CN110089666B - Lactobacillus plantarum and extracellular polysaccharide thereof - Google Patents
Lactobacillus plantarum and extracellular polysaccharide thereof Download PDFInfo
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- CN110089666B CN110089666B CN201910406712.6A CN201910406712A CN110089666B CN 110089666 B CN110089666 B CN 110089666B CN 201910406712 A CN201910406712 A CN 201910406712A CN 110089666 B CN110089666 B CN 110089666B
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- lactobacillus plantarum
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- metal lead
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- 235000013965 Lactobacillus plantarum Nutrition 0.000 title claims abstract description 24
- 229940072205 lactobacillus plantarum Drugs 0.000 title claims abstract description 24
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- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 22
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 22
- 229910001385 heavy metal Inorganic materials 0.000 claims abstract description 38
- 241000186660 Lactobacillus Species 0.000 claims abstract description 32
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 2
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/27—Removal of unwanted matter, e.g. deodorisation or detoxification by chemical treatment, by adsorption or by absorption
- A23L5/273—Removal of unwanted matter, e.g. deodorisation or detoxification by chemical treatment, by adsorption or by absorption using adsorption or absorption agents, resins, synthetic polymers, or ion exchangers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
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Abstract
The invention discloses lactobacillus plantarum and extracellular polysaccharide thereof, which are classified and named as Lactobacillus plantarum, have the strain number of Lactobacillus plantarum-2 and are preserved in China general microbiological culture collection center (CGMCC) No.17566, and the preservation date is 2019, 4 months and 12 days. The lactobacillus plantarum and the extracellular polysaccharide thereof provided by the invention have obvious effect of adsorbing heavy metal lead, and on one hand, the Lactobacillus plantarum18-2 strain with heavy metal adsorption effect can be used as a starter in food production to prevent accumulation of heavy metal lead; on the other hand, EPS produced by the Lactobacillus plantarum-2 strain has excellent effect of adsorbing heavy metal lead, can be used as a heavy metal lead adsorption antidote to be added singly or in a compounding way, prevents pollution and accumulation of the heavy metal lead, and ensures food safety.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to lactobacillus plantarum and extracellular polysaccharide thereof.
Background
Along with the promotion of industrialization progress, heavy metal pollution is more and more serious, and heavy metal pollution not only causes great economic loss, but also endangers human health, lead (Pb) is widely distributed in the environment as one of heavy metals with strongest toxicity to human bodies published by the joint national grain and agriculture organization (FAO), is easy to be absorbed and enriched by organisms, acts on human beings through an accumulation and amplification mechanism of a food chain, and further endangers human health.
Microorganisms in the environment cannot effectively degrade heavy metals and the heavy metals cannot be decomposed through chemical reactions, so that the toxicity of the heavy metals cannot be directly eliminated, and the toxic action of the heavy metals can be relieved only by changing the existing form of the heavy metals or removing the heavy metals from foods or human bodies. The main methods for reducing the heavy metal pollution hazard at the present stage are a physical method, a chemical method and a biological method. Physical means mainly include membrane separation method, active carbon adsorption method, etc., and chemical means mainly include precipitation method, electrolytic method, etc. Compared with the traditional physical and chemical methods, the microbial adsorbent has the advantages of rich strains, low cost, no secondary pollution and the like.
In the ecosystem, the main routes of heavy metal pollution are water and food. Aquatic animals, such as fish, are an effective source of animal proteins and thus one of the major sources of heavy metal contamination in humans. The pollution sources of heavy metals in food processing production mainly comprise polluted food raw materials, food additives, food packaging materials, heavy metal pollution of food during processing, storage and transportation, and the like. Lead is considered as a human carcinogen, and the respiratory and digestive systems are important pathways for lead contamination in humans, and exposure of humans to certain levels of lead can damage the nervous system, hematopoietic system, and cardiovascular system, and even lead to memory and mental decline. In the face of such serious health problems and food safety problems, the conventional control method is poor in effect and unsuitable for application in the food industry. Therefore, research into safe and effective food-grade control agents that adsorb heavy metals has been urgent.
Lactic acid bacteria are ubiquitous in the gastrointestinal tract, food and environment of animals and humans, are pathogen free, some have desirable health care effects and important physiological functions, and are considered safe (Generally regarded as safe, GRAS) food-grade microorganisms. Lactic acid bacteria are reported to have a certain adsorption effect on lead (Pb) and to protect the intestinal barrier of the host. Lactic acid bacteria Exopolysaccharide (EPS) is a type of saccharide compound secreted outside the cell wall and permeated into the culture medium during the growth and metabolism of lactic acid bacteria. The lactobacillus EPS can increase the viscosity, stability, emulsifying property and water retention of food when being added into the food, and can improve the mouthfeel, texture and flavor of the food. EPS has been reported to have an effect of anti-tumor, antioxidant, immune function regulating and the like which are significant for human health. However, the existing lactobacillus has very limited exopolysaccharide secretion, so that the adsorption effect of the lactobacillus heavy metal is not very good, and therefore, a biological adsorbent with good adsorption effect and sustainable development of the heavy metal needs to be developed.
Disclosure of Invention
The invention aims at the technical problems and provides lactobacillus plantarum and extracellular polysaccharide thereof. On the one hand, the Lactobacillus plantarum18-2 strain (CGMCC No. 17566) with heavy metal adsorption effect can be used as a starter in food production to prevent accumulation of heavy metal lead; on the other hand, EPS produced by the Lactobacillus plantarum-2 strain has excellent effect of adsorbing heavy metal lead, can be used as a heavy metal lead adsorption antidote to be added singly or in a compounding way, prevents pollution and accumulation of the heavy metal lead, and ensures food safety.
In order to achieve the above object, the present invention provides the following technical solutions:
the lactobacillus plantarum provided by the invention is classified and named as Lactobacillus plantarum, has a strain number of Lactobacillus plantarum-2, is preserved in China general microbiological culture collection center, has a preservation number of CGMCC No.17078, and has a preservation date of 2019, 4 and 23.
The Lactobacillus plantarum-2 is prepared by asepsis sampling from traditional dairy product milk bean curd of inner Mongolia, and diluting with 0.5% CaCO by a double-ratio dilution method 3 Is separated from the MRS agar medium, and is further purified by streaking culture of the MRS agar medium.
The gram staining microscopy of Lactobacillus plantarum18-2 was positive (cells stained purple as shown in FIG. 1).
Lactobacillus plantarum18 cell morphology and physicochemical properties are shown in the following Table:
the sequence of the 16s rDNA of Lactobacillus plantarum18-2 is different from other strains by sequencing, and the sequence is shown as SEQ ID No. 1.
SEQ ID No.1
ATACATGCAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTAAGGTGGGACAGATGATTAGGGTGAAGTCGTAACAAG
The invention also provides the extracellular polysaccharide of the lactobacillus plantarum, and the preparation method of the extracellular polysaccharide comprises the following steps: inoculating Lactobacillus plantarum-2 into MRS culture medium containing 2% sucrose, centrifuging to obtain supernatant, extracting crude EPS by alcohol precipitation water extraction, and dialyzing and Sepharose CL-6B chromatography to obtain pure EPS, wherein the EPS contains two components EPSA and EPSB (shown in figures 2 and 3); the EPSA and the EPSB both have polysaccharide characteristic absorption peaks (shown in figure 4 and figure 5) through infrared spectrum detection; after acid hydrolysis, EPSA is detected to be composed of galactose, glucose and xylose by high performance ion chromatography, the molar ratio is 1.0:5.1:3.8, EPSB is composed of galactose, glucose and xylose, the molar ratio is 1.0:3.2:6.8, and EPSB also contains 2.85% glucuronic acid (as shown in figures 6 and 7); the number average molecular weight of each of the obtained samples was 5800 daltons as measured by GPC using a Waters high performance liquid chromatograph (see FIGS. 8 and 9).
Compared with the prior art, the invention has the beneficial effects that:
the lactobacillus plantarum provided by the invention is derived from traditional dairy products in an inner Mongolian pasture area, has the advantages of nature, green and safety, and the screened Lactobacillus plantarum18-2 thalli and the purified extracellular polysaccharide EPS thereof have obvious adsorption effect on heavy metal lead, and can be applied to a starter and a heavy metal adsorption antidote.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings may be obtained according to these drawings for a person having ordinary skill in the art.
FIG. 1 is a gram microscopic photograph (MRS, 1000X) of Lactobacillus plantarum18-2 provided by the present invention;
FIG. 2 is a DEAE Sepharose Fast Flow column chromatography purification elution profile of Lactobacillus plantarum18-2 provided by the present invention;
FIG. 3 is a DEAE Sepharose CL-6B column chromatography purification elution profile of Lactobacillus plantarum18-2 provided by the present invention;
FIG. 4 is an EPSA infrared spectrum of Lactobacillus plantarum18-2 provided by the invention;
FIG. 5 is an EPSA infrared spectrum of Lactobacillus plantarum18-2 provided by the invention;
FIG. 6 is a high performance ion chromatogram of the EPSA hydrolysate of Lactobacillus plantarum18-2 provided by the invention;
FIG. 7 is a high-performance ion chromatogram of EPSB hydrolysate Lactobacillus plantarum18-2 provided by the invention;
FIG. 8 is a high performance liquid chromatography (EPSA) of Lactobacillus plantarum18-2 provided by the invention.
FIG. 9 is a high performance liquid chromatography (EPSB) of Lactobacillus plantarum18-2 provided by the invention.
Detailed Description
In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in further detail with reference to examples.
Example 1Lactobacillus plantarum18-2 heavy metal tolerance test
Lactobacillus plantarum18-2 is inoculated into MRS culture media with different heavy metal ion concentration gradients for culturing for 24 hours, the growth condition of each strain of lactobacillus is observed, and the highest ion concentration capable of tolerating heavy metals is determined. The results show (see Table 2) that Lactobacillus plantarum18-2 is tolerant to 200mg/L Cu 2+ ，1100mg/L Pb 2+ And 200mg/L Cd 2+ Has good application potential. In contrast, lactobacillus plantarum-2 can tolerate 200mg/L Cu 2+ ，600mg/L Pb 2+ And 900mg/L Cd 2+ . Lactobacillus plantarum18-2 thalli have obvious adsorption effect on heavy metal lead.
Table 2Lactobacillus plantarum for Pb at different concentrations 2+ Adsorption rate (unit:%)
EPS heavy metal lead adsorption test of example 2Lactobacillus plantarum18-2
To 9mL of EPS solution having a concentration of 0mg/mL, 1mg/mL, 1.5mg/mL, 2mg/mL, 2.5mg/mL and 3mg/mL, pb at a concentration of 1mg/mL was added, respectively 2+ Mixing with vortex oscillator, standing in a 37deg.C constant temperature water bath for 4 hr, stirring to allow adsorption, centrifuging at 4000rpm for 20min, collecting supernatant, and collecting Pb 2+ Is measured. As a result, it was found that 2-component EPS of Lactobacillus plantarum18-2 was effective against Pb at concentrations of 1mg/mL and 1.5mg/mL 2+ Shows a very high adsorption rate (see table 3).
TABLE 3 EPS vs Pb at various concentrations 2+ Adsorption rate (unit:%)
Therefore, EPSA and EPSB of the present invention have the best effect of adsorbing Pb at a concentration of 1mg/ml 2+ The highest adsorption rate of the strain (A) can reach 69.59% and 71.61%, and is superior to that of xanthan gum and extracellular polysaccharide of other strains with the same concentration.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; 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 solutions described in the foregoing embodiments may be modified or some technical features may be replaced with others, which may not depart from the spirit and scope of the technical solutions of the embodiments of the present invention. The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Sequence listing
<110> university of inner Mongolia
<120> a Lactobacillus plantarum and extracellular polysaccharide thereof
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<170> SIPOSequenceListing 1.0
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<213> Lactobacillus plantarum (Lactobacillus plantarum)
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atacatgcag tcgaacgaac tctggtattg attggtgctt gcatcatgat ttacatttga 60
gtgagtggcg aactggtgag taacacgtgg gaaacctgcc cagaagcggg ggataacacc 120
tggaaacaga tgctaatacc gcataacaac ttggaccgca tggtccgagt ttgaaagatg 180
gcttcggcta tcacttttgg atggtcccgc ggcgtattag ctagatggtg gggtaacggc 240
tcaccatggc aatgatacgt agccgacctg agagggtaat cggccacatt gggactgaga 300
cacggcccaa actcctacgg gaggcagcag tagggaatct tccacaatgg acgaaagtct 360
gatggagcaa cgccgcgtga gtgaagaagg gtttcggctc gtaaaactct gttgttaaag 420
aagaacatat ctgagagtaa ctgttcaggt attgacggta tttaaccaga aagccacggc 480
taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gatttattgg 540
gcgtaaagcg agcgcaggcg gttttttaag tctgatgtga aagccttcgg ctcaaccgaa 600
gaagtgcatc ggaaactggg aaacttgagt gcagaagagg acagtggaac tccatgtgta 660
gcggtgaaat gcgtagatat atggaagaac accagtggcg aaggcggctg tctggtctgt 720
aactgacgct gaggctcgaa agtatgggta gcaaacagga ttagataccc tggtagtcca 780
taccgtaaac gatgaatgct aagtgttgga gggtttccgc ccttcagtgc tgcagctaac 840
gcattaagca ttccgcctgg ggagtacggc cgcaaggctg aaactcaaag gaattgacgg 900
gggcccgcac aagcggtgga gcatgtggtt taattcgaag ctacgcgaag aaccttacca 960
ggtcttgaca tactatgcaa atctaagaga ttagacgttc ccttcgggga catggataca 1020
ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag 1080
cgcaaccctt attatcagtt gccagcatta agttgggcac tctggtgaga ctgccggtga 1140
caaaccggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca 1200
cacgtgctac aatggatggt acaacgagtt gcgaactcgc gagagtaagc taatctctta 1260
aagccattct cagttcggat tgtaggctgc aactcgccta catgaagtcg gaatcgctag 1320
taatcgcgga tcagcatgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc 1380
acaccatgag agtttgtaac acccaaagtc ggtggggtaa ccttttagga accagccgcc 1440
taaggtggga cagatgatta gggtgaagtc gtaacaag 1478
Claims (8)
1. A lactobacillus plantarum is classified and named as Lactobacillus plantarum, has a strain number of Lactobacillus plantarum-18-2 and is preserved in China general microbiological culture collection center (CGMCC) No.17566, and the preservation date is 2019, 4, 12.
2. Use of lactobacillus plantarum according to claim 1 for the preparation of a starter culture.
3. An extracellular polysaccharide of lactobacillus plantarum secreted by lactobacillus plantarum according to claim 1.
4. The extracellular polysaccharide of lactobacillus plantarum according to claim 3, wherein the extracellular polysaccharide has a number average molecular weight of 5800 daltons and consists of galactose, glucose and xylose in a molar ratio of 1.0:5.1:3.8.
5. The extracellular polysaccharide of lactobacillus plantarum according to claim 3, wherein the extracellular polysaccharide has a number average molecular weight of 5800 daltons, consists of galactose, glucose and xylose, has a molar ratio of 1.0:3.2:6.8 and further contains 2.85% glucuronic acid.
6. The extracellular polysaccharide of lactobacillus plantarum according to claim 4 or 5, characterized in that it is prepared by the following method: lactobacillus plantarum18-2 is inoculated into MRS culture medium containing 2% of sucrose, supernatant is centrifugated, crude extracellular polysaccharide is extracted by an alcohol precipitation water extraction method, and pure extracellular polysaccharide is obtained by dialysis and Sepharose CL-6B chromatography.
7. Use of the extracellular polysaccharide of lactobacillus plantarum according to any of claims 3 to 5 for the preparation of a heavy metal adsorption antidote.
8. Use of the extracellular polysaccharide of lactobacillus plantarum according to claim 7 for the preparation of a heavy metal adsorption antidote, characterized in that the heavy metal is lead.
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