CN116103193B - Probiotic powder and application thereof in relieving colonitis - Google Patents
Probiotic powder and application thereof in relieving colonitis Download PDFInfo
- Publication number
- CN116103193B CN116103193B CN202211430430.8A CN202211430430A CN116103193B CN 116103193 B CN116103193 B CN 116103193B CN 202211430430 A CN202211430430 A CN 202211430430A CN 116103193 B CN116103193 B CN 116103193B
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- lactobacillus plantarum
- probiotic powder
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- mice
- probiotic
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Abstract
The invention relates to the technical field of functional microorganism screening and application, in particular to probiotic powder and application thereof in relieving colonitis. The probiotic powder contains lactobacillus plantarum WW (Lactobacillus plantarum WW) which is preserved in China general microbiological culture Collection center (CGMCC No. 24189) in the year 2021, 12 and 24, and the viable count is more than 10 9 CFU/g can effectively relieve the symptoms of colonitis, can be widely used for preventing and treating colonitis, and has remarkable effect.
Description
Technical Field
The invention belongs to the technical field of screening and application of probiotics, and particularly relates to probiotic powder and application thereof in relieving colonitis.
Background
Probiotic preparations, also known as micro-ecological modulators and live bacteria preparations, refer to a class of microorganisms that are beneficial to human health after ingestion of a certain amount. They maintain the beneficial flora of intestinal microecological balance and play an irreplaceable role in human health (Gerritsen et al 2011). In recent years, probiotics have been widely focused and their function is increasingly known. A large number of researches show that probiotics have certain treatment effects on various diseases, and proper supplementation of probiotics is beneficial to maintaining the steady state of the environment in the intestinal tract and maintaining the health of organisms. Has effects of improving irritable bowel syndrome, enhancing host immune regulation, treating obesity, and preventing and treating cancer.
IBD, including CD and UC, is a chronic immune bowel disease with chronic diarrhea, bloody stool and intestinal microbial disorders as the major clinical symptoms (Xavier & Podolsky, 2007). Compared with CD, the incidence of UC is higher, and most cases have the characteristics of unknown etiology, easy recurrence, more complications and difficult cure, and more evidence indicates that chronic colon inflammation and persistent inflammatory bowel disease can lead to colorectal cancer (Gupta et al,2007;Rutter et al,2004). The occurrence of UC is thought to be related to genetic factors, intestinal mucosal immune dysfunction, changes in intestinal micro-ecological environment, and the like (Yadav et al, 2016). At present, the UC treatment method mainly adopts medicine treatment, and the medicine has certain treatment effect, but the problems of treatment limitation, large side effect and the like (Zheng Yuxing, 2020) still exist, and even nephritis, hepatitis, blood cachexia and the like are caused (Yuan Ziwen, 2020). In addition, biological agents (e.g., anti-TNF-alpha, alpha 4 beta 7 integrin, IL-12/IL-23p40 monoclonal antibodies, etc.) are also common drugs for UC. Biological agents are generally expensive and their efficacy and safety are compromised by immunogenicity when used. Numerous studies have shown that many probiotic strains are able to modulate the intestinal flora and have anti-inflammatory effects (Chen et al,2019a;Din et al,2020;Luo et al,2020;Chen et al,2020b), and that probiotics are less tolerant and adverse reactions and highly safe (Borriello et al,2003;Tan et al,2013), and are expected to be an important means for preventing or assisting in the treatment of UC (Zheng Yuxing, 2020).
The ingestion and colonic enrichment of probiotics can improve intestinal microecological balance, repair intestinal flora disorder caused by inflammation, and further improve colonitis symptoms (Zhou et al 2020 b). In addition, the supplementation of probiotics may also increase the production of beneficial metabolites in the colon. These short chain fatty acids also have a certain therapeutic effect on colonic inflammation. Therefore, the colonic colonization and proliferation of the colonic associated probiotics are expected to become a new approach for preventing and treating colonitis.
Disclosure of Invention
The invention aims to provide probiotic powder and application thereof in relieving colonitis. The probiotic powder has high viable bacteria content, can effectively relieve the symptoms of the colonitis, and can be widely used for preventing and treating the colonitis.
One aspect of the invention relates to a probiotic powder comprising lactobacillus plantarum (Lactobacillus plantarum).
The lactobacillus plantarum is lactobacillus plantarum WW (Lactobacillus plantarum WW) which is preserved in common microorganisms of China Committee for culture Collection of microorganisms in 2021, 12 and 24 days, and the preservation number is CGMCC No.24189.
The live bacteria amount in the probiotic powder is more than 10 9 CFU/g。
The invention relates to a preparation method of the probiotic powder, which comprises the following steps:
(1) Inoculating lactobacillus plantarum seed solution into MRS liquid culture medium at a volume ratio of 2-5%, and culturing at 37 ℃ for 24 hours to obtain fermentation liquor;
(2) Centrifuging lactobacillus plantarum fermentation liquor at 4 ℃ and 5000r/min for 10min, and collecting thalli to obtain lactobacillus plantarum bacterial sludge;
(3) Re-suspending the bacterial mud in a skim milk solution to obtain lactobacillus plantarum bacterial suspension;
(4) Freeze drying the bacterial suspension to obtain the probiotic powder.
The mass volume percentage of the skim milk solution in the step (3) is 10-20%.
The viable bacterial count of the lactobacillus plantarum suspension in the step (3) is 10 8 -10 9 CFU/mL。
The invention also relates to application of the probiotic powder in preparation of medicines for relieving or treating colonitis.
The invention also relates to application of the probiotic powder in preparation of medicines with antioxidant function.
The invention also relates to application of the probiotic powder in preparation of fermented foods or health care products.
Advantageous effects
The probiotic powder provided by the invention has a good relieving effect on the ulcerative colitis of mice induced by DSS, can effectively improve the symptoms of weight loss, colon length shortening, splenomegaly and the like of the colonitis mice, lighten the oxidative damage of colon tissues, regulate and control the levels of inflammatory factors in serum and colon of the mice, improve the intestinal flora disturbance of the colonitis mice caused by inflammation and improve the content of short-chain fatty acid.
Compared with the model (DSS) group, the Myeloperoxidase (MPO), NO and MDA content in colon tissue of mice in the probiotic (l.plantarum WW) group were significantly reduced, while GSH and SOD content was significantly increased. Therefore, the probiotic powder provided by the invention can effectively relieve damage of DSS-induced oxidative stress to the intestinal tract of mice through antioxidation.
The content of pro-inflammatory cytokines TNF-alpha, IL-1 beta and IL-6 in the serum of mice in the group of probiotics (L.plantarum WW) is obviously reduced (p < 0.05), the content of IL-10 is obviously increased (p < 0.05), and the content of pro-inflammatory cytokines TNF-alpha, IL-1 beta, IL-6 and IFN-gamma in colon tissues is obviously reduced (p < 0.05). Therefore, the probiotics powder provided by the invention has obvious anti-inflammatory effect and obvious relieving effect on acute colitis induced by DSS.
The probiotic (l.plantarum WW) group can significantly reduce the abundance of intestinal pathogens Alistipes, odoribacter, helicobacter, parasutterella and mucispirum, increasing the abundance of intestinal probiotics Akkermansia, lachnospiraceae, prevotellaceae _ucg_001, bifidobacterium, desulfovibrio and lactobacillus. In addition, the probiotic bacteria lactobacillus in the intestinal tract of mice of the group of probiotic (l.plantarum WW) were most abundant in each group. Therefore, the probiotic powder provided by the invention can relieve the colonitis symptoms of mice by regulating and controlling intestinal flora.
The total content of short chain fatty acids in the colon content of mice of the probiotic (l.plantarum WW) group was significantly increased, with the content of acetic acid and butyric acid being significantly higher than that of the healthy (CD) group, and the content of propionic acid being substantially comparable to that of the healthy (CD) group. Therefore, the probiotic powder provided by the invention can obviously improve the content of short chain fatty acid in colon of colonitis mice, and further effectively relieve the symptoms of DSS-induced colonitis.
The probiotic powder can be widely used for preventing and treating colonitis and has wide application prospect.
Drawings
FIG. 1 is a diagram showing the morphology of Lactobacillus plantarum WW colonies;
FIG. 2 is a gram of Lactobacillus plantarum WW;
FIG. 3 is a phylogenetic tree of Lactobacillus plantarum WW based on the 16S rDNA gene sequence;
FIG. 4 is a graph showing the bacteriostatic effect of Lactobacillus plantarum WW;
FIG. 5 shows the body weight change (A) and DAI score (B) for each group of mice;
FIG. 6 is a comparison of colon length for each group of mice;
FIG. 7 is a comparison of spleen weights for each group of mice;
FIG. 8 is a graph of colon tissue HE staining of each group of mice; wherein a is a health (CD) group; b is a model (DSS) group; c is a probiotic (L.plantarum WW) group;
FIG. 9 is a comparison of MPO activity in colon tissue of each group of mice;
FIG. 10 is a comparison of colon tissue antioxidant levels for each group of mice;
FIG. 11 is a comparison of TNF- α, IL-1β, IL-6 and IL-10 levels in serum from mice of each group;
FIG. 12 is a comparison of TNF- α, IL-1β, IL-6 and IFN- γ levels in colon tissue of each group of mice;
FIG. 13 is a graph showing a differential intestinal flora analysis of mice in each group based on genus level;
fig. 14 is a comparison of the short chain fatty acid content of colon contents of each group of mice.
Detailed Description
The lactobacillus plantarum WW (Lactobacillus plantarum WW) provided by the invention meets the regulation requirement, can be used as a food raw material source, and has no side effect and excessive risk after long-term administration. The lactobacillus plantarum WW is a newly discovered strain through heterogeneous taxonomy identification. The lactobacillus plantarum WW provided by the invention can effectively relieve the symptoms of ulcerative colitis and has important application value.
The applicant reserves the lactobacillus plantarum WW (Lactobacillus plantarum WW) in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCCNo.24189 in the 12 th month of 2021.
The screening method of the present invention is not limited to the examples, but known screening methods can be used to achieve the screening purpose, and the screening description of the examples is only illustrative of the present invention and is not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The invention is further described in conjunction with the following detailed description.
Example 1 isolation, screening and identification of strains
1. Sample source
The invention screens out a strain W8 with the strongest inhibiting effect on escherichia coli from traditional fermented soybean paste collected in Shenyang city of Liaoning province.
As shown in fig. 1 and 2, the W8 strain is a gram-positive bacterium, facultative anaerobic, spore-free, and motionless; the bacterial colony is smaller and uniform in size, neat in edge, smooth and fine in surface, white or milky white; the thalli are in a short rod shape and are arranged in pairs or chains.
The physiological and biochemical properties of the W8 strain are shown in Table 1.
TABLE 1 physiological and biochemical characteristics
Note that: "+" indicates positive reaction or growth, and "-" indicates negative reaction or no growth.
The similarity of the 16S rDNA of the W8 strain to Lactobacillus plantarum (Lactobacillus plantarum) was found to be highest by NCBI BLAST alignment. Moreover, it can be seen from the phylogenetic tree shown in FIG. 3 that the W8 strain has a recent homology with Lactobacillus plantarum.
In summary, in combination with the colony morphology, physiological and biochemical characteristics and molecular biology identification result of the W8 strain, the applicant determines that the screened W8 strain is a novel lactobacillus plantarum, which is named as lactobacillus plantarum WW (Lactobacillus plantarum WW) and is preserved in China general microbiological culture Collection center (CGMCC) for 12 months and 24 days in 2021, wherein the address is North Chen West Lu No. 1, 3 in the Korean region of Beijing, and the preservation number is CGMCC No.24189.
Example 2 antibacterial Effect experiment of Lactobacillus plantarum WW
1. Preparation of indicator bacteria:
taking five indicator strain freezing tubes of salmonella, escherichia coli, pseudomonas aeruginosa, enterobacter sakazakii and shigella to melt at room temperature.
Lactobacillus plantarum WW and Lactobacillus plantarum ATCC8014 seed solutions were inoculated into MRS medium at an inoculum size of 1% (V/V), respectively, and cultured at 37℃for 24 hours.
Respectively inoculating salmonella, escherichia coli, pseudomonas aeruginosa, enterobacter sakazakii and shigella in an inoculum size of 1% (V/V) to a liquid culture medium, and culturing at a constant temperature of 37 ℃ overnight to obtain a culture solution.
2. Experiment of inhibition zone
Respectively regulating the concentration of the suspension of five indicator bacteria of salmonella, escherichia coli, pseudomonas aeruginosa, enterobacter sakazakii and shigella to 10 by using sterile physiological saline 6 cfu/mL。
100 mu L of the indicating fungus suspension is uniformly coated on the solid culture medium, and the sterile oxford cup is vertically placed into the solid culture medium for punching and then is taken out. 200. Mu.L of Lactobacillus plantarum WW supernatant was added to the well and incubated at 37℃for 48h. And observing whether a transparent ring exists or not, and detecting the diameter of the transparent ring by using a vernier caliper respectively. The specific results are shown in FIG. 4.
As can be seen from fig. 2, the lactobacillus plantarum WW provided by the invention has a strong inhibition effect on salmonella, escherichia coli, pseudomonas aeruginosa, enterobacter sakazakii and shigella, wherein the inhibition effect on salmonella, enterobacter sakazakii and shigella is strongest, and the diameter of a inhibition zone exceeds 18mm.
3. Minimum Inhibitory Concentration (MIC) determination
And determining the minimum inhibitory concentration of the lactobacillus plantarum WW on five indicator bacteria of salmonella, escherichia coli, pseudomonas aeruginosa, enterobacter sakazakii and shigella by adopting a double-ratio dilution method.
Centrifuging lactobacillus plantarum WW fermentation culture solution at 4deg.C and 10000r/min for 10min, removing thallus to obtain supernatant, spin-evaporating the supernatant at 45deg.C, lyophilizing to obtain lyophilized powder, and storing.
After the lactobacillus plantarum WW supernatant freeze-dried powder is dissolved by using sterile water, respectively adding fermentation culture solutions of indicator bacteria, so that the final concentration of the freeze-dried powder reaches 1024 mug/mL, 512 mug/mL, 256 mug/mL, 128 mug/mL, 64 mug/mL, 32 mug/mL, 16 mug/mL, 8 mug/mL and 4 mug/mL, and setting a blank control group as the freeze-dried powder without adding the supernatant, and culturing for 24 hours at 37 ℃.
And respectively measuring OD600 values of indicator bacteria treated by supernatants with different concentrations, observing the growth condition of the strain, and obtaining the concentration of the supernatant which does not grow to the naked eye and is closest to the OD600 value of a blank control group as the Minimum Inhibitory Concentration (MIC). The specific results are shown in Table 2.
TABLE 2 determination of minimum inhibitory concentration of Lactobacillus plantarum WW
As can be seen from the results in Table 2, the minimum inhibitory concentrations of Lactobacillus plantarum WW against Salmonella, escherichia coli, pseudomonas aeruginosa, enterobacter sakazakii, and Shigella were 64. Mu.g/mL, 128. Mu.g/mL, 64. Mu.g/mL, and 32. Mu.g/mL, respectively.
EXAMPLE 3 Lactobacillus plantarum WW tolerance test to the reverse digestive tract Environment
3.1 acid resistance test
(1) Control group: inoculating activated lactobacillus plantarum WW bacterial liquid into liquid MRS culture medium (pH 6.7) according to the proportion of 3% (V/V);
(2) Experimental group: inoculating activated lactobacillus plantarum WW bacterial liquid into liquid MRS culture medium with pH value of 2.0 and 3.0 according to the proportion of 3% (V/V);
after stationary culture for 24 hours at 37 ℃, 10-time gradient dilution is carried out by using sterile physiological saline, 200 mu L of bacteria liquid with proper dilution is respectively taken for mixed bacteria counting operation, each dilution is repeated for 3 times, and after stationary culture for 24 hours at 37 ℃, viable bacteria colony counting is carried out on the flat plate.
The number of viable bacteria measured in the control group was represented by N', the number of viable bacteria measured in the experimental group was represented by N ", and the survival rate of Lactobacillus plantarum WW under acidic conditions was calculated, and the results are shown in Table 3.
The calculation formula is as follows:
survival (%) = lg cfu N "/lg cfu N' ×100%.
TABLE 3 tolerance of Lactobacillus plantarum WW to acidic conditions
From the data in Table 3, the Lactobacillus plantarum WW provided by the invention has strong tolerance to acidic environment, and the survival rate is still as high as 61.5% after 24 hours of treatment under the acidic condition of pH 2.0, so that the effect is remarkable.
3.2 bile salt resistance test
(1) Control group: inoculating activated lactobacillus plantarum WW bacterial liquid into a liquid MRS culture medium (pH 6.7) without bovine bile salt according to the proportion of 3% (V/V);
(2) Experimental group: inoculating activated lactobacillus plantarum WW bacterial liquid into liquid MRS culture medium (pH 6.7) containing 0.3%, 0.5%, 1.0% and 1.5% of ox gall salt according to the proportion of 3% (V/V);
after stationary culture for 24 hours at 37 ℃, 10-time gradient dilution is carried out by using sterile physiological saline, 200 mu L of bacteria liquid with proper dilution is respectively taken for mixed bacteria counting operation, each dilution is repeated for 3 times, and after stationary culture for 24 hours at 37 ℃, viable bacteria colony counting is carried out on the flat plate.
The number of viable bacteria measured in the control group was represented by N', the number of viable bacteria measured in the experimental group was represented by N ", and the survival rate of Lactobacillus plantarum WW was calculated, and the results are shown in Table 4.
Survival (%) = lg cfu N "/lg cfu N' ×100%.
TABLE 4 tolerance of Lactobacillus plantarum WW to bile salts
From the data in Table 4, it is clear that Lactobacillus plantarum WW provided by the invention has strong tolerance to bile salts, and the survival rate is as high as 67.2% after 24 hours of treatment in 1.5% bile salts, thus unexpected technical effects are obtained.
3.3 Artificial gastric juice test
Taking 10mL of lactobacillus plantarum WW fermentation liquor, and centrifuging at 5 ℃ for 10min at 5000g to obtain bacterial sludge; after 3 times of flushing with PBS buffer solution, the bacterial sludge is resuspended in 10mL of artificial gastric juice; digesting for 3h at 37 ℃, and sampling and measuring the number of viable bacteria at 0h and 3h respectively.
The initial viable count in simulated gastric fluid is denoted by N', the viable count in simulated gastric fluid after digestion for 3 hours is denoted by N ", and the survival rate of Lactobacillus plantarum WW in simulated gastric fluid is calculated, and the results are shown in Table 5. The calculation formula is as follows:
survival (%) = lg cfu N "/lg cfu N' ×100%.
TABLE 5 tolerance of Lactobacillus plantarum WW to gastric juice artifacts
From the data in Table 5, the Lactobacillus plantarum WW strain provided by the invention has strong survival ability in artificial gastric juice, the viable bacteria log of the Lactobacillus plantarum WW strain slightly decreases after 3h digestion, the survival rate reaches 94.1%, and the effect is remarkable.
3.4 Artificial intestinal juice test
Taking 10mL of lactobacillus plantarum WW fermentation liquor, and centrifuging at 5 ℃ for 10min at 5000g to obtain bacterial sludge; after 3 times of flushing with PBS buffer solution, the bacterial sludge is resuspended in 10mL of artificial intestinal juice; digesting for 3h at 37 ℃, and sampling and measuring the number of viable bacteria at 2h and 4h respectively.
The initial viable count in the artificial intestinal juice is represented by N', the viable count in the artificial intestinal juice after 2h and 4h digestion is represented by N ", and the survival rate of lactobacillus plantarum WW in the artificial intestinal juice is calculated, and the result is shown in Table 6. The calculation formula is as follows:
survival (%) = lg cfu N "/lg cfu N' ×100%.
TABLE 6 tolerance of Lactobacillus plantarum WW to artificial intestinal juice
From the data in Table 6, it is clear that the Lactobacillus plantarum WW provided by the invention has strong tolerance to artificial intestinal juice, and the survival rate is not only reduced but also improved to 107.9% when the Lactobacillus plantarum WW is digested for 2 hours and 4 hours. Therefore, the strain can realize effective proliferation in artificial intestinal juice and has unexpected technical effects.
The experimental results show that the lactobacillus plantarum WW provided by the invention can effectively resist the influence of gastrointestinal fluid, low-acidity environment and high-bile-salt environment, and keep higher activity.
EXAMPLE 4 antioxidant Property test of Lactobacillus plantarum WW
4.1 protease Activity
The protease activity of the strain is screened and evaluated by adopting an o-phthalaldehyde method, and the o-phthalaldehyde method is a widely accepted protease hydrolysis activity measuring method. The principle is that alpha-amino acid is released through hydrolysis reaction of phthalic dicarboxaldehyde and beta-mercaptoethanol, a strong absorption peak is formed at the ultraviolet wavelength of 340nm, and the proteolytic capability of lactobacillus is reflected through measuring the content of alpha-amino acid.
(1) Reagent preparation
Preparation of a tyrosine solution of 100. Mu.g/mL: the tyrosine is baked to constant weight in a baking oven at 105 ℃, 0.1000g of tyrosine with constant weight is accurately weighed, 6mL of hydrochloric acid with the concentration of 1mol/L is gradually added to dissolve the tyrosine, 0.2mol/L of hydrochloric acid is used for fixing the volume to 100mL, at the moment, the concentration of the tyrosine solution is 1000 mug/mL, 10mL of the solution is absorbed, 0.2mol/L of hydrochloric acid is used for fixing the volume to 100mL, and the tyrosine solution with the concentration of 100 mug/mL is prepared, and the solution is timely used or timely put into a refrigerator for storage after being prepared.
The preparation of the OPA reagent comprises the steps of mixing 62.5mL of 0.1M sodium tetraborate solution, 6.25mL of 20% SDS solution, 100mg of phthalic dicarboxaldehyde, 5mL of methanol solution and 250uL of beta-mercaptoethanol solution, and fixing the volume to 250mL to prepare the OPA reagent, wherein the OPA reagent needs to be prepared at present and stored in a dark place.
(2) Determination of a Standard Curve
The tyrosine solutions (concentration: 100. Mu.g/mL) were measured at 0, 1mL, 2mL, 3mL, 4mL, and 5mL, respectively, and ddH was used 2 Supplementing the solution to 5mL, and uniformly mixing for later use; taking 150uL of tyrosine solution with different concentrations, adding 3mLOPA reagent, gently shaking uniformly, reacting at room temperature for 2min, and measuring OD 340 Values of OD on the abscissa of tyrosine concentration 340 The values are the ordinate to make a standard curve: y=0.0143x+0.0027 (R 2 ＝0.9903)。
(3) Sample measurement
Inoculating activated lactobacillus plantarum WW bacterial liquid into MRS culture medium according to an inoculum size of 3% (V/V), and culturing at 37 ℃ for 24 hours; 1.25mL of the bacterial liquid was taken and 0.3mL of ddH was added 2 O,3mL of 0.75M trichloroacetic acid, and standing at room temperature for 10min after uniform mixing; centrifuging at 4deg.C for 6min at 6000r/min, and collecting supernatant; taking 150 mu L of supernatant, adding 3mL of OPA reagent, gently oscillating, standing at room temperature for 2min for reaction, and measuring the absorbance of the solution at the wavelength of 340 nm; the OD to be obtained 340 The values were compared with a standard curve and protease activity was calculated. Lactobacillus plantarum ATCC8014 was also purchased as a control. The specific results are shown in Table 7.
TABLE 7 protease production by Lactobacillus plantarum
As shown in Table 7, the proteolytic activity of the lactobacillus plantarum WW fermentation supernatant provided by the invention is obviously higher than that of the lactobacillus plantarum ATCC8014 serving as a control bacterium, and the proteolytic activity reaches 215.48 mug/mL.
4.2 superoxide dismutase
The kit adopts a double-antibody one-step sandwich method enzyme-linked immunosorbent assay (ELISA). Adding a sample, a standard substance and an HRP-marked detection antibody into a coated micropore which is pre-coated with a superoxide dismutase (SOD) antibody, and thoroughly washing after incubation. The color is developed with the substrate TMB, which is converted to blue under the catalysis of the peroxidase and to the final yellow under the action of the acid. The color shade and superoxide dismutase (SOD) in the sample are positively correlated. Measuring absorbance (OD value) at 450nm wavelength with enzyme-labeled instrument, and calculating superoxide dismutase activity and OD in lactobacillus plantarum WW fermentation supernatant 450 The values are the ordinate to make a standard curve: y= 135.03X-12.061 (R 2 = 0.9946). Lactobacillus plantarum ATCC8014 was also purchased as a control. The specific results are shown in Table 8.
TABLE 8 TABLE 7 case where Lactobacillus plantarum produces superoxide dismutase
As can be seen from the results in Table 8, the hydrolysis activity of superoxide dismutase (SOD) in the supernatant of the WW fermentation of Lactobacillus plantarum of the invention is significantly higher than that of Lactobacillus plantarum ATCC8014, reaching 187.66. Mu.g/mL.
4.3DPPH radical scavenging test
Lactobacillus plantarum WW and Lactobacillus plantarum ATCC8014 seed solutions of control bacteria were inoculated into liquid MRS medium at an inoculum size of 1% (V/V), and cultured at 37℃for 24 hours, respectively.
Preparing trichloroacetic acid (TCA) solution with the mass concentration of 15%, and mixing lactobacillus plantarum fermentation liquor with TCA solution 1:1, mixing uniformly, standing for 20min at room temperature, centrifuging for 10min at 4 ℃, filtering the supernatant after centrifugation by a water phase membrane of 0.45 mu m, freeze-drying to obtain crude peptide, putting 250mg of crude peptide into 1mL of artificial gastric juice and artificial intestinal juice respectively, performing simulated digestion for 1h and 2h at 37 ℃ and 120r/min, and respectively measuring the clearance rate of the lactobacillus plantarum fermentation liquor after simulated digestion on DPPH free radicals.
Preparation of 1×10 with absolute ethanol -4 Taking 2mL of DPPH solution and 2mL of solution to be tested in a test tube, uniformly mixing by vortex, carrying out light-shielding reaction for 30min at room temperature, and measuring the absorbance of a sample at 517 nm. DPPH radical scavenging was calculated according to the following formula. The specific results are shown in Table 9.
DPPH clearance% = [1- (A) 1 -A 2 )/A 3 ]×100％。
Wherein: a is that 1 Adding an absorbance value of the DPPH solution to the sample; a is that 2 Absorbance values for the sample solution; a is that 3 Absorbance of DPPH solution was added to ethanol.
TABLE 9 scavenging of DPPH free radical by Lactobacillus plantarum WW
As shown in the data of Table 9, the Lactobacillus plantarum WW provided by the invention has strong antioxidant capacity, can effectively remove DPPH free radicals, has a clearance rate of 40.22% -52.94%, and has an improved 21.8% -30.8% compared with the Lactobacillus plantarum ATCC8014, thus achieving unexpected technical effects.
In conclusion, the lactobacillus plantarum WW strain obtained by screening has obvious inhibition effect on common gram-negative pathogenic bacteria such as salmonella, escherichia coli, pseudomonas aeruginosa, enterobacter sakazakii and shigella. The strain can effectively resist the influence of gastrointestinal fluid, low-acidity environment and high-bile-salt environment, and keep higher activity. The hydrolytic activity of protease and superoxide dismutase (SOD) in the fermentation supernatant of lactobacillus plantarum WW strain is obviously higher than that of lactobacillus plantarum ATCC8014, and the scavenging rate of DPPH free radical is high. The strain can be widely applied to the field of food or medicine production and has wide prospect.
Example 5 preparation of probiotic powder
Inoculating lactobacillus plantarum WW seed solution into MRS liquid culture medium at a volume ratio of 2-5% under aseptic condition, and culturing at 37deg.C for 24 hr; after fermentation is completed, centrifuging lactobacillus plantarum WW fermentation liquor for 10min at 4 ℃ under 5000r/min, and collecting thalli to obtain lactobacillus plantarum WW bacterial sludge; resuspending the bacterial mud in 10% -20% skim milk solution to obtain the bacterial strain with the living bacterial load of 10% 8 -10 9 CFU/mL of lactobacillus plantarum WW bacterial suspension; freeze drying the bacterial suspension to obtain the probiotic powder.
The viable count in the probiotic powder is more than 10 9 CFU/g。
Example 6 application of probiotic powder to alleviation of ulcerative colitis in mice
1. Grouping, modeling and administration of animals
Mice were divided into 3 groups of 6 mice each, acclimatized for 1 week, and group modeling and dosing began on day 8. In addition to the healthy group, the other groups had Dextran Sodium Sulfate (DSS) added to the mice' drinking water at 3% (w/v) while the experimental group was gavaged 1 time daily and the mice were sacrificed on day 8 of gavage. The specific grouping and stomach filling doses are as follows:
(1) Health (CD) group: normal drinking water (without DSS), 500 μl of PBS was infused daily;
(2) Model (DSS) group: 100-500 mu L of PBS for stomach irrigation every day;
(3) Probiotic (l.plantarum WW) group: 500 mu L of the gastric lavage probiotic powder solution is filled every day; the preparation method of the probiotic powder solution comprises the following steps: the probiotic powder and sterile PBS solution were mixed at a ratio of 1:100 Mixing the components in the ratio of (v/v).
2. Sample collection
(1) Fecal sample collection
Collecting mouse feces, and storing at-80deg.C.
(2) Serum collection
The abdominal aorta was bled and serum was isolated and stored at-80 ℃.
(3) Colon tissue and content harvesting
Mice were sacrificed after blood collection and colorectal was collected from each group of mice. The anus to ileocecal junction distance was measured and the mouse colorectal was fixed in 10-20% neutral formalin solution.
The other mice took out the colon content, rinsed the colon with sterile physiological saline, blotted dry with filter paper, and transferred the colon content and colon tissue rapidly into liquid nitrogen, and later transferred to-80 ℃ for preservation.
3. Index detection
3.1 weight change and disease Activity index calculation
During the test period, mice were monitored for water consumption and weight changes.
In addition, the fecal state of the mice was observed, and the fecal occult blood condition of the mice was detected.
Hematochezia conditions are classified into five classes: the color is not developed and is negative; secondly, the yellow-green color is the positive (+) of occult blood; thirdly, the blue-green color is positive with occult blood (++); (IV) the dark blue-green color is positive in occult blood (++); fifth, blood is visible to the naked eye, and then hematochezia is visible to the naked eye.
Finally, the disease activity index (Disease activity index, DAI) of the mice was calculated according to the scoring criteria of table 10.
TABLE 10 disease Activity index (Disease activity index, DAI) scoring criteria
3.2 measurement of colon length in mice
The anus to the cecum end whole section of the colon of the mice in different groups were taken, the length was measured, and photographing and recording were performed.
3.3 colon HE staining and tissue injury scoring
Colon paraffin sections were prepared and HE stained, and HE stained sections were scanned using a section scanner and scored for tissue damage. The scoring criteria are shown in Table 11.
TABLE 11 tissue injury scoring Table
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3.4 detection of MPO Activity in colon tissue
Myeloperoxidase (MPO) is one of the markers of oxidative stress, and massive neutrophil influx into tissues after inflammation of the colon results in a significant increase in MPO viability.
10-20% colon tissue homogenate was prepared. The homogenate was used for protein concentration determination according to BCA protein assay kit (soribao) instructions. The homogenate is taken to detect the MPO activity of the colon tissue of the mouse according to the instruction of MPO (Myeloperoxidase) detection kit (Nanjing built).
3.5 detection of antioxidant indicators in colon tissue
Colon tissue homogenate samples were prepared and assayed for NO, MDA, GSH and SOD content according to the kit (built in south kyo) instructions.
3.6ELISA determination of cytokine content in colon and serum
The content of TNF-alpha, IL-1 beta, IL-6 and IL-10 in the serum of the mice is determined according to the instruction of the kit. Taking colon tissue of a mouse at a distance of 1-2cm from cecum, placing the colon tissue into centrifugal tubes, placing 4 zirconium beads into each centrifugal tube, adding pre-cooled sterile PBS (phosphate buffer solution) with a volume of 9 times, placing the mixture into a tissue grinding homogenizer for grinding, centrifuging at 12000g and 4 ℃ for 15min, and collecting supernatant for cytokine detection. The levels of cytokines TNF- α, IL-1β, IL-6 and IFN- γ in the colon of the mice were determined according to the ELISA kit instructions.
3.7 effects of probiotic powder on the intestinal flora diversity and the modulation of the metabolite SCFAs in mice with colon inflammation
(1) Intestinal flora diversity analysis
Colon content samples of each group of mice according to the followingTotal bacterial DNA was extracted from the colon contents using the Soil DNAKit (Omega Bio-Tek, USA) kit instructions. DNA integrity was checked by agarose gel electrophoresis and the DNA concentration of each set of samples was checked using a NanoDrop2000 (Thermo, USA). Then, using the upstream primer 338F: ACTCCTACGGGAGGCAGCA and downstream primer 806R: GGACTACHVGGGTWTCT AAT PCR amplification was performed on the V3 to V4 hypervariable regions of the 16S r RNA genes of each set of DNA samples. PCR products were identified and purified using agarose gel electrophoresis detection and AxyPrep DNAGel Extraction Kit (Axygen, USA), using Quantus TM The Fluorometer (Promega, USA) performs PCR quantification. Finally use->The Rapid DNA-Seq Kit (bio Scientific, USA) and Miseq PE300 platform from Illumina corporation were pooled and sequenced. All of the above procedures, including DNA extraction and sequencing, were delegated to be done by shanghai meg corporation.
(2) Detection of short chain fatty acids in the colon contents of mice
Taking 100-200mg of colon contents of each group of mice, adding 500-1000 mu l of diethyl ether, sufficiently shaking and uniformly mixing, then carrying out ultrasonic treatment for 30min, and detecting the content of short chain fatty acid by using GC-MS through a 0.22 mu m organic filter membrane.
4. Experimental results
The detection results are shown in fig. 4-12:
from fig. 5-7, it can be seen that the symptoms of weight loss, injury to colon tissue, reduced colon length, and splenomegaly were significantly alleviated in the group of colitis mice with the gastric lavage probiotic powder (l.plan WW) compared to the model (DSS) group.
As shown in fig. 8, the colonic mucosa of healthy (CD) mice were intact and continuous, the glands were aligned, the crypts were normal, and no inflammatory cell infiltration was observed. The colon injury degree of the mice in the model (DSS) group is serious, bleeding and deep ulcers are visible, obvious acute inflammatory reaction is formed, and a large amount of inflammatory cells infiltrate the mucous membrane and submucosa. The colon of the probiotic (l.plantarum WW) group was least damaged.
As shown in FIG. 9, myeloperoxidase (MPO) levels were different in colon tissues of different groups of mice. Wherein the MPO value is highest in the model (DSS) group and is significantly higher than in the healthy (CD) group, and the MPO value is significantly lower in the probiotic (L.plantarum WW) group than in the model (DSS) group. Thus, the symptoms of the colonitis of the mice are obviously relieved by the gastric lavage probiotic powder.
As shown in fig. 10, the levels of oxidative stress products NO and MDA in colon tissues were significantly increased (p < 0.05) in mice in the model (DSS) group compared to the healthy (CD) group, and the levels of GSH and SOD for antioxidant effect were significantly reduced, indicating that the level of oxidative stress in DSS-induced acute colitis mice was significantly increased. Compared to the model (DSS) group, the NO and MDA content in the colon tissue of the probiotic (l.plantarum WW) group was significantly reduced, while the GSH and SOD content was significantly increased. Therefore, the probiotic powder provided by the invention can effectively relieve damage of DSS-induced oxidative stress to the intestinal tract of mice through antioxidation.
As shown in FIG. 11, the proinflammatory cytokines TNF- α, IL-1β, IL-6 were highest in the serum of mice in the group of model (DSS) groups, with the interleukin IL-10 content being lowest. Compared with the model (DSS) group, the content of TNF-alpha, IL-1 beta and IL-6 in the serum of mice in the probiotic (L.plantarum WW) group is obviously reduced (p < 0.05), and the content of IL-10 is obviously improved (p < 0.05). Therefore, the probiotics powder provided by the invention has obvious anti-inflammatory effect and obvious relieving effect on acute colitis induced by DSS.
As shown in FIG. 12, the proinflammatory cytokines TNF- α, IL-1β, IL-6 and IFN- γ were highest in colon tissue of mice in the model (DSS) group in each group. Compared to the model (DSS) group, the levels of TNF- α, IL-1β, IL-6 and IFN- γ in colon tissue were significantly reduced in mice from the probiotic (l.plantarum WW) group (p < 0.05). Therefore, the probiotics powder provided by the invention has obvious anti-inflammatory effect and obvious relieving effect on acute colitis induced by DSS.
As shown in fig. 13, the abundance of pathogenic bacteria Alistipes, odoribacter and Helicobacter in the gut was significantly increased (p < 0.05) in the mice in the model (DSS) group compared to the healthy (CD) group, while the abundance of pathogenic bacteria parisutterella and mucispirum associated with Inflammatory Bowel Disease (IBD) was significantly increased (p < 0.05) and the abundance of pathogenic bacteria romidoutsia was slightly increased (p > 0.05) in the model (DSS) group; while the intestinal probiotics Akkermansia, prevotellaceae _ucg001, bifidobacterium, desulfovibrio and lactobacillus decreased in abundance, indicating that the intestinal flora of mice in model (DSS) group had been disturbed. Compared with the model (DSS) group, the probiotic bacteria (L.plantarum WW) group can remarkably reduce the abundance of intestinal pathogenic bacteria Alistipes, odoribacter, helicobacter, parasutterella and Mucispirum, and increase the abundance of intestinal probiotic bacteria Akkermansia, lachnospiraceae, prevotellaceae _UCG_001, bifidobacterium, desulfovibrio and Lactobacillus. In addition, the probiotic bacteria lactobacillus in the intestinal tract of mice of the group of probiotic (l.plantarum WW) were most abundant in each group. Therefore, the probiotic powder provided by the invention can relieve the colonitis symptoms of mice by regulating and controlling intestinal flora.
As shown in fig. 14, the total short chain fatty acid content in the colon content was significantly reduced (p < 0.05) in the mice in the model (DSS) group compared to the healthy (CD) group; whereas the total content of short chain fatty acids in the colon content of mice in the probiotic (l.plantarum WW) group was significantly increased, with the content of acetic acid and butyric acid being significantly higher than in the healthy (CD) group, and the content of propionic acid being substantially comparable to that of the healthy (CD) group. Therefore, the probiotic powder provided by the invention can obviously improve the content of short chain fatty acid in colon of colonitis mice, and further effectively relieve the symptoms of DSS-induced colonitis.
In conclusion, the probiotic powder provided by the invention has a remarkable anti-inflammatory effect, can effectively improve the abundance of beneficial bacteria and the content of medium and short chain fatty acids in the intestinal tract of a mouse, further remarkably relieves the symptoms of ulcerative colitis of the mouse, relieves intestinal injury, and can be widely used for preventing and treating enteritis.
Claims (8)
1. A probiotic powder is characterized in that the probiotic powder comprises lactobacillus plantarumLactobacillus plantarum) WW; the lactobacillus plantarum WW is preserved in China general microbiological culture Collection center (CGMCC) for 24 days in 12 months of 2021, and the preservation number is CGMCC No.24189.
2. The probiotic powder of claim 1, wherein the live bacteria content of the probiotic powder is more than 10 9 CFU/g。
3. The probiotic powder according to claim 1 or 2, characterized in that the preparation method of the probiotic powder comprises the following steps:
(1) Inoculating lactobacillus plantarum WW seed solution into MRS liquid culture medium at a volume ratio of 2-5%, and culturing at 37 ℃ for 24h to obtain fermentation liquor;
(2) Centrifuging lactobacillus plantarum WW fermentation liquor at 4 ℃ and 5000r/min for 10min, and collecting thalli to obtain lactobacillus plantarum bacterial sludge;
(3) Re-suspending the bacterial sludge in a skim milk solution to obtain lactobacillus plantarum WW bacterial suspension;
(4) Freeze drying the bacterial suspension to obtain the probiotic powder.
4. A probiotic powder according to claim 3, characterized in that the mass volume percentage of the skim milk solution in step (3) is 10% -20%.
5. The probiotic powder of claim 4, wherein the live bacterial load of the lactobacillus plantarum bacterial suspension of step (3) is 10 8 -10 9 CFU/mL。
6. Use of a probiotic powder according to any one of claims 1 to 5 for the preparation of a medicament for the alleviation or treatment of colitis.
7. Use of the probiotic powder of any one of claims 1-5 for the preparation of a medicament having an antioxidant function.
8. Use of the probiotic powder of any one of claims 1-5 for the preparation of a fermented food or a health product.
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| CN114231443A (en) * | 2021-11-29 | 2022-03-25 | 大连工业大学 | Lactobacillus plantarum complex bacteria and application thereof in preparation of complex probiotics for relieving intestinal inflammation or treating ulcerative colitis |
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