CN113564095B - Application of exogenous nucleotide in promoting lactobacillus casei to antagonize salmonella enterica - Google Patents
Application of exogenous nucleotide in promoting lactobacillus casei to antagonize salmonella enterica Download PDFInfo
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- 125000003729 nucleotide group Chemical group 0.000 title claims abstract description 62
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Abstract
The invention discloses application of exogenous nucleotide in promoting lactobacillus casei to antagonize salmonella enterica, and belongs to the technical field of microorganisms. The invention verifies that 5 '-cytidine monophosphate (5' -CMP), 5 '-adenosine monophosphate (5' -AMP) and 5 '-disodium uridylate (5' -UMPNa) are used for the growth test, the signal molecule generation test, the mobility test, the clustering test and the capability test of the eosinophil to generate intestinal pathogenic bacteria by the Lactobacillus casei antagonism 2 ) And 5 '-guanylate disodium (5' -GMPNa) 2 ) Promote the growth of lactobacillus casei and inhibit the growth of salmonella enterica. The invention provides a theoretical basis for exploring the use of exogenous nucleotide for promoting lactobacillus casei to antagonize enteropathogenic bacteria and exogenous nucleotide as a potential prebiotic.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to application of exogenous nucleotide in promoting lactobacillus casei to antagonize salmonella enterica.
Background
The intestinal tract is the largest immune organ and the largest micro-ecosystem of the human body. 500 or more than ten trillion different bacteria inhabit in the intestinal tract of a normal human body, and are divided into two major types of beneficial bacteria and harmful bacteria, and the two types of bacteria are coordinated and mutually restrained to form a complex and stable microecological environment. However, due to the change of certain physiological conditions of human body, food, pathogenic bacteria infection, use of antibiotics and the like can cause intestinal flora disorder, a series of symptoms such as diarrhea, constipation, gastroenteritis and the like can occur, the health of the human body is affected, and probiotics are required to be supplemented at the moment. Probiotics inhibit the growth and proliferation of harmful flora by competing with the nutrient and oxygen, and the production of intestinal putrefying substances. However, probiotics are easy to inactivate, so finding a substance which can promote the activity of the probiotics and can promote the probiotics to antagonize enteropathogenic bacteria is a problem to be solved urgently.
The nucleotide is an extremely important low-molecular compound in organisms and has the functions of promoting growth and development, regulating lipid metabolism and the like. It has been found that many tissues of mammals which are actively metabolized, such as intestinal, lymphoid, myeloid cells, etc., have a lack or limited ability to synthesize nucleotides, and that the presence of nucleotides synthesized in vivo does not meet the growth requirements of these important tissues, thus requiring the supplementation of exogenous dietary nucleotides. The nucleotide has high safety, and is added into infant formula milk powder at present.
Disclosure of Invention
The present invention addresses the above problems by providing the use of exogenous nucleotides to promote antagonism of salmonella casei against salmonella enterica. By studying 4 exogenous nucleotides, namely cytidine 5 '-monophosphate (5' -CMP), adenosine 5 '-monophosphate (5' -AMP), disodium 5 '-uridylate (5' -UMPNa) 2 ) And 5 '-guanylate disodium (5' -GMPNa) 2 ) The application of the exogenous nucleotide in promoting the antagonism of lactobacillus casei to enteropathogenic bacteria (enterosalmonella) is found to promote the growth and proliferation of lactobacillus casei and promote the growth, signal molecule generation, mobility, clustering and ferrite generation capacity of lactobacillus casei to antagonize enteropathogenic bacteria, so that the exogenous nucleotide is knownThe acid has potential as a novel prebiotic and a high quality nitrogen source and can play a great role in promoting the growth of probiotics and regulating intestinal flora.
The invention aims to achieve the aim, and the aim is achieved by the following technical scheme:
the application of exogenous nucleotide in promoting Lactobacillus casei antagonism to intestinal pathogenic bacteria.
Further, the enteropathogenic bacteria include salmonella enterica.
Further, the exogenous nucleotide includes cytidine 5 '-monophosphate (5' -CMP), adenosine 5 '-monophosphate (5' -AMP), disodium 5 '-uridylate (5' -UMPNa) 2 ) And 5 '-guanylate disodium (5' -GMPNa) 2 ) One or two or more of them.
Further, the exogenous nucleotide is composed of cytidine 5 '-monophosphate (5' -CMP), adenosine 5 '-monophosphate (5' -AMP), disodium 5 '-uridylate (5' -UMPNa) 2 ) And 5 '-guanylate disodium (5' -GMPNa) 2 ) Composition is prepared.
Further, in the exogenous nucleotide, cytidine 5 '-monophosphate (5' -CMP), adenosine 5 '-monophosphate (5' -AMP), disodium 5 '-uridylate (5' -UMPNa) 2 ) And 5 '-guanylate disodium (5' -GMPNa) 2 ) The molar ratio of (1-5) to (1-5).
Further, the application is verified by a lactobacillus casei antagonism enteropathogenic bacteria growth test, a signal molecule generation test, a mobility test, a clustering test and a ferrite production capability test.
Further, the specific process of the lactobacillus casei antagonizing enteropathogenic bacteria growth test is to prepare a co-culture system of lactobacillus casei and salmonella enterica, add exogenous nucleotide, measure the quantity of lactobacillus casei and salmonella enterica by using a dilution plate counting method, and draw a bacterial growth curve.
Further, the concentration of the exogenous nucleotide in the co-culture system is 1.0g/100 mL-5.0 g/100mL.
Further, the specific process of the signal molecule generation test is to detect the change condition of the AI-2 type signal molecule in a co-culture system made of lactobacillus casei and salmonella enterica through vibrio harveyi Vibrio harveyi BB.
Further, the swimming performance test and the clustering test are specifically to inoculate intestinal pathogenic bacteria in a swimming performance culture medium and a clustering culture medium respectively, and measure the diffusion distance of the intestinal pathogenic bacteria after putting the swimming performance and the clustering flat plate in an incubator for culturing for 12-48 hours.
Further, the test of the production capacity of the mesophilic bacteria comprises the steps of adding lactobacillus casei extract or lactobacillus casei crude extract after exogenous nucleotide culture into intestinal pathogenic bacteria bacterial liquid, centrifuging the bacterial liquid after overnight culture to obtain supernatant, taking 50-200 mu L of the supernatant, adding the supernatant into a CASAD mesophilic bacteria plate, and measuring the secretion condition of the mesophilic bacteria of the bacterial strain.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention is carried out by studying 4 exogenous nucleotides, namely, cytidine 5 '-monophosphate (5' -CMP), adenosine 5 '-monophosphate (5' -AMP), disodium 5 '-uridylate (5' -UMPNa) 2 ) And 5 '-guanylate disodium (5' -GMPNa) 2 ) In promoting Lactobacillus casei antagonizing enteropathogenic bacteria (Salmonella enterica), exogenous nucleotide is found to promote Lactobacillus casei growth and proliferation, and promote Lactobacillus casei antagonizing Salmonella enterica growth, signal molecule production, mobility, clustering and ferrite production, and antagonism is mainly caused by the phenotype of Salmonella enterica population system regulation. Thus, exogenous nucleotides have the potential to act as novel prebiotics and superior nitrogen sources, and will play a tremendous role in promoting the growth of probiotics and regulating intestinal flora.
(2) The exogenous nucleotide has important effects of improving the activity of probiotics, promoting the probiotics to antagonize intestinal pathogenic bacteria and regulating intestinal flora. Because the exogenous nucleotide has very reliable safety, the normal people and special people can reasonably use the exogenous nucleotide. In addition, the exogenous nucleotide has excellent physiological function, so that the intake of the exogenous nucleotide is purposefully improved in daily diet, the content of beneficial bacteria in intestinal tracts can be stably proliferated, and the intestinal flora can keep a good microecological balance. The effects of disease prevention and health care without disease are achieved. The invention provides reference for the production of various products such as medicines, health products and the like by utilizing exogenous nucleotide in future development.
Drawings
FIG. 1 is the effect of exogenous nucleotides on the growth curve of Lactobacillus casei and Salmonella enterica co-culture.
FIG. 2 is the effect of exogenous nucleotides on AI-2 signaling molecules during co-cultivation of Lactobacillus casei and Salmonella enterica.
FIG. 3 is the effect of exogenous nucleotides on antagonizing Salmonella enterica clustering and mobility of crude extracts of Lactobacillus casei.
FIG. 4 is the effect of exogenous nucleotides on the secretion of ferrite after Lactobacillus casei and Salmonella culture.
The specific embodiment is as follows:
the invention is further illustrated below in connection with specific examples which should be construed as merely illustrative, and not a limitation of the scope of the invention in any way whatsoever. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples are commercially available unless otherwise specified.
Example 1: effect of exogenous nucleotide on growth curves of Lactobacillus casei and Salmonella enterica Co-culture Process
Lactobacillus casei is inoculated into MRS liquid culture medium, and is cultured for 24 hours at 37 ℃ in a shaking way to activate the strain. V. harveyi BB170 was inoculated into AB broth, shake cultured overnight at 30 ℃ for activation, salmonella enterica was inoculated into LB broth, and shake cultured at 28 ℃. And (5) continuously subculturing each activated strain for two generations to obtain the final product for subsequent experiments.
Lactobacillus casei and salmonella enterica are shake cultured to the same concentration, lactobacillus casei is centrifuged for 10min at 4 ℃ and 10,000r/min, the supernatant is discarded, and the suspension is prepared into heavy suspension by using the same amount of sterile water after being washed for 3 times. Salmonella enterica and fresh LB broth were mixed at a volume of 1:100 (v/v) and 0.5mL of Lactobacillus casei heavy suspension was added to make two typesCo-culture system of strains, 4.0g/100mL of exogenous nucleotide mixture (5 ' -CMP, 5' -AMP, 5' -UMPNa) was added 2 And 5' -GMPNa 2 Molar ratio of 1:1:1:1), culture system without exogenous nucleotide mixture was used as control group. Static culture was carried out at 28℃for 24 hours, 3 replicates per group, and Lactobacillus casei and Salmonella enterica under the same culture conditions were used as control groups.
The amounts of lactobacillus casei and salmonella enterica were determined by dilution plate counting. Wherein, the total number of the two bacteria is measured by adopting a plate counting agar culture medium in the co-culture system, and the number of the lactic acid bacteria is counted by using a lactic acid bacteria selective culture medium for the lactic acid bacteria. The plates were placed in a 37℃incubator for 24 hours, the number of strains in each culture system was measured every 4 hours and bacterial growth curves were drawn.
The effect of exogenous nucleotides on lactobacillus casei and salmonella enterica during co-cultivation is shown in figure 1, and it can be seen that the growth curves of both lactobacillus casei and salmonella enterica are typical "S" type curves. After co-cultivation, the number of cells of lactobacillus casei is obviously increased, which indicates that the growth of lactobacillus casei is not affected after co-cultivation, and the number of salmonella is opposite to that, and the tendency of obvious decline is shown. It is presumed that the possible cause is that Lactobacillus casei secretes an inhibiting factor detrimental to the growth of Salmonella enterica when co-cultured.
Example 2: effect of exogenous nucleotide on AI-2 Signal molecule during Co-culture of Lactobacillus casei and Salmonella enterica
Most gram-positive strains possess both intraspecies and interspecies quorum sensing systems, and the signal molecule AI-2 of the interspecies quorum sensing system is a common language for bacterial interspecies communication. The detection of the signal molecule by the biological luminescence method of the harveyi BB170 is simple in operation, wide in application range and high in detection sensitivity, and is the most commonly used method for detecting the activity of the signal molecule AI-2.
Salmonella enterica and fresh LB broth were mixed in a volume of 1:100 (v/v), and then 0.5mL of Lactobacillus casei heavy suspension was added to prepare a co-culture system of the two strains, and 4.0g/100mL of an exogenous nucleotide mixture (5 ' -CMP, 5' -AMP, 5' -UMPNa) was added 2 And 5' -GMPNa 2 The molar ratio of (1:1:1:1), the culture system without adding the exogenous nucleotide mixture is used as a control, the culture system is subjected to stationary culture at 28 ℃ for 24 hours, culture solutions of the culture system are respectively taken at 0, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours and 24 hours, lactobacillus casei and salmonella enterica are obtained by separation, the culture medium is inoculated, the shaking culture is continued at 30 ℃ for 2 hours, and the supernatant to be tested is obtained after centrifugation. And lactobacillus casei and salmonella enterica under the same culture conditions were used as blank controls.
V. harveyi BB170 was inoculated in AB medium and shake-cultured overnight at 30℃to OD 600 1, diluting the culture solution into a fresh AB culture medium according to a ratio of 1:5000 (v/v), and shaking and uniformly mixing to obtain a diluted bacterial solution.
Mixing the obtained supernatant to be tested with the V.harveyi BB170 diluted bacterial liquid according to the ratio of 10:90 (v/v), taking 100 microliters to a 96-well plate, carrying out light shaking culture at 30 ℃ for 2.5 hours, selecting an OPS mode by a fluorescence microplate reader, and detecting a bioluminescence result by the aid of the microplate reader. Wherein Vibrio harveyi BB152 (AI-2) + ) The positive control was made with AB medium, the negative control was made with medium control in AU for each system. The activity of the signal molecule AI-2 is expressed by relative fluorescence intensity, and the calculation formula is as follows, based on the time point when the fluorescence intensity value of the negative control group reaches the minimum value:
relative fluorescence intensity of sample to be measured = fluorescence intensity value of sample to be measured/medium contrast fluorescence intensity value
Positive control relative fluorescence intensity value = positive control fluorescence intensity value/negative control fluorescence intensity value
As shown in FIG. 2, both strains co-cultured were able to induce luminescence of V.harveyi BB170, indicating that both strains can produce AI-2 signaling molecules. The AI-2 signal molecule of Lactobacillus casei starts to increase with the extension of the culture time, and slowly decreases after the middle and late period of the index reaches the maximum. After co-culture with Salmonella enterica, the content of AI-2 produced by Lactobacillus casei is increased under the action of exogenous nucleotide, which indicates that exogenous nucleotide can stimulate QS system of Lactobacillus casei, thereby promoting secretion of signal molecule. Instead, the yield of Salmonella enterica signal molecules decreased, indicating that Lactobacillus casei or its secreted metabolites inhibited Salmonella enterica growth after co-cultivation, resulting in decreased AI-2 yield.
Example 3: effect of exogenous nucleotide on Lactobacillus casei crude extract on antagonizing Salmonella enterica aggregation and mobility
Preparing a swimming culture medium: 1g tryptone, 0.5g sodium chloride, 0.3g agar and 100mL distilled water.
Preparing a clustering medium: 1g peptone, 0.5g sodium chloride, 0.3g agar, 0.5g D-fructose and 100mL distilled water. The culture medium is sterilized at 121deg.C for 15min, cooled to 45deg.C, and poured into flat plate.
Respectively inoculating Salmonella enterica 15. Mu.L with exogenous nucleotide (5 ' -CMP, 5' -AMP, 5' -UMPNa) and 4 parts of swimming culture medium plate and 4 parts of clustered culture medium plate 2 And 5' -GMPNa 2 Salmonella enterica, salmonella enterica added with crude Lactobacillus casei extract, and exogenous nucleotide (5 ' -CMP, 5' -AMP, 5' -UMPNa) added at a molar ratio of 1:1:1:1) 2 And 5' -GMPNa 2 Salmonella enterica bacteria of the crude Lactobacillus casei extract cultured at a molar ratio of 1:1:1:1) were centered on plates, labeled a, b, c and d, respectively, and plates of the colonic medium were labeled e, f, g and h, respectively. The strain diffusion distance was measured after placing the mobility and clustering plates in an incubator for 24 hours of culture.
The specific results are shown in FIG. 3, wherein (a, e) is Salmonella enterica without addition of exogenous nucleotide; (b, f) Salmonella enterica bacteria added with exogenous nucleotide; (c, g) salmonella enterica to which a crude lactobacillus casei extract has been added; (d, h) Salmonella enterica added with exogenous nucleotide cultured Lactobacillus casei crude extract. As can be seen from the figure, the crude extract of Lactobacillus casei has an inhibitory effect on the clustering (a-d) and mobility (e-h) of Salmonella enterica, and the size of colonies formed on the clustering and mobility plates is significantly reduced after the crude extract of Lactobacillus casei is added, indicating that the Lactobacillus casei secreted metabolites have the ability to inhibit movement of Salmonella enterica. When exogenous nucleotide is added into the culture medium of lactobacillus casei, the inhibition capability of the crude extract to salmonella enterica is increased, which indicates that the exogenous nucleotide promotes the generation of substances of lactobacillus casei antagonizing the motility of salmonella enterica.
Example 4: effect of exogenous nucleotide on ferrite secretion after Co-culture of Lactobacillus casei and Salmonella
CASAD siderophore plate configuration: solution a:0.07g of chrome azure S with 50mL of dH 2 O was dissolved, and 10mL of FeCl containing 10mmol/L hydrochloric acid at 1mmol/L was added 3 Stirring uniformly for later use; solution B: 0.06g of HDTMA was treated with 40mL of dH 2 O is dissolved for standby; slowly adding the solution A into the solution B, and uniformly mixing to obtain a solution C. Solution C was sterilized at 121℃for 15min. Subsequently, 0.2mL of 1mmol/L CaCl was added 2 0.2mL of 1mmol/L MgSO 4 ·7H 2 O, 2g of agar, 6mL of 10% (w/v) acid hydrolyzed casein solution (sterilized alone at 121 ℃) were mixed, and then pH was adjusted to 6.8-7.0 with Pipes buffer, and the volume was set to 100mL. The culture was sterilized at 121℃for 15min.
When the temperature of the culture medium is reduced to about 50 ℃, 10mL of solution C is added, the mixture is uniformly mixed, a plate (the diameter of the plate is 90 mm) is poured, and the oxford cup is perforated. Salmonella enterica is inoculated in fresh LB broth culture medium according to 1% inoculum size (v/v), lactobacillus casei crude extract and lactobacillus casei crude extract after 4.0g/100mL exogenous nucleotide culture are respectively added into an salmonella enterica culture system, and after standing overnight at 37 ℃, bacterial liquid is centrifuged to obtain supernatant, and culture supernatant is prepared. To each well of the solidified ferrite-plate medium, 100. Mu.L of Lactobacillus casei supernatant, salmonella enterica supernatant with crude Lactobacillus casei extract after exogenous nucleotide culture, and blank were added. The plates were placed in an incubator for 24h to determine the size of yellow halo in the siderophore plates.
The results are shown in FIG. 4, wherein (a) is Lactobacillus casei supernatant; (b) salmonella enterica supernatant; (c) Adding salmonella enterica supernatant of lactobacillus casei crude extract; (d) Salmonella enterica supernatant to which the crude Lactobacillus casei extract after exogenous nucleotide culture has been added; (e) is a blank.
As can be seen from the figure, due to HDTMA, CAS and Fe 3+ The presence of the ternary complex, the siderophore detection plate appeared blue. When bacteria produce ferrite, a yellowish region occurs in the blue plate due to the ferrite vs. Fe 3+ Has high affinity. After injection of the bacterial culture supernatant of salmonella enterica in the siderophore plate, a yellowish zone appeared, indicating that salmonella enterica was able to secrete siderophores, whereas lactobacillus casei bacteria did not. When Salmonella enterica is added to the crude extract of Lactobacillus casei, the yellowish area is reduced, indicating that the crude extract of Lactobacillus casei inhibits the ability of Salmonella enterica to secrete ferriphilic bacteria. When the coarse extract of the lactobacillus casei after the exogenous nucleotide is added for culture, the content of the ferrite generated by the salmonella enterica is obviously reduced, which indicates that the exogenous nucleotide can promote the lactobacillus casei to generate substances for inhibiting the induction of the salmonella enterica group.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (6)
1. Application of exogenous nucleotide in preparing medicine for promoting lactobacillus casei antagonizing intestinal pathogenic bacteria;
the enteropathogenic bacteria are salmonella enterica causing food poisoning;
the exogenous nucleotide consists of 5 'cytidine monophosphate, 5' adenosine monophosphate, 5 'disodium uridylate and 5' disodium guanylate, and the molar ratio of the components is 1:1:1:1.
2. the use according to claim 1, characterized in that said use is verified by a lactobacillus casei antagonizing enteropathogenic bacteria growth test, a signal molecule production test, a mobility test, a clustering test and a siderophilic production test.
3. The use according to claim 2, wherein the lactobacillus casei antagonizing the enteropathogenic bacteria is performed by preparing lactobacillus casei and salmonella enterica into a co-culture system, adding exogenous nucleotides, measuring the quantity of lactobacillus casei and salmonella enterica by a dilution plate count method, and plotting a bacterial growth curve.
4. The use according to claim 2, wherein the signal molecule is produced by the process of Vibrio harveyiVibrio harveyiBB170 detects changes in AI-2 type signal molecules in co-culture systems made of Lactobacillus casei and Salmonella enterica.
5. The use according to claim 2, wherein the swimming test and the colonization test are specifically to inoculate the enteropathogenic bacteria in a swimming medium and a colonization medium, respectively, and to measure the diffusion distance of the enteropathogenic bacteria after placing the swimming and colonization plates in an incubator for culturing 12-48 h.
6. The use according to claim 2, wherein the test of the production capacity of the mesophilic bacteria is characterized in that lactobacillus casei extract or lactobacillus casei crude extract after being added with exogenous nucleotide culture is added into intestinal pathogenic bacteria bacterial liquid, after being cultured overnight, the bacterial liquid is centrifuged to obtain supernatant, 50-200 mu L of the supernatant is added into a CASAD mesophilic bacteria plate, and the secretion situation of the mesophilic bacteria is measured.
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