culture medium, and the bacterial liquid is cultured for 48h at 37 ℃. After the culture is finished, selecting a plate with 50-150 single colonies growing in an agar culture medium, picking typical colonies, carrying out streaking purification on an MRS agar plate for many times until the colony forms on the whole plate are consistent, and picking single colonies to an MRS liquid culture medium for enrichment culture. The obtained strains were all stored in MRS liquid medium containing 40% glycerol at-80 deg.C by freezing. The strain ZJUIDS09 was obtained.
2. Identification of Lactobacillus johnsonii ZJUIDS09
2.1 characteristics of the colonies
After lactobacillus johnsonii ZJUIDS09 is cultured in MRS agar medium for 48 hours, the diameter is 0.3-1.5 mm, the colony is circular, the edge is neat, the color is white, and the surface is moist and smooth, as shown in figure 1.
2.2 microscopic morphology:
colony smear of lactobacillus johnsonii zjuid 09: gram-positive, non-sporulating, rectus rotundus, single, paired, or short chain, see fig. 2.
2.3 probiotic API identification results
The strain is identified by adopting API 50CH (Merrier, France) physiological and biochemical test paper, and the result shows that the strain is Lactobacillus johnsonii.
TABLE 1 physiological and biochemical characteristics of Lactobacillus johnsonii ZJUIDS09
Note: + indicates a positive reaction and-indicates a negative reaction.
2.416S rDNA identification
Extracting the target strain genome DNA by using an Ezup column type bacterial genome DNA extraction kit, taking the extracted lactobacillus genome DNA as a template for PCR amplification, carrying out 16S rDNA PCR experiment by using bacterial universal primers 27F and 1492R, and after the PCR amplification reaction is finished, taking a PCR product to carry out agarose gel detection and photographing, wherein the length of an amplified fragment is about 1.5kb, and the figure is 3. The PCR product was sent to Biotechnology engineering (Shanghai) Co., Ltd for sequencing, and the results are shown in SEQ ID NO: 1, BLAST sequence alignment on the NCBI website showed that the sequence has over 99% homology to the 16S rDNA sequence of Lactobacillus johnsonii.
Combining the sequence alignment result of the strain ZJUIDS09 with the physiological and biochemical results to determine that the screened Lactobacillus ZJUIDS09 is Lactobacillus johnsonii (Lactobacillus johnsonii).
The strain ZJUIDS09 of the invention has the preservation information as follows: the preservation name is: lactobacillus johnsonii; the preservation unit: china general microbiological culture Collection center, preservation Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, on Beijing, with a deposit number: CGMCC NO.22608, preservation time 2021 year 05 month 26 days.
Example 2 confirmation of the in vitro hypoglycemic Capacity of Lactobacillus johnsonii ZJuIDS09
1. DPP-IV inhibition rate
Preparation of Strain fermentation broth
The strain ZJUIDS09 preserved in a glycerol tube is firstly streaked and activated for 2-3 times on an MRS agar plate, then a single colony is picked up and is subjected to amplification culture in an MRS liquid culture medium for 18 hours, and the concentration of the bacterial liquid reaches 107CFU/mL or so as to be used as bacterial suspension. Inoculating the bacterial suspension into MRS liquid culture medium with blood sugar (water solubility) concentration of 100 mug/mL according to the inoculation amount of 2 percent (volume percent), and culturing at 37 ℃ for 48 hours to obtain strain fermentation liquor. The fermentation broth was centrifuged (at 12000 rpm for 20 minutes) to obtain a supernatant and cells, and the cells were disrupted by a cell disruptor to prepare a content.
The measurement method is as follows:
mu.l glycine-paranitroaniline (0.2mM) and 25. mu.l of the bacterial sample (supernatant, bacterial suspension, contents, respectively) were added to a 96-well microplate and preincubated at 37 ℃ for 10 minutes. Thereafter, 50. mu.l DPP-IV (0.01U/ml) was added and incubated at 37 ℃ for 60 minutes. The reaction was stopped by adding 100. mu.l of sodium acetate buffer (1M, pH 4.0), and the absorbance of the sample was measured at 405 nm. The result is A in the formula of "DPP-IV inhibition rate".
The result obtained by changing "50. mu.l DPP-IV (0.01U/ml)" to "50. mu.l sterile water" in the same manner as above was B in the formula "DPP-IV inhibitory rate".
The "25. mu.l of the bacterial sample" was changed to "25. mu.l of PBS solution (concentration: 0.1mol/L, pH 6.8)", and the result was C in the formula "DPP-IV inhibitory ratio" in the same manner as above.
The results obtained in the same manner as above were obtained for "DPP-IV inhibition ratio" D in the formula "25. mu.l of the bacterial sample" was changed to "25. mu.l of PBS" and "50. mu.l of DPP-IV (0.01U/ml)" was changed to "50. mu.l of sterile water".
DPP-IV inhibition rate is [1- (A-B)/(C-D) ]. times.100%.
Each strain was run in 3 replicates and each experiment was repeated 3 times with PBS control and lactobacillus rhamnosus ATCC53103 as a positive control strain.
TABLE 2 DPP-IV inhibition ratio (%)
Marked by significant difference, P <0.05
As shown in Table 2, the Lactobacillus johnsonii ZJUIDS09 of the invention is significantly better than the standard strain Lactobacillus rhamnosus ATCC53103 in terms of thallus inhibitory rate, supernatant inhibitory rate, content inhibitory rate and the like. The lactobacillus johnsonii ZJUIDS09 is shown to have excellent DPP-IV inhibition effect and can be used for screening strains with the function of reducing blood sugar.
2. Alpha-glucosidase inhibition rate
The fermentation supernatant, the cells and the cell contents were prepared as above.
To a total volume of 205. mu.L, 50. mu.L of PBS solution (concentration 0.1mol/L, pH6.8) was added, 50. mu.L of p-nitrophenol-alpha-D-glucopyranoside (PNPG) solution (concentration 20 mmol/L) and 25. mu.L of sample (supernatant, bacterial suspension, content, respectively) were added, the mixture was incubated at 37 ℃ for 10min, 30. mu.L of alpha-glucosidase solution (20U/mL) was added, reaction was continued for 20min, and 50. mu.L of Na (concentration 1 mol/L) was added2CO3As a reaction termination solution, the light absorption value of the reaction solution is measured at 405nm and is in direct proportion to the free amount of the p-nitrophenol PNP. The result is a in the formula of "α -glucosidase inhibition rate".
The "50. mu.L of a p-nitrophenol-. alpha. -D-glucopyranoside (PNPG) solution having a concentration of 20 mmol/L" was changed to "50. mu.L of sterile water", and the other procedure was the same as above, and the result was B in the "alpha-glucosidase inhibition ratio" formula.
The 25. mu.l sample to be tested was changed to 25. mu.l PBS solution, and the results obtained were C in the formula "alpha-glucosidase inhibition ratio" in the same manner as above.
The "25. mu.l of the sample to be tested" was changed to "25. mu.l of PBS solution", and the "50. mu.l of a solution of p-nitrophenol-. alpha. -D-glucopyranoside (PNPG) having a concentration of 20 mmol/L" was changed to "50. mu.l of sterile water", and the results were obtained as D in the "alpha-glucosidase inhibition ratio" formula in the same manner as above.
Alpha-glucosidase inhibition rate ═ 100%
Each strain was run in 3 replicates and each experiment was repeated 3 times, with PBS as control and lactobacillus rhamnosus ATCC53103 as positive control strain.
TABLE 3 alpha-glucosidase inhibition ratio (%)
Marked by significant difference, P <0.05
As shown in Table 3, the inhibition rate of the lactobacillus johnsonii ZJUIDS09 on alpha-glucosidase of thallus and broken content is obviously higher than that of the standard strain lactobacillus rhamnosus ATCC53103, and the inhibition rate of the supernatant is not obviously different from that of the standard strain. This indicates that the cells of Lactobacillus johnsonii ZJUIDS09 have excellent alpha-glucosidase inhibitory activity.
The two tests show that the Lactobacillus johnsonii ZJUIDS09 has good effects on DPP-IV and alpha-glucosidase inhibition, has obvious advantages compared with a standard strain Lactobacillus rhamnosus ATCC53103, and can be used for the later-stage blood sugar reducing effect research and product development.
Example 3 confirmation of bile salt hydrolase Activity of Lactobacillus johnsonii ZJuuds 09
1. Qualitative determination of Lactobacillus johnsonii ZJUIDS09 bile salt hydrolase
Adding 0.3% (m/v, namely 3g/1000ml) of sodium deoxytaurocholate, 0.2% (m/v) of sodium thioglycolate and 0.37g/L of CaCl into a freshly prepared MRS agar culture medium2It was completely dissolved. Sterilizing at 121 deg.C for 15min, pouring into sterile plate, placing sterile filter paper sheet uniformly into the plate after solidification, and adding 10 μ L of Lactobacillus johnsonii ZJUIDS09 bacterial suspension (about 10 μ L) dropwise onto the filter paper sheet7CFU/mL) with 10 μ L of sterile phosphate buffer added dropwise as a blank. The plates were incubated anaerobically in anaerobic jars (OXOID) at 37 ℃ for 72 h. Bile salt hydrolase activity is considered to be present if there is a white precipitate around the filter paper sheet.
2. Quantitative determination of bile salt hydrolase activity of Lactobacillus johnsonii ZJUIDS09
Taking Lactobacillus johnsonii ZJUIDS09 fermentation broth (about 10)7CFU/mL)10mL in a centrifuge tube. Centrifuging at 4 deg.C and 8000r/min for 10min, discarding supernatant, and collecting thallus precipitate. The cells were rinsed 2 times with sterile physiological saline and suspended in 30mL of 0.1mol/L phosphate buffer (pH 7.0). The bacterial suspension was sonicated in an ice bath for 20min (100W, working time/gap time 3: 5, protection temperature 37 ℃). Then, the mixture is centrifuged at 10000r/min at 4 ℃ for 10min, and the supernatant is the bile salt hydrolase solution of the lactobacillus johnsonii ZJUIDS 09.
Taking 0.1mL of the bile salt hydrolase solution of Lactobacillus johnsonii ZJUIDS09, adding 1.8mL of 0.1mol/L phosphate buffer solution and 0.1mL of 6mmol/L sodium taurocholate; the resulting mixture was incubated at 37 ℃ for 30min, after which 0.5mL of 15% trichloroacetic acid was added to stop the enzymatic reaction. The mixture was centrifuged, and 0.5mL of the supernatant was added to 1mL of ninhydrin color-developing solution. Mixing with vortex, and boiling for 35 min. After cooling, the absorbance at 570nm was determined. One bile salt hydrolase activity unit is defined as the amount of enzyme required to release 1. mu. moL taurine from the substrate per minute.
3. The activity of the bile salt hydrolase was measured using Lactobacillus rhamnosus ATCC53103 as a positive control.
4. Table 4 shows the results of quantitative determination of the activities of bile salt hydrolase of the two strains. As can be seen from the table, the bile salt hydrolase activity of both strains is above 1.0U/mg, and the bile salt hydrolase activity of Lactobacillus johnsonii ZJUIDS09 is 1.25U/mg, which is not obviously different from that of the control strain. The experimental result shows that the lactobacillus johnsonii ZJUIDS09 has higher bile salt hydrolase activity.
TABLE 4 bile salt hydrolase assay results of the strains
Note: + indicates the formation of a precipitation ring, -indicates the absence of a precipitation ring.
5. The bile salt hydrolase can hydrolyze in-vivo bound bile salt into free bile salt, and the free bile salt does not participate in liver and intestine circulation and is discharged out of a body along with excrement, so that the bile salt hydrolase activity is a key factor for reducing in-vivo blood fat. The lactobacillus johnsonii ZJUIDS09 provided by the invention has stronger bile salt hydrolase activity and can reduce blood fat in vivo.
Example 4 confirmation of acid and bile salt resistance of Lactobacillus johnsonii ZJuuds 09
1. Acid resistance test
Selecting a single colony of lactobacillus johnsonii ZJUIDS09 to perform amplification culture in an MRS liquid culture medium for 18h, inoculating the expanded bacterial suspension into the MRS liquid culture medium in an amount of 1%, and culturing at 37 ℃ for 18 h. The culture was centrifuged at 8000r/min at 4 ℃ for 5min to collect the cells, which were washed 2 times with phosphate buffer (pH 6.8). Thallus suspensionFloating in MRS liquid medium at pH 3.0, the initial viable count was corrected to about 108CFU/mL, first cultured at 37 ℃ for 3 h. Then counting viable bacteria in the 0h and 3h samples by adopting a pour plate method, and determining the survival rate of the viable bacteria, wherein the survival rate calculation formula is as follows:
in the above formula, N0The number of viable bacteria (CFU/mL) of the test strain is 0 h; n is a radical oftTo test the viable count of the strain for 3h (CFU/mL).
2. Bile salt resistance test
Inoculating 1% of activated and expanded Lactobacillus johnsonii ZJUIDS09 bacterial suspension into MRS liquid culture medium, culturing at 37 deg.C for 18h, mixing by vortex, and correcting initial viable count to about 109CFU/mL. The culture was inoculated in an amount of 10% to MRS liquid medium containing 0.3% (m/v) of bovine bile salt (control was MRS liquid medium containing no bovine bile salt), and cultured at 37 ℃ for 3 hours. The number of viable bacteria in the sample was then counted using the pour plate method. The bile salt tolerance of the strain is expressed as the logarithm of the difference between the viable count per mL of medium containing bile salts and the viable count per mL of medium without bile salts at 3h (. DELTA.logCFU/mL).
3. The acid and bile salt resistance measurements described above were carried out with Lactobacillus rhamnosus ATCC53103 as a control.
4. As shown in Table 5, the acid and bile salt resistance of Lactobacillus johnsonii ZJUIDS09 was significantly better than that of the control strain ATCC 53103. Its survival rate in MRS medium with pH 3.0 is as high as 81.74%. The viable count in the environment containing 0.3% of ox bile salt still reaches 106CFU/mL or above indicates that the bile salt tolerance is better. Experiments prove that the Lactobacillus johnsonii ZJUIDS09 has higher gastrointestinal tract viability.
TABLE 5 results of the strains' tolerance to acids and bile salts
| Bacterial strains | Acid resistance (%) | Bile salt tolerance ability (Δ Log CFU/mL) |
| Lactobacillus johnsonii ZJUIDS09 | 81.74±0.41* | 1.48±0.11* |
| Lactobacillus rhamnosus ATCC53103 | 62.12±0.18 | 1.03±0.23 |
Marked by significant difference, P <0.05
5. Probiotics must be able to survive a range of adverse environments such as gastric acid and bile in the gastrointestinal tract to exert their probiotic effects. The lactobacillus johnsonii ZJUIDS09 provided by the invention can grow and proliferate under the condition of pH 3.0, and can smoothly pass through the acidic environment in the stomach to reach the small intestine. Meanwhile, the lactobacillus johnsonii ZJUIDS09 can resist bile salt, can survive in intestinal tracts and further can effectively play a role in reducing blood sugar.
Example 5 confirmation of the hydrophobic Capacity of Lactobacillus johnsonii ZJuIDS09
1. Measurement of hydrophobicity
The Lactobacillus johnsonii ZJuuds 09 pellet was washed twice with clean PBS broth and resuspended to OD610The absorbance of the lactobacillus is about 0.5 to obtain lactobacillus suspension; medium bacterial suspension preparation was made in accordance with the examples above.
Thoroughly mixing 2ml of lactobacillus suspension and 2ml of xylene, shaking in water bath at 37 deg.C for 5min, and measuring OD of water phase after 0h and 2h respectively610And (4) light absorption value.
A0Absorbance of 0h, AtTh absorbance.
The results obtained are shown in table 6 below.
TABLE 6 surface hydrophobicity of different strains (%)
2. Analysis of results
The hydrophobicity of lactobacillus johnsonii zjuid 09 was measured to be 51.43% and significantly higher than the control standard strain. The result shows that the strain has stronger adhesive capacity, can be adhered to the intestinal tract of a human and improves the health of intestinal flora.
Example 6 confirmation of antibiotic susceptibility of Lactobacillus johnsonii ZJuIDS09
Culturing for 18h at a concentration of about 107CFU/mL Lactobacillus johnsonii ZJUIDS09 suspension was added at 1% to sterilized MRS agar medium cooled to about 45 deg.C, mixed well, and dosed at 15 mL/dish. After coagulation, the drug sensitive paper is taken out with tweezers and placed on the culture medium. The plate was placed right side up in a 37 ℃ incubator for 24 h. Paper without antibiotic was used as a blank control. And measuring the diameter of the inhibition zone. Each was repeated three times.
The diameters of the zones of inhibition of the L.johnsonii ZJUIDS09 on antibiotic sensitivity are shown in Table 7. With reference to CLSI (2017) drug susceptibility test standards, Lactobacillus johnsonii ZJUIDS09 shows sensitivity to penicillin G, ampicillin, cefazolin, amikacin, gentamicin, erythromycin, sulfamethoxazole, and chloramphenicol. Present as intermediaries for ciprofloxacin, norfloxacin. The experimental results show that lactobacillus johnsonii zjuid 09 is sensitive to common antibiotics.
TABLE 7 results of susceptibility of Lactobacillus johnsonii ZJUIDS09 to antibiotics
Note: s, sensitivity; i, an intermediary; r, drug resistance
With the wide application of antibiotics in clinical treatment, the drug resistance of lactic acid bacteria is more and more serious, and the intake of drug-resistant lactic acid bacteria for a long time brings great difficulty to clinical treatment. The lactobacillus johnsonii ZJUIDS09 provided by the invention is sensitive to common antibiotics and cannot cause harm to human health.
Example 7 confirmation of the inhibitory Activity against pathogenic bacteria of Lactobacillus johnsonii ZJuuds 09
The antibacterial activity of the lactic acid bacteria is determined by adopting an international universal agar diffusion method. 10mL of LB agar medium was poured into a sterile petri dish, and cooled to prepare a lower layer medium. The concentration of the bacteria is about 10 after 18h of culture7CFU/mL indicator suspension was added at 1% to sterilized LB agar medium cooled to about 45 deg.C, mixed well and dosed to 10 mL/dish. Placing the sterilized oxford cup on the upper surface. After the upper medium was condensed, the Oxford cup was gently pulled out. A sample of fermentation supernatant (i.e., supernatant obtained in example 2) was added to Lactobacillus johnsonii ZJUIDS09 at 100. mu.L/well and phosphate buffer (pH6.8) was used as a control. The strains with obvious inhibition zones around the small holes are selected, the diameters of the inhibition zones are measured, and each is repeated three times. FIG. 4 is an example of a plate of Lactobacillus johnsonii ZJUIDS09 for its inhibitory effect on four pathogenic bacteria.
As shown in Table 8, the metabolite of Lactobacillus johnsonii ZJUIDS09 has certain inhibitory effect on Staphylococcus aureus, Escherichia coli, Salmonella enteritidis and Listeria monocytogenes, and is superior to the bacteriostatic effect of ATCC 53103. The metabolite of the strain can be seen to have bacteriostatic properties.
TABLE 8 results of the inhibitory potency of the strains on pathogenic bacteria
Staphylococcus aureus is the most common pathogenic bacterium in human pyogenic infection, some Escherichia coli can cause severe diarrhea and septicemia, Salmonella species can also cause human food poisoning, and Listeria monocytogenes is also an important human pathogenic bacterium. Bacteriocin, organic acid, hydrogen peroxide and other bacteriostatic substances generated by the metabolism of the lactic acid bacteria can inhibit the growth of the pathogenic bacteria individually or jointly. The metabolite of lactobacillus johnsonii ZJUIDS09 provided by the invention has certain antagonistic action on the three pathogenic bacteria, plays an important role in maintaining intestinal microecological balance and has a health promotion effect.
Comparative example lactobacillus as described in table 9 below was tested with reference to the above example and the results compared to the present invention are shown in table 9 below:
TABLE 9
Lactobacillus johnsonii MH-68 has a bile salt hydrolase activity of about 0.7U/mg and is far less tolerant to bile salts than ZJUIDS09 of the present invention.
Example 8 preparation of functional fermented yogurt Using Lactobacillus johnsonii ZJUIDS09
Processing technological process and operation key points of yoghourt
(1) Raw materials: 2L of whole UHT sterilized milk or fresh cow milk;
(2) preheating: putting into a container and heating to 63 ℃;
(3) homogenizing: homogenizing in homogenizer under pressure of 15-25MPa), pouring the mixed solution into iron tank, adding 100g white sugar, and sterilizing in 90 deg.C water bath environment for 10 min;
(4) blending: adding 5% white sugar into milk, 100g and dissolving;
(5) and (3) sterilization: sterilizing the sweetened cow milk in water bath at 90 deg.C for 10 min;
(6) and (3) cooling: cooling the sterilized milk to 40-50 ℃ for later use;
(7) preparing a leaven: the strain of Lactobacillus johnsonii ZJUIDS09 is inoculated in a test tube filled with sterilized skimmed milk (12%, w/v) under an aseptic environment and cultured for 20 hours at 37 ℃; the inoculation amount of each passage is 2-4% (v/v), the vitality is restored after 2-3 passages, and the mixture is placed in a refrigerator at 4 ℃ for preservation;
(8) inoculation and fermentation: inoculating activated Lactobacillus johnsonii ZJUIDS09 under aseptic condition, wherein the inoculation amount is 2-4% (v/v). Fermenting at 42 deg.C for 6-10 h;
(9) after-ripening: after fermentation, putting the mixture into a refrigerator with the temperature of 4 ℃ for after-ripening for 12-24 h;
(10) filling and refrigerating: after the after-ripening was completed, the mixture was filled into 250mL sterilized glass bottles and sent to a freezer for refrigeration.
Example 9 preparation of functional fermented fruit and vegetable juice Using Lactobacillus johnsonii ZJUIDS09
1. Processing technological process of fermented fruit and vegetable juice
Raw material → cleaning → flash evaporation → pulping → blending → homogenizing → sterilizing → cooling → inoculating → closed fermentation → after-ripening → filling → refrigeration;
2. the key points of the operation
(1) Raw materials: selecting fresh pumpkin and dragon fruit.
(2) Cleaning and cutting: cleaning, peeling (removing pulp from fructus Cucurbitae Moschatae), and cutting into small pieces.
(3) Flash evaporation: and (3) inactivating enzyme by adopting a flash evaporation method, treating for 0.5-1 min at 121 ℃, and quickly exhausting gas.
(4) Pulping: according to the weight ratio of 1: 1, the pumpkin and water are gradually put into a colloid mill to be ground, and coarse grinding and fine grinding are carried out once respectively. Pulping the dragon fruit by a pulping machine until the pulp is uniform and has no blocks.
(5) Blending and homogenizing: regulating soluble solid content to 10% with sucrose according to pumpkin juice 15%, dragon fruit juice 30%, and sugar°Brix, adding 0.2% of CMC (carboxymethyl cellulose) as a stabilizer, uniformly mixing, and adopting a two-stage homogenization method, wherein the low pressure (15MPa) is firstly adopted, and the high pressure (25MPa) is secondly adopted, so that the particle diameter of the melon pulp particles is 2-3 mu m.
(6) And (3) sterilization and cooling: keeping the temperature of the blended composite fruit and vegetable juice at 100 ℃ for 10min, and cooling to about 40 ℃.
(7) Inoculation and fermentation: inoculating activated Lactobacillus johnsonii ZJUIDS09 under aseptic condition, and controlling initial bacterial count at 107CFU/mL. Constant temperature at 37 DEG CFermenting for 24 h.
(8) After-ripening: after the fermentation is finished, putting the mixture into a refrigerator with the temperature of 4 ℃ for 3 hours.
(9) Filling and refrigerating: after the after-ripening was completed, the mixture was filled into 250mL sterilized glass bottles and sent to a freezer for refrigeration.
Example 10 preparation of functional fermented sour meat Using Lactobacillus johnsonii ZJUIDS09
The processing flow of the fermented sour meat is as follows:
(1) slicing: commercially available fresh streaky pork was cut into 3cm square slices.
(2) Mixing glutinous rice flour: 1000g of raw meat is mixed with 250g of glutinous rice flour, and 1% of glucose is added.
(3) Inoculation and fermentation: inoculating activated Lactobacillus johnsonii ZJUIDS09 under aseptic condition, and controlling initial bacterial count at 107CFU/mL. Fermenting at 37 deg.C for 18 h.
(4) Pickling: adding 2% salt, and pickling at 25 deg.C for 20 d.
Example 11 preparation of hypoglycemic powder Using Lactobacillus johnsonii ZJuuds 09
1. Preparation of lactobacillus johnsonii ZJUIDS09 bacterial paste
A single colony of Lactobacillus johnsonii ZJUIDS09 was picked and inoculated into 50mL of MRS liquid medium, and placed in an incubator at 37 ℃ for 18 h. Then activated in 250mL MRS liquid culture medium according to the inoculum size of 5 percent, and cultured for 24h in an incubator at 37 ℃. Finally, the activated Lactobacillus johnsonii ZJUIDS09 was cultured in a 10L fermentor at 5% inoculum size for high density anaerobic culture at 37 ℃ and pH6.8 for 18 h. Then centrifuging at 8000r/min and 4 deg.C for 15min, discarding supernatant, collecting thallus precipitate, and rinsing thallus with sterile phosphate buffer (pH 7.0) for 2 times. Thus obtaining the lactobacillus johnsonii ZJUIDS09 bacterial mud.
2. Preparation of the protective agent
The freeze-drying protective agent contains 15% of skim milk powder, 5% of trehalose, 3% of sodium glutamate, 1% of glycerol and 0.5% of cysteine hydrochloride; the balance being water (as solvent). Sterilizing at 110 deg.C for use. The above% is by mass.
3. Preparation of Lactobacillus johnsonii ZJUIDS09 powder
And (3) fully and uniformly mixing the prepared lactobacillus johnsonii ZJUIDS09 thallus precipitate with a protective agent solution according to the mass ratio of 1: 5. Pre-freezing for 5h at-40 ℃ to uniformly freeze the lactobacillus johnsonii on the inner wall of the container, then carrying out vacuum freeze drying, and drying for 18-20 h to obtain lactobacillus johnsonii ZJUIDS09 bacterial powder. The bacterial powder of lactobacillus johnsonii ZJUIDS09 is washed twice after being rehydrated by normal saline, and the number of the living bacteria in the bacterial powder of lactobacillus johnsonii ZJUIDS09 is 1.0 multiplied by 1011～5×1011CFU/g。
Example 12 preparation of silage Using Lactobacillus johnsonii ZJUIDS09
The steps for preparing alfalfa silage from lactobacillus johnsonii ZJUIDS09 are as follows:
(1) preparing raw materials: after the alfalfa is harvested, cleanness is guaranteed, no putrefaction and deterioration occur, and the alfalfa is cut into 1-2 cm.
(2) Preparation of silage: controlling the water content of the alfalfa grass to be 60-65% during ensiling, weighing 200g of alfalfa grass raw materials, filling the alfalfa grass raw materials into a vacuum packaging bag, inoculating the lactobacillus johnsonii ZJUIDS09 bacterial powder prepared in example 9 into the raw materials according to the inoculation amount of 0.02 per mill, carrying out vacuum packaging by using a vacuum packaging machine, and then placing the packaged powder into a storage room for fermentation.
(3) Storage conditions were as follows: the temperature is 15-45 ℃ and the time is 30-60 days.
Example 13 preparation of probiotic milk powder for pets Using ZJuIDS09
1. Preparation of Lactobacillus johnsonii ZJUIDS09 powder
Reference example 11 preparation of lyophilized powder of Lactobacillus fermentum ZJUIDS09 having a viable count of 1.0X 1011-5×1011CFU/g。
2. Preparation of pet formula powder
Primary selection of raw materials: milk powder, fish meal, bone meal, grains, vegetable oil and additives: vitamins, trace elements, functional factors, and others;
automatic batching: putting the obtained material raw materials into a material bin according to a formula;
crushing: crushing the weighed materials by a crusher;
mixing: adding vegetable oil and trace elements into the crushed materials, and adding the materials into a mixer for uniformly mixing;
puffing: the mixed materials are made into granular materials through a bulking machine;
drying: drying the mixed materials by a dryer, and controlling the temperature to be 65-70 ℃;
grading and screening: the material flow passes through a grading sieve, and the particle size is controlled between 2.5 and 5 mm.
3. Preparation of probiotic formula powder for pets
Mixing the bacterial powder prepared in the step 1 and the pet feed prepared in the step 2 according to the ratio of 1: 1000, and the live bacteria leaving the factory in the final product are 108CFU/g is higher than the standard. And (5) after filling, storing and selling the product.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Sequence listing
<110> Zhejiang university
<120> hypoglycemic lactobacillus ZJUIDS09 and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1277
<212> DNA
<213> Lactobacillus johnsonii
<400> 1
tgctcaggac gaacgctggc ggcgtgccta atacatgcaa gtcgagcgag cttgcctaga 60
tgattttagt gcttgcacta aatgaaacta gatacaagcg agcggcggac gggtgagtaa 120
cacgtgggta acctgcccaa gagactggga taacacctgg aaacagatgc taataccgga 180
taacaacact agacgcatgt ctagagtttg aaagatggtt ctgctatcac tcttggatgg 240
acctgcggtg cattagctag ttggtaaggt aatggcttac caaggcgatg atgcatagcc 300
gagttgagag actgatcggc cacattggga ctgagacacg gcccaaactc ctacgggagg 360
cagcagtagg gaatcttcca caatggacga aagtctgatg gagcaacgcc gcgtgagtga 420
agaagggttt cggctcgtaa agctctgttg gtagtgaaga aagatagagg tagtaactgg 480
cctttatttg acggtaatta cttagaaagt cacggctaac tacgtgccag cagccgcggt 540
aatacgtagg tggcaagcgt tgtccggatt tattgggcgt aaagcgagtg caggcggttc 600
aataagtctg atgtgaaagc cttcggctca accggagaac tgcatcagaa actgttgaac 660
ttgagtgcag aagaggagag tggaactcca tgtgtagcgg tggaatgcgt agatatatgg 720
aagaacacca gtggcgaagg cggctctctg gtctgcaact gacgctgagg ctcgaaagca 780
tgggtagcga acaggattag ataccctggt agtccatgcc gtaaacgatg agtgctaagt 840
gttgggaggt ttccgcctct cagtgctgca gctaacgcat taagcactcc gcctggggag 900
tacgaccgca aggttgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat 960
gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcca gtgcaaacct 1020
aagagattag gtgttccctt cggggacgct gagacaggtg gtgcatggct gtcgtcagct 1080
cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgtca ttagttgcca 1140
tcattaagtt gggcactcta atgagactgc cggtgacaaa ccggaggaag gtggggatga 1200
cgtcaagtca tcatgcccct tatgacctgg gctacacacg tgctacaatg gacggtacaa 1260
cgagaagcga acctgcg 1277
Claims (5)
1. Lactobacillus johnsonii ZJUIDS09, characterized by a collection number of CGMCC NO. 22608.
2. The Lactobacillus johnsonii ZJUIDS09 according to claim 1, wherein: the 16S rDNA full sequence of Lactobacillus johnsonii ZJUIDS09 is SEQ ID No: 1 is shown.
3. Use of lactobacillus johnsonii ZJUIDS09 according to claim 1 or 2 for the preparation of a product with hypoglycemic effect.
4. Use according to claim 3, characterized in that: the product with the function of reducing blood sugar comprises a bacterial powder preparation of Lactobacillus johnsonii (Lactobacillus johnsonii) ZJUIDS 09.
5. Use according to claim 4, characterized in that: in the preparation of powder of Lactobacillus johnsonii ZJUIDS09, the viable count of Lactobacillus johnsonii ZJUIDS09 is 1.0 × 1011～5×1011CFU/g。
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