CN112501045B - Lactobacillus fermentum capable of degrading biogenic amine and resisting salt and application thereof - Google Patents
Lactobacillus fermentum capable of degrading biogenic amine and resisting salt and application thereof Download PDFInfo
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Images
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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
- A23L5/28—Removal of unwanted matter, e.g. deodorisation or detoxification using microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/143—Fermentum
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of microorganisms, and discloses a lactic acid bacterium capable of degrading biogenic amine and resisting salt and application thereof. The Lactobacillus is named as Lactobacillus fermentum SL611 (Lactobacillus fermentum SL 611) and is preserved in China center for type culture collection at the eight-path No. 299 of the Wuchang district, wuhan City, hubei province; the preservation number is CCTCC M2019758, and the preservation date is 2019, 9 and 26 days. The lactobacillus fermentum SL611 can be applied to the production of pepper sprouts, cheese, fermented soya beans and other foods, can degrade biogenic amine in the foods, has high degradation rate, and has the degradation rates of cadaverine, histamine and tyramine respectively reaching 55.35%, 82.95% and 71.17%. This not only expands the application range of lactobacillus fermentum SL611 and improves its utility value, but also brings new hopes for the production of fermented foods with low biogenic amine.
Description
Technical Field
The invention relates to a lactobacillus fermentum capable of degrading biogenic amine and resisting salt and application thereof, belonging to the technical field of microorganisms.
Background
Lactic acid bacteria are bacteria which can ferment carbohydrates into lactic acid, gram-positive, prokaryotic organisms, facultative anaerobic type or anaerobic type, are widely existed in various fermented foods, inhabit intestinal tracts and other organs of people and various animals as dominant bacteria, are nontoxic and harmless to human bodies, and can improve the flavor of foods, improve the storability of the foods and regulate normal flora of gastrointestinal tracts of organisms due to metabolic activity of most of the lactic acid bacteria. In recent years, people have been intensively researched on lactic acid bacteria, and the research finds that the lactic acid bacteria have the effects of regulating the immune system, regulating the balance of intestinal flora, delaying the aging of organisms, reducing serum cholesterol, reducing tumor risk and the like on human health. Due to the unique physiological characteristics, the lactic acid bacteria have wide application in industry, agriculture, medicine and health and daily life in China and have extremely high application value.
Yak yoghourt in Qinghai-Tibet plateau is an indispensable delicious food for common people in China. The traditional solidified yak yoghourt is distributed in Qinghai-Tibet areas, gannan Tibet areas, sichuan Tibet areas and Diqing Tibet areas in China. Compared with common cow milk, the yak milk is rich in protein, essential amino acid, fat, lactose and mineral substances, so that the yak milk has extremely high nutritional value. The herdsman continues to process dairy products such as yak yoghourt and the like by using a traditional and ancient method, can better preserve beneficial microorganisms (particularly lactic acid bacteria) in the local natural environment, and provides conditions for the development of wild lactic acid bacteria strains.
Biogenic amines are nitrogen-containing low molecular mass active organic compounds, which are formed mainly from specific biogenic amine decarboxylases by converting specific amino acids. When the intake of human body is too high, pathological reactions such as dizziness, blood pressure change, nausea and the like can be generated, and some biogenic amines can form carcinogenic nitrosamine. Histamine is most toxic in biogenic amines, and excessive histamine can cause headache, digestive disorder, abnormal blood pressure and even certain neurotoxicity. Tyramine is less toxic and overdose can also cause headache and hypertension. Cadaverine and putrescine are less toxic in nature, but both can inhibit the activity of metabolic enzymes related to histamine and tyramine to further increase the content of histamine and tyramine, thereby enhancing the discomfort symptoms of human body, and the 2 biogenic amines can react with nitrite to generate nitrosamine, and have potential carcinogenicity. At present, the main biological amine control means include irradiation, high hydrostatic pressure treatment, addition of food additives and preservatives and the like, but the biological amine control means may have potential harm to human bodies. In recent years, safe and environment-friendly biological methods are more and more accepted by consumers, such as adding a strain for degrading the biogenic amine in the food fermentation process to control the content of the biogenic amine. Therefore, the method has important practical significance for researching and developing functional lactic acid bacteria starter, establishing lactic acid bacteria resource library and solving food safety problem to a certain extent.
Disclosure of Invention
The invention aims to screen and obtain a lactobacillus fermentum which can be used for reducing the content of biogenic amines such as cadaverine, histamine, tyramine and the like in traditional high-salt fermented food such as pepper sprouts, fermented soybeans, bean cotyledons and the like.
In order to achieve the above purpose, the invention provides the following technical scheme:
yak yogurt is type IV fermented milk prepared by old and traditional methods along with herdsmen in Qinghai-Tibet plateau. The Lactobacillus fermentum SL611 (Lactobacillus fermentum SL 611) is separated from traditional fermented yak yogurt collected from herders in the autonomous region of Shangri Shangrira in the Di Qing and Tibetan province of Yunnan, and is preserved in a China center for type culture Collection (CCTCC for short, address: eight-way 299 Wuhan university school in Wuhan district, wuhan City, hubei province) in 26 months in 2019, and the preservation number is CCTCC M2019758.
The Lactobacillus fermentum SL611 (Lactobacillus fermentum SL 611) with the preservation number of CCTCC M2019758 is used as a leaven in food production. The lactobacillus fermentum can degrade biogenic amines in food.
Further, the biogenic amine is any one or combination of cadaverine, histamine and tyramine.
The lactobacillus fermentum can also be used in the fermentation production with the NaCl concentration of 2.00-7.00%.
The results of the biogenic amine determination tests show that the lactobacillus fermentum SL611 can obviously reduce three biogenic amines and can better grow and propagate in an environment with a salt content of 7.00%. Therefore, the lactobacillus fermentum SL611 can be used for producing fermented foods such as pepper sprouts, cheese, bean paste or fermented soya beans.
The invention has the beneficial effects that: the invention provides application of lactobacillus fermentum SL611 in preparing fermented foods for degrading biogenic amine, such as cheese, fermented soya beans and the like, wherein the biogenic amine in the foods is degraded, the degradation rate is high, and the degradation rates of cadaverine, histamine and tyramine respectively reach 55.35%, 82.95% and 71.17%. Not only expands the application range of the lactobacillus fermentum SL611 and improves the utilization value of the lactobacillus fermentum, but also brings new hope for the production of fermented food with low biogenic amine.
Drawings
FIG. 1 shows a colony morphology of Lactobacillus fermentum SL611 (a) and gram stain results (b);
FIG. 2 shows the result of API50CH reaction of Lactobacillus fermentum SL 611;
FIG. 3 is a diagram of a chromogenic medium;
FIG. 4 is a spectrum of a biogenic amine HPLC standard;
FIG. 5 is a graph of a standard curve of biogenic amine content;
FIG. 6 shows the salt tolerance (OD) of Lactobacillus fermentum SL611 at different salt contents 600nm ) And (5) a result chart.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
1. Identification of lactobacillus fermentum SL611, biogenic amine degradation and salt tolerance experiment
1 materials of the experiment
Lactic acid bacteria are obtained by separating and screening traditional fermented yak yogurt collected from herdsmen in the self-curing prefecture of prefecture Shanglira, dianzench, yunnan province.
2 method of experiment
2.1 Strain identification test
Inoculating lactobacillus preserved in ampoule tube into MRS broth, activating for two generations, performing plate streaking and gram staining microscopy, and extracting DNA of strain with bacterial genome DNA extraction kit if the strain is pure. PCR amplification is carried out by adopting a 25 mu L reaction system, and agarose gel electrophoresis is used for detection after the reaction is finished. After the PCR amplification product is qualified, the Huada Gene science and technology company is entrusted to carry out bidirectional sequencing on the PCR amplification product, and the sequencing result is subjected to homology comparison analysis through a BLAST program in NCBI.
2.2 API kit identification
Culturing the strain at 37 ℃ for 18h, centrifuging under 4000r/min and 10min to collect thalli, washing the thalli with sterile normal saline, and then suspending the thalli into a bacterial suspension. Refer to the API kit instructions for the procedure.
2.3 chromogenic culture test
Inoculating the activated bacterial suspension into a chromogenic culture medium with the inoculation amount of 2%, culturing for 48h at 37 ℃, recording the experimental result, and determining that the bacteria are positive when showing purple and negative when showing yellow without changing color. Parallel 3 groups were made and a blank medium was used as a control group. If the result is negative, the next experiment is carried out.
2.4 biogenic amine assay
2.4.1 Pre-column derivatization
Adding 1mL of 2mol/L NaOH solution and 10 mu L of benzoyl chloride into 2mL of the standard mixed solution of biogenic amine, then carrying out vortex oscillation for 30s, placing the mixture in a water bath condition at the temperature of 30 ℃ and carrying out dark reaction for 40min, and oscillating the mixture once every 10 min. After completion of the reaction, the reaction was interrupted with 2mL of saturated NaCl solution. Then extracting with 3mL of ether, centrifuging at 1200r/min for 10min, removing the ether layer with a pipette, repeating the steps again, transferring the ether layer into a 10mL centrifuge tube, repeating the operations once, drying with nitrogen at 35 ℃, finally dissolving with 1mL of methanol (chromatographic grade), filtering with a 0.22 mu m filter membrane, placing in a 10mL centrifuge tube, and storing in a 4 ℃ refrigerator for testing.
2.4.2 Standard Curve plotting
Accurately weighing 50mg of cadaverine, histamine and tyramine respectively, and diluting to 50mL by using 0.1mol/LHCl solution to prepare a mixed standard solution with the concentration of 1000 mu g/mL for later use. Taking the above standard stock solutions, respectively, and preparing into mixed standard solutions with concentrations of 1.0, 2.0, 5.0, 10, 20, 40, and 80 μ g/mL with 0.1mol/LHCl solution. Then 2.3.1 pre-column derivatization treatment is carried out, and a standard curve is drawn after analysis and detection.
2.4.3 determination of the content of biogenic amines
Respectively inoculating the activated bacterial suspension into MRS liquid culture media containing 500 mu g/mL of three kinds of biogenic amines (tyramine, cadaverine and histamine) by using the inoculation amount of 2%, culturing for 48h at 37 ℃, respectively adding 1mL of bacterial liquid into 4mL of 0.1mol/LHCl solution, performing 2.3.1 pre-column derivatization, and finally performing high performance liquid chromatography to determine the biogenic amine content. Parallel 3 groups were made and blank liquid medium was used as a control group.
2.4.4 chromatographic conditions
The column was AgilentXDB-C18 (250minX4.6mm, 5 μm), mobile phase A was ultrapure water, mobile phase B was methanol, and gradient elution was performed according to the elution procedure shown in the following Table. The flow rate is 1.0mL/min, the ultraviolet detection wavelength is 254nm, and the sample injection amount is 20 mu L.
TABLE 1 gradient elution schedule
2.5 salt tolerance test
The activated suspension of the negative strain was inoculated at an inoculum size of 2% into each of 0.00%, 5.00%, 7.00%, 9.00%, 11.00%, and 13.00% NaCl in a liquid MRS medium, and after culturing at 37 ℃ for 48 hours, the OD at 600nm was measured and plotted.
2.6 statistical analysis
Experimental data were processed and plotted by Excel software.
(W 0 The content of biogenic amine in the control is mu g/mL; w is a group of 1 The content of biogenic amine in the experimental group, μ g/mL)
3 results and analysis
3.1 identification of the Strain
The activated SL611 forms a single colony in an MRS culture medium, the colony morphology is almost consistent, most of the colony forms a round shape, is milky white, and has a smooth and wet surface. The blue-violet cell morphology was observed under a microscope after gram staining, and the cells were judged as gram-positive bacteria (G) + ). Among them, the colony morphology and gram staining of the strain of SL611 are shown in fig. 1. The results of the 16S rDNA homology analysis showed that the sequence of the amplification product of the 16S rDNA gene of SL611 had 100% homology to Lactobacillus fermentum (Lactobacillus fermentum) known in the GeneBank database.
3.2 Biochemical characterization of the Strain
Phenotypic identification at the lactobacillus species level is based primarily on carbohydrate fermentation assays. The API50CH kit was identified by the utilization of 49 different carbohydrates by the strain.
Figure 2 shows the API50CH reaction results for strain No. SL 611. Table 2 shows the results of the fermentation test of strain number SL611 on 49 carbohydrates. As can be seen from fig. 2 and table 2, among the 49 carbon sources tested, 14 carbohydrates among them were available for the strain numbered SL 611. Finally identified by API lab plus system, the strain with number SL611 is Lactobacillus fermentum (Lactobacillus fermentum) with ID value of 76.60% and T value of 0.33.
TABLE 2 SL611 results of fermentation test on 49 carbohydrates
Note: "+" indicates positive reaction; "-" indicates negative reaction.
3.3 results of color development test
In order to maintain self-reproduction and development of lactic acid bacteria under relatively low acid or nutrient-deficient conditions, strains having amino acid decarboxylase activity, which are regulated by a stress mechanism, decarboxylate amino acids to form the corresponding biogenic amines (a basic substance). Bromocresol purple shows yellow under acidic conditions, indicating that the strain does not have amino acid decarboxylase activity, i.e., a negative strain that does not produce biogenic amine, and shows purple under alkaline conditions, indicating that the strain has amino acid decarboxylase activity, i.e., a positive strain that produces biogenic amine, as shown in fig. 3 (purple on the left and yellow on the right).
The lactobacillus fermentum SL611 was preliminarily judged as a negative strain according to the chromogenic status of the liquid medium.
3.4 biogenic amine assay results
3.4.1 drawing of Standard Curve
The standard solution was derivatized as described above, and the 7 mixed standard solutions were repeatedly assayed 3 times. The concentration of the three biogenic amines is used as the abscissa, the peak area is used as the ordinate, linear regression is carried out, and a standard curve is drawn and is shown in figure 5.
As can be seen from fig. 4, the various biogenic amine standards are clearly separated on the high performance liquid chromatography, the peak-forming speed is fast, and all the three biogenic amines are formed within 20 minutes, which are respectively: cadaverine peaked at about 7.1 minutes, histamine peaked at about 16.7 minutes, and tyramine peaked at about 18.9 minutes. From FIG. 5 (the three standard curves are tyramine, cadaverine and histamine from top to bottom), it can be seen that the peak areas of the three biogenic amines and the corresponding concentrations thereof respectively present good linear relations, linear regression equation and related coefficient R 2 See Table 2 for the correlation coefficient R 2 All are more than 0.99, which proves that the method has good reliability for determining the three biogenic amines.
TABLE 2 Linear regression equation Table
3.4.2 biogenic amine degradability results
The color test is a preliminary screening method, and HPLC is a method for quantitatively detecting biogenic amine, so that the accuracy and the rigor of the experimental result are ensured, and the capability of the biogenic amine degrading strain for degrading biogenic amine can be accurately evaluated.
The results are shown in table 4, lactobacillus fermentum SL611 has good degradation capability on three biogenic amines, and is a more ideal high-efficiency biogenic amine degrading strain.
TABLE 4 degradation rate of the biogenic amines by Lactobacillus fermentum SL611
3.5 salt tolerance test results
Most fermented foods in China have high-salt environment, so if the biological amine degrading strain is applied to the fermented foods, the strain is required to be capable of adapting to the environment with high salt and high osmotic pressure. The strain was subjected to a salt tolerance test to determine its OD 600nm It was found that the growth of the strain gradually underwent as the salt concentration increasedAnd (4) inhibiting. The strain is inhibited because the environment with high salt content has salt stress effect on the growth and the reproduction of the lactic acid bacteria, inhibits the growth and the reproduction of the lactic acid bacteria and influences the survival rate of the lactic acid bacteria. Meanwhile, the high-concentration environment with salt content can increase the osmotic pressure of the solution, so that water in the lactic acid bacteria cells flows out, and the cytoplasm of the cells is separated, thereby causing structural and physiological damage to the cells, and causing the cells to stop growing and even die.
As can be seen from FIG. 6, lactobacillus fermentum SL611 is able to grow and propagate well in the environment with salt content of 0.00% -7.00%. The common fermented food has salt content in 2.50-10.00%, and SL611 can grow well in the range, so that the common fermented food can be applied to fermented food.
Finally, it is noted that the above-mentioned embodiments illustrate rather than limit the invention, and that, while the invention has been described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
SEQUENCE LISTING
<110> agriculture rural committee of Jiangjin area of Chongqing city, southwest university
<120> lactobacillus fermentum capable of degrading biogenic amine and resisting salt and application thereof
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1403
<212>DNA
<213>Lactobacillus fermentum SL611
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ttgattgatg gtgcttgcac ctgattgatt ttggtcgcca acgagtggcg gacgggtgag 60
taacacgtag gtaacctgcc cagaagcggg ggacaacatt tggaaacaga tgctaatacc 120
gcataacaac gttgttcgca tgaacaacgc ttaaaagatg gcttctcgct atcacttctg 180
gatggacctg cggtgcatta gcttgttggt ggggtaacgg cctaccaagg cgatgatgca 240
tagccgagtt gagagactga tcggccacaa tgggactgag acacggccca tactcctacg 300
ggaggcagca gtagggaatc ttccacaatg ggcgcaagcc tgatggagca acaccgcgtg 360
agtgaagaag ggtttcggct cgtaaagctc tgttgttaaa gaagaacacg tatgagagta 420
actgttcata cgttgacggt atttaaccag aaagtcacgg ctaactacgt gccagcagcc 480
gcggtaatac gtaggtggca agcgttatcc ggatttattg ggcgtaaaga gagtgcaggc 540
ggttttctaa gtctgatgtg aaagccttcg gcttaaccgg agaagtgcat cggaaactgg 600
ataacttgag tgcagaagag ggtagtggaa ctccatgtgt agcggtggaa tgcgtagata 660
Tatggaagaa caccagtggc gaaggcggct acctggtctg caactgacgc tgagactcga 720
aagcatgggt agcgaacagg attagatacc ctggtagtcc atgccgtaaa cgatgagtgc 780
taggtgttgg agggtttccg cccttcagtg ccggagctaa cgcattaagc actccgcctg 840
gggagtacga ccgcaaggtt gaaactcaaa ggaattgacg ggggcccgca caagcggtgg 900
agcatgtggt ttaattcgaa gctacgcgaa gaaccttacc aggtcttgac atcttgcgcc 960
aaccctagag atagggcgtt tccttcggga acgcaatgac aggtggtgca tggtcgtcgt 1020
cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tgttactagt 1080
tgccagcatt aagttgggca ctctagtgag actgccggtg acaaaccgga ggaaggtggg 1140
gacgacgtca gatcatcatg ccccttatga cctgggctac acacgtgcta caatggacgg 1200
tacaacgagt cgcgaactcg cgagggcaag caaatctctt aaaaccgttc tcagttcgga 1260
ctgcaggctg caactcgcct gcacgaagtc ggaatcgcta gtaatcgcgg atcagcatgc 1320
cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccatga gagtttgtaa 1380
cacccaaagt cggtggggta acc 1403
Claims (5)
1. A lactobacillus fermentum with biogenic amine degradation and salt tolerance is characterized in that: the Lactobacillus fermentum SL611 is classified and named as Lactobacillus fermentum and is preserved in China center for type culture collection (CCTCC NO): m2019758 with a preservation date of 2019, 9 and 26.
2. Use of a strain of lactobacillus fermentum having biogenic amine degrading and salt tolerance properties, wherein the lactobacillus fermentum of claim 1 is used as a starter to degrade biogenic amine in food products.
3. Use of a lactobacillus fermentum with biogenic amine degrading and salt tolerant properties according to claim 2, characterized in that: the biogenic amine is any one or combination of more of cadaverine, histamine and tyramine.
4. Use of lactobacillus fermentum with biogenic amine degrading and salt tolerant properties according to any one of claims 2-3, wherein: the food is fructus Zanthoxyli bud vegetable, cheese, bean or semen Sojae Preparatum.
5. Use of lactobacillus fermentum with biogenic amine degrading and salt tolerant properties according to any one of claims 2-3, wherein: the concentration of NaCl in the food is 2.00-7.00%.
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