CN115058367B

CN115058367B - Lactobacillus pentosus (Lactobacillus pentosus) 068-1 and application thereof

Info

Publication number: CN115058367B
Application number: CN202210799704.4A
Authority: CN
Inventors: 薛文娇; 刘晨; 刘瑶; 丁浩; 安超; 马赛箭; 张琪雯
Original assignee: Microbiology Institute Of Shaanxi
Current assignee: Microbiology Institute Of Shaanxi
Priority date: 2022-07-06
Filing date: 2022-07-06
Publication date: 2023-06-16
Anticipated expiration: 2042-07-06
Also published as: CN115058367A

Abstract

The invention discloses lactobacillus pentosus 068-1 with a preservation number of CGMCC No.24424, which can convert rutin into quercetin by whole cells and improve the bioavailability of rutin. And has good probiotics performance, and the gastrointestinal fluid tolerance, self-polymerization capability, caco-2 adhesion capability and antibacterial performance are all superior to those of the commercial strain lactobacillus rhamnosus GG (LactobacillusrhamnosusGG). The strain of the invention can be used as a novel dietary supplement for regulating intestinal flora and improving the bioavailability of rutin for individuals. The strain can also be used for fermenting rutin-containing edible and medicinal plants or fruits and vegetables, and can be used for in vitro improving the content of quercetin in the fermented product, thereby improving the activity and bioavailability of the product.

Description

Lactobacillus pentosus (Lactobacillus pentosus) 068-1 and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to lactobacillus pentosus (Lactobacillus pentosus) 068-1 and application thereof.

Background

Rutin is a flavonoid compound widely existing in the roots, stems, leaves, flowers, fruits and seeds of edible or medicinal plants. Some common traditional Chinese medicines, such as flos Sophorae Immaturus, herba Rutaceae, fructus Hordei vulgaris, fructus Hippophae, semen Ginkgo, fructus Lycii, herba Leonuri, bupleuri radix, prunellae Spica, aloe, folium Eucalypti Globueli and tobacco leaf, etc., contain rutin. As a safe and low-toxicity natural active ingredient, rutin has various effects of resisting oxidation, reducing blood fat, resisting cancer, resisting inflammation and the like, and plays an important role in improving and preventing various metabolic diseases such as obesity, diabetes, cancer and the like. However, a great deal of human body studies have shown that rutin is absorbed and utilized with a large individual difference.

Rutin is not hydrolyzed and absorbed by beta-glucosidase in the small intestine in vivo, and thus almost entirely accumulates in the large intestine. In the large intestine, rutin must be hydrolyzed by alpha-L-rhamnosidase (hydrolyzed rutin to isoquercetin) and beta-glucosidase (hydrolyzed isoquercetin to quercetin) or rutin enzyme (directly hydrolyzed rutin to quercetin) metabolized by intestinal microorganisms to remove glycoside, and aglycone is further hydrolyzed, absorbed and utilized. A plurality of researches prove that obvious individuation differences exist in intestinal flora, so that the conversion efficiency of rutin is greatly different, the individuation difference phenomenon of rutin utilization is improved, and the problem that the strain with high rutin conversion efficiency and strong intestinal colonization capability is needed to be solved by the technicians in the field is provided.

Disclosure of Invention

In view of this, the present invention provides a strain of Lactobacillus pentosus (Lactobacillus pentosus) 068-1 and its uses. Lactobacillus is a widely used type of probiotic bacteria due to its safety, among which Lactobacillus is a recently re-described type of Lactobacillus with great probiotic potential. Lactobacillus is generally derived from plants, is a main starter for fermenting plant food, has safe and good probiotics potential and has biological effects of probiotics and flavonoid natural products when being used together with rutin, and has good application prospect in functional foods such as fermented foods and novel dietary supplements.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

lactobacillus pentosus (Lactobacillus pentosus) 068-1 with a preservation number of CGMCC No.24424. The microbial strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection) for 23 days in 2022, and has a preservation address of North Xielu No. 1, 3, a division name of Lactobacillus pentosus (Lactobacillus pentosus) 068-1 in the Korean region of Beijing city.

The strain can transform rutin into quercetin by whole cells, has good probiotics performance, and has better gastrointestinal fluid tolerance, self-polymerization capability, caco-2 adhesion capability and antibacterial performance than those of commercial strain lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG).

A method for culturing lactobacillus pentosus (Lactobacillus pentosus) 068-1 comprises culturing lactobacillus pentosus (Lactobacillus pentosus) 068-1 in MRS culture medium at 37 ℃ for 24-48 h.

A microbial inoculum comprises the lactobacillus pentosus (Lactobacillus pentosus) 068-1.

A food comprises the above Lactobacillus pentosus (Lactobacillus pentosus) 068-1.

A health product comprises the above Lactobacillus pentosus (Lactobacillus pentosus) 068-1.

A medicament comprising lactobacillus pentosus (Lactobacillus pentosus) 068-1 as described above.

Use of Lactobacillus pentosus (Lactobacillus pentosus) 068-1 in preparing product from rutin-containing plant source is provided.

An application of lactobacillus pentosus (Lactobacillus pentosus) 068-1 in preparing a product in cooperation with a rutin-containing natural product.

Preferably, the product is a food, a health product or a medicament.

A method for improving rutin bioavailability comprises treating rutin or rutin-containing plant source with Lactobacillus pentosus (Lactobacillus pentosus) 068-1.

The beneficial effects are that: the invention discloses a lactobacillus pentosus (Lactobacillus pentosus) 068-1 strain which can convert rutin into quercetin by whole cells, so that the activity of a product is improved, and the bioavailability of the rutin by an individual is improved. The strain has good probiotics performance, and has excellent gastrointestinal fluid tolerance, self-polymerization capability, caco-2 adhesion capability and antibacterial performance.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 is a colony morphology of strain 068-1;

FIG. 2 is a gram of strain 068-1;

FIG. 3 is a phylogenetic tree of strain 068-1; description: based on the whole gene sequencing result, the taxonomic status of the Lactobacillus pentosus is changed, and the original root Lactobacillus is called as Lactiplantibacillus by La Ding Wenming;

FIG. 4 is an HPLC chart of rutin converted by strain 068-1 to generate quercetin;

FIG. 5 shows simulated gastric juice tolerance performance of strain 068-1;

FIG. 6 shows simulated intestinal juice tolerance performance of strain 068-1;

FIG. 7 shows the self-polymerizing ability of strain 068-1;

FIG. 8 shows the adhesion rate of strain 068-1 to Caco-2 cells;

FIG. 9 shows the results of a hemolysis test of strain 068-1, wherein a is strain 068-1, b is LGG, and c is S.aureus.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The embodiment of the invention discloses lactobacillus pentosus (Lactobacillus pentosus) 068-1, the reagents involved in the embodiment are all available through conventional purchase, the sources of the reagents are not limited, details which are not mentioned in the related method are all conventional operations, and the details are not repeated.

Example 1

Isolation and identification of Strain 068-1

(1) Separating and purifying strains: taking 0.5g of traditional fermented food slurry in the western security area of Shaanxi, cutting the sample into small pieces of 0.3 multiplied by 0.3cm by using sterile scissors, adding 10ml of sterile physiological saline, and performing vortex oscillation for 3min for gradient dilution to 10 ^-7 Respectively absorb 10 ^-4 ～10 ^-7 100 mu L of the diluent is coated on MRS solid culture medium added with 2% calcium carbonate, and the mixture is subjected to stationary culture at 37 ℃ for 48 hours; picking colonies with calcium-dissolving rings, streaking purified gram stain on MRS solid medium and observing under a microscope; gram-positive strains (No. 068-1) were selected and stored frozen at 25% glycerol at-80℃for further use (see FIG. 1 for colony culture of 068-1, see FIG. 2 for gram staining).

(2) Identification of strains: strain 068-1 was subjected to whole genome sequencing using Illumina NovaSeq and Oxford Nanopore ONT platform (Shanghai penoxen) and phylogenetic tree was constructed based on whole genome sequence using TYGS (https:// types.dsmz. De) (see fig. 3), which showed that the strain was lactobacillus pentosus (Lactobacillus pentosus) deposited at the general microbiological center of the chinese microbiological bacterial deposit management committee at 2022 month 23, with the deposit number CGMCC No.24424, with the deposit address of the north chen west road No. 1, the region of korea, and the taxonomic designation: lactobacillus pentosus (Lactobacillus pentosus).

Example 2

Conversion and detection of rutin

The 068-1 bacterial liquid after two generations of transfer is inoculated into a sugar-free MRS culture medium added with 1mM rutin according to the inoculation amount of 10 percent. After incubation of the cultures for 72 hours at 37 ℃, the fermentation broth was centrifuged at 8000rpm for 5min at 4 ℃, the supernatant was collected, 1ml of supernatant was extracted with three volumes of water saturated n-butanol, well mixed and allowed to stand for stratification, the upper organic phase was carefully aspirated, evaporated in a fume hood at room temperature and redissolved with 1ml of methanol. After filtration through a 0.22 μm organic phase filter, the mixture was added to a HPLC dedicated bottle and detected by high performance liquid chromatography.

Chromatographic conditions: c18 (4.6 mm. Times.250 mm,5 μm) column; the mobile phase was methanol/0.4% phosphoric acid (60/40, v/v), the flow rate was 1.0mL/min, the column temperature was 30deg.C, and the detection wavelength was 256nm.

The results show that (see figure 4), the strain 068-1 can transform rutin into quercetin by whole cells.

Example 3

Probiotic performance of Strain 068-1

1. Strain 068-1 gastrointestinal fluid tolerance

The strain 068-1 frozen and preserved at-80 ℃ is continuously activated for two generations, inoculated into MRS liquid culture medium with 1% (v/v) inoculum size, kept stand and cultured for 18 hours at 37 ℃, centrifugally collected thalli (8000 Xg, 4 ℃ and 10 min), resuspended in PBS and the concentration of the thalli is adjusted to 10 percent ⁹ cfu/ml. Respectively adding 9mL of artificial gastric juice (3 g/L pepsin, pH 3.0) or artificial intestinal juice (1 g/L trypsin, 0.3% bovine bile salt, pH 8.0) into 1mL of the bacterial suspension, standing and culturing at 37 ℃ for 3h, sampling in the process, centrifuging, discarding supernatant, serially diluting with 0.85% sterilized normal saline, coating and counting on MRS flat plates, counting the initial viable count without being treated by gastrointestinal simulant, counting the viable count after being treated as Nt, and taking three groups of bacteria (taking commercial strain lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG strain CGMCC 1.3724.3724) as a control)]The survival rate calculation formula is as follows:

percent survival = (log cfu/ml Nt)/(log cfu/ml N0) ×100

The results show that (see figure 5 and figure 6) the survival rate of 068-1 bacteria is above 96% and the number of viable bacteria is 10 when the gastric juice is simulated for 3 hours ⁸ cfu/ml or more; in the simulated intestinal juice, the survival rate of 068-1 is 66% at 3h, and the viable count is 10 ⁵ cfu/ml or so. The strain 068-1 has better tolerance to simulated gastrointestinal fluids than LGG.

2. Self-polymerizing ability

The strain 068-1 frozen and preserved at the temperature of 80 ℃ below zero is continuously activated for two generations, inoculated into MRS liquid culture medium with an inoculum size of 1% (v/v), and collected by centrifugation (8000 g,4 ℃ C., 10 min); after washing 2 times with PBS (pH 7.4), the bacterial liquid concentration was adjusted to 10 by re-suspending in PBS ⁸ cfu/mL, taking 50ul of fungus suspension, adding into 150ul of PBS, mixing uniformly, and measuring the absorption at 600nmThe light value is recorded as A0; loading 4mL of equal amount of cell bacterial suspension into a 5mL centrifuge tube, mixing uniformly, standing At 37 ℃ for 28h, taking 50ul of upper bacterial suspension At different times in the process, adding into 150ul of PBS, mixing uniformly, measuring the absorbance At 600nm, and recording as At [ with commercial strain lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG) as a control ]]。

The aggregation rate (a%) was calculated as: a% = (A0-At)/A0.times.100%

The results showed (see FIG. 7) that the self-polymerization rate of 068-1 reached 80.79% when left for 20h and 81.09% when left for 28h, which was about twice that of LGG (41.77%). The result shows that the strain 068-1 has extremely strong self-aggregation capability, and has good potential for adhesion and colonization in intestinal tracts and inhibition of pathogenic bacteria.

3. Adhesion property of small intestine cell Caco-2

10% fetal bovine serum, 1% nonessential amino acids (N1250, soxhibao) and 1% green-streptomycin (10000 IU/ml,10000 ug/ml) were additionally added to a high-sugar DMEM medium (HyClone, USA) to prepare a DMEM complete medium.

The Caco-2 cell freezing tube taken out from the liquid nitrogen tank is immediately placed into a water tank with the temperature of 37 ℃ for quick thawing, the cell suspension after thawing is slowly added into a cell culture dish, then 10mLDMEM complete culture medium is added into the culture dish, and the mixture is uniformly mixed and placed into a water tank with the temperature of 37 ℃ and CO of 5% ₂ Is cultured in a constant temperature incubator. After 2-3 days, after the cells cover 80% -90% of the culture dish, the cells are digested by trypsin for passage. After three passages, the cells were transferred to 24-well plates with an inoculum size of 10 per well ⁵ And 37 ℃,5% CO ₂ The culture medium is changed every two days in a constant temperature incubator for 21 days, and the differentiated monolayer Caco-2 cells are obtained. Before 4 hours of the adhesion experiment, the culture broth was replaced with a double antibody-free DMEM broth (high sugar DMEM supplemented with 10% fetal bovine serum, 1% non-essential amino acids), and 1ml of 068-1 strain suspension ((1×10) resuspended in double antibody-free DMEM broth) was added to each well ⁸ CFU/ml))37℃，5％CO ₂ Culturing for 1h under conditions followed by pre-heated DPBS (pH 7.2, ca+ and Mg+ -free); procall) is washed twice to wash off unadsorbed bacterial cells. Dissolving with DPBS containing 1% (v/v) Triton X-100Adsorbed cells for 10min. And (3) collecting thalli, and calculating the adhesion rate of 068-1 to Caco-2 cells by a plate counting method.

Adhesion% = number of viable bacteria adhered/number of viable bacteria added × 100%

The results showed (see FIG. 8) that strain 068-1 had an adhesion rate to Caco-2 cells of 9.79% which was 1.3 times higher than that of commercial strain LGG (4.19%), showing excellent intestinal adhesion colonization potential.

4. Antagonistic properties of Strain 068-1

(1) Bacterial inhibition assay

The culture conditions of the indicator bacteria are 37 ℃ and 180rpm for shake culture, and the indicator bacteria are used for bacteriostasis test after two generations of activation; after two generations of activation of strain 068-1 and LGG (control), the supernatant was centrifuged at 8000rpm at 4℃for 5 min. Mu.l of the indicator suspension was added to 200ml MRS agar, mixed well and poured into a petri dish. A well of 8mm in diameter was punched out on the plate with a punch, 80. Mu.l of the supernatant of the test strain was added to each well, and the plate was incubated overnight at 37 ℃. The zone of inhibition was observed and its diameter (mm) measured with an electronic vernier caliper, see table 1.

TABLE 1

Note that: "-" is no inhibition; "+" indicates that the bacteriostasis area is 0-3 mm; the "++" indicates that the bacteriostasis area is 3-6 mm; "+". ++'s indicating bacteriostasis the circle is larger than 6mm. The diameter of the hole (8 mm) was subtracted.

(2) Test for inhibition of fungi

Fungus 7d was cultivated in an inverted position at 28℃using PDA agar plates. Selecting a proper amount of C.Albicans thalli in sterile water, and uniformly mixing to obtain bacterial suspension; the remaining four plates of indicator bacteria were rinsed separately with sterile water to obtain spore suspensions. After two generations of activation of strain 068-1 and LGG (control), the bacterial solution was dipped, a short line of about 2cm was drawn on an MRS agar plate, and the plate was placed at 37℃for inverted culture for 48 hours. The spore suspension or fungus suspension is put into PDA agar, spread on MRS plate of cultured test strain after mixing, after it is solidified, inverted culture is carried out at 28 deg.C for 48h, and the size of bacteriostasis circle is observed and recorded, see Table 2.

TABLE 2

The results show (Table 1& Table 2) that strain 068-1 exhibited good antagonistic activity against all indicator bacteria including fungi and bacteria. Wherein 068-1 has the highest bacteriostatic activity on E.coli, S.separator phiB, S.flexneri, S.aureus and F.oxysporum, LGG only has the bacteriostatic activity on eight indicator bacteria, and only has the highest bacteriostatic activity on S.aureus.

5. Antibiotic resistance characteristics

Experiments were performed in 96-well plates, and the antibiotics were each diluted in a gradient such that the concentration of the diluted antibiotics was 1024. Mu.g/ml, 512. Mu.g/ml, 256. Mu.g/ml, 128. Mu.g/ml, 64. Mu.g/ml, 32. Mu.g/ml, 16. Mu.g/ml, 8. Mu.g/ml, 4. Mu.g/ml, 2. Mu.g/ml. Adding 40 μl of the antibiotic solution after gradient dilution, 158 μl of MRS liquid culture medium and 2 μl of 068-1 bacteria liquid after two generations of activation into the well respectively; the experiments set up negative controls (sterile water instead of bacterial fluid) and positive controls (sterile water instead of antibiotics). After incubation at 37℃for 24h, the OD600 absorbance was measured using an enzyme-labeled instrument, as shown in Table 3.

TABLE 3 Table 3

Note that: resistance reference breakpoint values (Bories G2008; EFSA 2012). R, drug resistance; s, sensitivity.

The results show that the strain 068-1 has drug resistance to vancomycin, kanamycin, carbenicillin sodium and gentamicin, and can be used together with the antibiotics; is sensitive to tetracycline, erythromycin, clindamycin and chloramphenicol.

6. Hemolysis test

After the strain 068-1 is activated for two generations, streaking on a blood plate by using an inoculating loop, and after inverted culture for 48 hours at 37 ℃, observing whether a hemolytic loop appears or not, wherein the non-transparent loop does not have beta-hemolytic activity, namely has no effect on hemolysis or does not have hemolysis. Commercial strain LGG was used as negative control and strain s.aureus with β -hemolytic activity was used as positive control.

The results show (see fig. 9) that s.aureus produces transparent circles on the blood platelets with β -hemolytic activity; 068-1 and LGG cells have no transparent rings around them and therefore do not have beta-hemolytic activity.

In conclusion, the lactobacillus pentosus (Lactobacillus pentosus) 068-1 provided by the invention can transform rutin in whole cells, can resist the extreme environment of the stomach and intestine, has good surface adhesion characteristics, strong colonisation ability in the intestinal tract, good antibacterial performance, definite antibiotic resistance characteristics, no hemolytic activity, and multiple performances superior to those of the lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG), can be used as a novel dietary supplement to regulate intestinal flora, and can improve the bioavailability of rutin for individuals. The strain can also be used for fermenting rutin-containing edible and medicinal plants or fruits and vegetables, and can be used for in vitro improving the content of quercetin in the fermented product, thereby improving the activity and bioavailability of the product.

In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Lactobacillus pentosus strainLactobacillus pentosus) 068-1, characterized in that the preservation number is CGMCC No.24424.

2. The lactobacillus pentosus strain according to claim 1Lactobacillus pentosus) 068-1, which is characterized by comprising the steps of culturing lactobacillus pentosusLactobacillus pentosus) 068-1 is cultured for 24-48 h at 37 ℃ in MRS culture medium.

3. A microbial inoculum, which is characterized by comprising the lactobacillus pentosus strain as claimed in claim 1Lactobacillus pentosus) 068-1。

4. A medicament comprising the Lactobacillus pentosus strain of claim 1Lactobacillus pentosus) 068-1。

5. The lactobacillus pentosus strain according to claim 1Lactobacillus pentosus) Application of 068-1 in preparing medicine with rutin plant source.

6. The lactobacillus pentosus strain according to claim 1Lactobacillus pentosus) Application of 068-1 in preparing medicine with rutin-containing natural product.

7. A method for improving bioavailability of rutin, which is characterized in that lactobacillus pentosus as defined in claim 1 is utilizedLactobacillus pentosus) 068-1 treating rutin or rutin-containing plant source.