CN104928219B - A kind of lactobacillus plantarum TH103 and application thereof - Google Patents
A kind of lactobacillus plantarum TH103 and application thereof Download PDFInfo
- Publication number
- CN104928219B CN104928219B CN201510385613.6A CN201510385613A CN104928219B CN 104928219 B CN104928219 B CN 104928219B CN 201510385613 A CN201510385613 A CN 201510385613A CN 104928219 B CN104928219 B CN 104928219B
- Authority
- CN
- China
- Prior art keywords
- lactobacillus plantarum
- culture
- spice
- ensilage
- temperature
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention discloses a kind of lactobacillus plantarum TH103 and application thereof; the ability of lactobacillus plantarum TH103 acidproof, bile tolerance with higher, resistance to NaCl; there is stronger inhibiting effect to salmonella typhimurium growth in vitro; it is inhibited to the growth of salmonella typhimurium in food system, there is protective effect to Salmonella Typhimurium Infection Intestinal epithelium cell HT-29 cell.The lactobacillus plantarum TH103 can be used for the food such as fermented vegetable, preparation production ensilage, ferment sausage.
Description
Technical field
The invention belongs to microorganisms technical field, it is related to a kind of to inhibit salmonella typhimurium growing plants lactobacillus
(Lactobacillus plantarum) TH103, further relates to the purposes of the lactobacillus plantarum TH103.
Background technique
Salmonella typhimurium (Salmonella typhimurium) is the important enteropathic for endangering human and animal
Bacterium, enteric infection and grice diarrhoea etc. as caused by mankind's food source contact scar.Salmonella typhimurium is entered small by alimentary canal
First then adherency and definite value invade cell, mainly cause enterocolitis, can lead to host's abdomen in small intestine epithelium surface after intestines
It rushes down, a series of lesions such as bacteremia, is most common person in children's salmonella infection.Salmonella typhimurium nature epidemic disease source is wide
General, many poultry, domestic animal, mouse, bird and cold-blooded animal are its natural reservoir (of bird flu viruses), and fly, flea can carry disease germs propagation, often by pollute water, ox
Milk and food peroral infection.According to statistics, salmonella typhimurium infects 1/5 that quantity accounts for about salmonella infection amount every year, is
Threaten one of the main pathogenic fungi of human health.
Probiotics is to refer to survive and colonize with certain amount in host intestine, by adjusting intestinal flora balance,
Pathogen in antagonism enteron aisle, to play host health the active beneficial microorganism general name of beneficial effect.Have in probiotics
Much belong to lactic acid bacteria, lactic acid bacteria is that a group can be with fermentable carbohydrates, and generate the Gram-positive bacillus of a large amount of lactic acid
Or the general designation of coccus.In 400 multiple-microorganism of gastrointestinal tract, lactic acid bacteria accounts for about 10%, is the Major Members of normal intestinal flora.
As the normal flora of humans and animals enteron aisle, lactic acid bacteria, which has, to prevent pathogen to the invasion of enteron aisle and colonizes, and inhibits pathogen
With it is anti-infective, maintain the prebiotic function such as microecological balance of enteron aisle.Lactic acid bacteria and its metabolite may be to Salmonella typhimuriums
The pollution of bacterium has certain prevention or intervention effect, there is the potential of the auxiliary therapeutical agent as antibiotic, and screening has excellent
The lactic acid bacteria of antagonistic properties is necessary for inhibiting the pollution of salmonella typhimurium.
Summary of the invention
The purpose of this section is to summarize some aspects of the embodiment of the present invention and briefly introduce some preferable implementations
Example.It may do a little simplified or be omitted to avoid our department is made in this section and the description of the application and the title of the invention
Point, the purpose of abstract of description and denomination of invention it is fuzzy, and this simplification or omit and cannot be used for limiting the scope of the invention.
The problem of in view of above-mentioned and/or existing lactobacillus plantarum TH103 and application thereof, propose the present invention.
Therefore, it is an object of the present invention to provide a kind of lactobacillus plantarum TH103, and the bacterial strain is in April, 2015
In China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, the number that preservation is registered on the books was on 23rd
CGMCC10739, classification naming are as follows: lactobacillus plantarum Lactobacillus plantarum.
A kind of application it is a further object to provide lactobacillus plantarum TH103 in fermented vegetable field.
As lactobacillus plantarum TH103 of the present invention the application in fermented vegetable field a kind of preferred embodiment, in which:
Including, vegetables are cleaned drain after by the vegetables peeling, cutting, be added according to the vegetables poidometer 1.0%~1.2%
Sucrose and 6%~8% salt, stir evenly in the pre- marinated 36~48h of room temperature, drain away the water, obtain a kind of pre- Pickle；
Edible spice is weighed, use uses 8~10 times of water in terms of spice weight to extract 4~6h at 75~80 DEG C of temperature by described,
The white wine that edible spice poidometer 10% is added after cooling, is uniformly mixed so as to obtain a kind of spice juice；By lactobacillus plantarum TH103 with
Fluid nutrient medium stereometer 1~3% is inoculated in fluid nutrient medium, is carried out under conditions of 37 DEG C of cultures 20~28h, 4500rpm
It is centrifuged 10~15min, abandons supernatant, the bacterium mud collected is resuspended with sterile water, so that the viable bacteria concentration of lactobacillus plantarum TH103
Reach 108Cfu/mL or more, to obtain a kind of bacteria suspension；Addition is in the pre- Pickle with the pre- Pickle weight
The spice juice of meter 10~12% and 2~5% the bacteria suspension, mix tinning, ferment at room temperature after sealing
Until fermentation liquid pH value up to 3.0~4.0, obtains fermented vegetable.
As lactobacillus plantarum TH103 of the present invention the application in fermented vegetable field a kind of preferred embodiment, in which:
The vegetables are for one of Chinese cabbage, asparagus lettuce, cowpea, carrot, white radishes, asparagus lettuce, cabbage, pimento, onion or celery
Or it is several.
As lactobacillus plantarum TH103 of the present invention the application in fermented vegetable field a kind of preferred embodiment, in which:
The spice is one or more of capsicum, Chinese prickly ash, ginger, garlic, illiciumverum, fennel, orange peel, spiceleaf.
A kind of application it is also another object of the present invention to provide lactobacillus plantarum TH103 in working stock culture field,
The concentration of lactobacillus plantarum TH103 is 10 in the working stock culture9Cfu/g or more.
As lactobacillus plantarum TH103 of the present invention the application in working stock culture field a kind of preferred embodiment,
In: including according to 1~2% inoculum concentration in terms of culture medium weight, lactobacillus plantarum TH103 original strain is inoculated in culture medium
In, 20~28h is cultivated under conditions of 37 DEG C and is activated, is continuously carried out in the same manner activation 2~3 times, and activation training is obtained
Supporting lactobacillus plantarum TH103 viable count in object is not less than 108cfu/mL；In the condition of 2~6 DEG C of temperature and revolving speed 4500rpm
Under carry out 10~15min of centrifugation, abandon supernatant, the bacterium mud collected is rinsed 3~4 times with the buffer of pH7.2, then, toward institute
Freeze drying protectant suspension is added in the bacterium mud stated, adjustment bacterial concentration is not less than 109Cfu/mL carries out vacuum refrigeration after mixing
It is dried to obtain the lactobacillus plantarum TH103 leavening, wherein the composition of the freeze drying protectant suspension is sugarcane by weight
Sugared 2%, the water of skimmed milk 15%, lactose 5%, glycerol 1% and 75%.
A kind of application it is a further object to provide lactobacillus plantarum TH103 in fermentation ensilage field,
It includes, and ensilage raw material is rubbed stand-by after cutting section；50~60kg ensilage raw material is weighed, by 0.5~0.6g as weighed
Benefit require 7 described in lactobacillus plantarum TH103 leavening be uniformly mixed with 200~250mL sterile water, be sprayed onto ensilage original
It in material, turns to be fitted into after mixing in ensilage bucket and is pressed and sealed, contained after being stored 1~2 month under normal temperature condition
The ensilage of lactobacillus plantarum TH103.
A kind of application it is also an object of the present invention to provide lactobacillus plantarum TH103 in ferment sausage field,
Including the meat selected from pork, beef or mutton being shredded, after carrying out 24~36h of pre-freeze in 5 DEG C~8 DEG C of refrigerating chamber, with food
Salt, white granulated sugar, sodium nitrite are sufficiently mixed according to weight ratio 100:4:1.5:0.015；Mixture is placed at 0~4 DEG C of temperature
Marinated 24~48h, is added according to 5~10% spice of meat poidometer, is stirred, is then added according to claim 7
Lactobacillus plantarum TH103 leavening, additional amount be 0.5~0.8% in terms of meat weight, after mixing containing work send out
The meat of ferment agent is filled into casing, obtained bowel lavage under conditions of 14 DEG C~15 DEG C of temperature is with relative humidity 80% fermentation 25~
30 days；It is toasted at 65~70 DEG C of temperature after fermentation, the ferment sausage is obtained after cooling.
Lactobacillus plantarum TH103 of the invention in vitro significantly inhibits salmonella typhimurium, to people
Work gastric juice, simulated intestinal fluid have strong tolerance, and tolerance NaCl ability is up to 10%, can significantly press down in actual food product system
The growth of salmonella typhimurium processed.There is protective effect to Salmonella Typhimurium Infection Intestinal epithelium cell HT-29 cell.
The lactobacillus plantarum TH103 can be used for the products such as fermented vegetable, preparation production ensilage, ferment sausage.
Detailed description of the invention
Fig. 1 is under the conditions of having TH103 and its to be added without growth of the salmonella typhimurium in cow's milk under the conditions of TH103
Curve.
Specific embodiment
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, right with reference to the accompanying drawings of the specification
A specific embodiment of the invention is described in detail.
In the following description, numerous specific details are set forth in order to facilitate a full understanding of the present invention, but the present invention can be with
Implemented using other than the one described here other way, those skilled in the art can be without prejudice to intension of the present invention
In the case of do similar popularization, therefore the present invention is not limited by the specific embodiments disclosed below.
Secondly, " one embodiment " or " embodiment " referred to herein, which refers to, may be included at least one realization side of the invention
A particular feature, structure, or characteristic in formula." in one embodiment " that different places occur in the present specification not refers both to
The same embodiment, nor the individual or selective embodiment mutually exclusive with other embodiments.
The present invention is described in more detail below.
The lactobacillus plantarum TH103 is obtained using following screening techniques, and is studied its property.
One, it screens
The specific implementation step of screening technique is as follows:
1, sample acquires
Primary sample picks up from Handan She County and the homemade spontaneous fermentation pickles of abundant village's local farmers.It is acquired with sterile triangular flask
Sample, the sample collected are stored under conditions of 0 DEG C of temperature, and the separation of sample analysis and lactic acid bacteria is carried out in sampling 48h.
2, the separating lactic acid bacterium from sample
10g sample is placed in the 90mL physiological saline with bead, vibrates 30min, then using physiological saline according to body
Product carries out serial dilution than 1:10.Using spread plate method known to those skilled in the art, by each dilution gradient
Bacterium solution 0.1mL be respectively coated on following three kinds of culture medium flat plates: containing NaCl concentration be 4% and addition bromocresol purple instruction
The MRS culture medium (for cultivating the culture medium of lactobacillus) of agent, 37 DEG C of culture 48h.From the plate with 30~300 bacterium colonies
Single bacterium colony is separated, with the random picking of oese 5~10 bacterium colonies therein in the MRS fluid nutrient medium of 4%NaCl, and
It cultivates at corresponding temperature for 24 hours, then crosses on the MRS solid plate of 4%NaCl.Isolate is continuously crossed to be obtained three times
Then pure single colonie carries out Gram's staining and catalase test known to those skilled in the art.By gram
Stained positive, catalase experiment access in MRS fluid nutrient medium for negative bacterial strain, 37 DEG C of stationary cultures for 24 hours, then according to
Culture medium poidometer 2-4% inoculum concentration is inoculated in respectively in MRS fluid nutrient medium, 37 DEG C of stationary cultures 18~activated for 24 hours,
Continuous switching 3 times obtains the good lactic acid bacteria of activity.It purifies bacterial strain in 30% sterile glycerol solution at -80 DEG C of temperature
Under the conditions of freeze.
Using addition 4%NaCl and bromocresol purple MRS agar medium on isolate 46 separation strains.By bacterium colony
Form, Gram's staining, catalase test eliminate 3 Gram-negatives, 1 contact enzyme positive and 2 yeast.In this way, point
From having obtained 32 plants of lactobacillus after purification.
3, there is the lactic acid bacteria of antagonism salmonella typhimurium outside screen body
By above-mentioned separating lactic acid bacterial strain according in terms of culture medium weight 2-4% inoculum concentration be inoculated in MRS fluid nutrient medium,
Fermented and cultured 18-24h is carried out under condition of culture of the anaerobism with 37 DEG C of temperature, obtains streptococcus acidi lactici fermented solution.
Then, Yihong methylene blue culture medium (for cultivating salmonella typhimurium) of sterilizing is poured into plate and (is often put down
Plate 15mL, plate are 9cm dressing plate), salmonella typhimurium (S.typhimurium) ATCC14028 is as indicator strain
(bacterial strain is bought from American type culture collection ATCC), and every plate is put into 4 Oxford cups (diameter 10mm), is added in Oxford cup
Enter above-mentioned 150 μ L of streptococcus acidi lactici fermented solution, be placed at 4 DEG C of temperature and spread 6h, is then placed in incubator and is cultivated at 37 DEG C of temperature
Measure inhibition zone size afterwards for 24 hours.Using MRS fluid nutrient medium as negative control, lactobacillus plantarum ST-III as positive control,
Test is repeated 3 times, every time 3 it is parallel.Filter out the lactic acid bacteria TH103 with inhibition zone bigger than positive control.These tests
As a result it is listed in table 1.
Inhibiting effect of 1 TH103 of table to salmonella typhimurium
Lactobacillus plantarum (Lactobacillus plantarum) TH103 is pair it can be seen from the result that table 1 is listed
Salmonella typhimurium growth has the lactic acid bacteria of obvious inhibiting effect.
4, the 16s rDNA Sequence Identification of TH103 bacterial strain
By lactobacillus plantarum TH103 according in terms of culture medium weight 2-4% inoculum concentration be inoculated in MRS fluid nutrient medium,
37 DEG C of culture 12h, take the fresh bacterium solution of 1~1.5mL, extract for bacterial genomes DNA.Using genomic DNA as template, use
Bacteria Identification universal primer 27F and 1492R well known in the art are primer, and 50 μ L reaction systems carry out PCR amplification.
The examining order of target gene pcr amplification product is completed by Shanghai biotechnology Services Co., Ltd.By plant
The 16S rDNA sequencing result of lactobacillus TH103 is compared with NCBI nucleic acid database, and final result shows: of the invention
TH103 and lactobacillus plantarum ST-III homology are up to 99%, therefore, TH103 bacterial strain of the invention are accredited as plant cream
Bacillus is named as Lactobacillus plantarum TH103, protects on April 23rd, 2015 in Chinese microorganism strain
The preservation of administration committee's common micro-organisms center is hidden, the number that preservation is registered on the books is CGMCC10739.
It is further to note that being obtained in first time screening and after plant identification lactobacillus TH103, every several times
(45 days, 3 months, 5 months, 7 months) remove acquisition sample site acquisition pickles sample again, obtain together by screening with identification
The bacterial strain of sample.
Two, the microbiological property of lactobacillus plantarum TH103
1, the measurement of lactobacillus plantarum TH103 acid resistance
It prepares simulated gastric fluid: taking 0.1mol/L hydrochloric acid 16.4mL that water is added to shake up, extremely with concentrated hydrochloric acid or 10%NaOH tune pH value
2.0, it is then settled to 1000mL, sterilize 30min at 121 DEG C, and commodity pepsin system then is added according to 1g/100mL ratio
Agent is uniformly mixed so as to obtain the simulated gastric fluid.
The measurement of lactobacillus plantarum TH103 acid resistance: the lactobacillus plantarum TH103 of preservation is cultivated into base weight according to MRS
Meter 2-4% inoculum concentration is inoculated in MRS culture medium, is cultivated for 24 hours at 37 DEG C of temperature, uses MRS culture solution in the same manner
Then switching culture 2~3 times takes activation bacteria suspension 40mL to be added in 50mL centrifuge tube, 4500rpm is centrifuged 5min and collects bacterium
Body, the simulated gastric fluid of the addition above-mentioned preparation of 40mL after then wash 3 times with PBS buffer solution (pH be 7.2 similarly hereinafter), in 37 DEG C of water-baths
3h is handled, is shaken up 1 time every 30min, viable bacteria is carried out using dressing plate colony counting method after handling 0h, 1.5h, 3h respectively
It counts.Lactobacillus plantarum TH103 acid-resistant property measurement result is listed in table 2.
2, the measurement of lactobacillus plantarum TH103 bile tolerance performance
It prepares simulated intestinal fluid: taking KH2PO46.8g add distilled water 500mL dissolve, with 0.4%NaOH solution by weight by its
PH is adjusted to 6.8, and then plus water is settled to 1000mL, according to the ratio addition pig gall for adding 0.4g Pig cholate in every 100mL solution
Salt, sterilize at 115 DEG C of temperature 15min after completely dissolution, obtains the simulated intestinal fluid.
Thallus is collected using with mentioned-above same method.The thallus of collection washs 3 with PBS buffer solution (pH 7.2)
The simulated intestinal fluid of the above-mentioned preparation of 40mL is added after secondary, handles 3h in 37 DEG C of temperature of water-bath, is shaken up once every 30min, point
Count plate is carried out using dressing plate colony counting method not after handling 0h, 1.5h, 3h.Lactobacillus plantarum TH103 bile tolerance
The measurement result of performance is listed in table 3.
3, the measurement of the resistance to NaCl performance of lactobacillus plantarum TH103
The lactobacillus plantarum TH103 that -80 DEG C of glycerol tubes save is crossed on MRS solid plate respectively, 37 DEG C of inversion trainings
It supports to growing single colonie；It chooses single colonie and is inoculated into MRS fluid nutrient medium, be after continuously transferring after 37 DEG C of stationary culture 12h 3 times
Pre-culture solution.
It is respectively 4%, 6%, 8%, 10% that the pre-culture solution of above-mentioned preparation is inoculated in salinity with 2% inoculum concentration respectively
In NaCl and MRS fluid nutrient medium without containing NaCl, 37 DEG C of stationary cultures, every 2h surveys a light absorption value, is drawn according to light absorption value
Growth curve processed.The measurement result of lactobacillus plantarum TH103 tolerance NaCl performance is listed in table 4.
4, lactobacillus plantarum TH103 adheres to the measurement of intestinal epithelial cell HT-29
(1) recovery of cell, cultivate and freeze
HT-29 cell cryopreservation tube is quickly put into 37 DEG C of water-baths, rear 800rpm centrifugation 5min is dissolved, is used after removing supernatant
Cell is resuspended in fresh culture solution, is dispersed in it in culture bottle, in 5%CO2With 37 under the gas condition of 95% air
It DEG C is cultivated, when recovery, every 48h was replaced culture solution 1 time.It is molten with pancreas enzyme -EDTA when cell well-grown (70% fusion)
Liquid digests HT-29 cell under conditions of temperature is 37 DEG C, 1:3 sub-bottle culture.Add in complete culture solution when freezing
Enter 10% methylene, two maple (DMSO), Liquid nitrogen storage.It is 2 × 10 that cell concentration will be adjusted when adhesion experiment after cell dissociation5A/
It is long to degrees of fusion up to 80% to cell to be inoculated in the 6 orifice plates containing coverslip culture by mL.
(2) adhesion experiment
4500rpm centrifugation 5min collects the thallus grown in corresponding culture medium；With weight after brine thallus 3 times
Outstanding thallus, and adjusting cell concentration is 108cfu/mL；Above-mentioned bacteria suspension 2mL is added containing the long HT-29 cell lid to single layer
In the 6 orifice plates of slide, in 5%CO237 DEG C of incubation 2h in incubator；It is washed 3 times after incubation with sterile PBS；0.4% paraformaldehyde
Gram's staining after fixed 0.5h.
(3) adherency observation and counting
After dyeing, the oily microscopic observation bacterial adhesion the case where, and take pictures.It is random under each salinity when micro- sem observation
About 100 cells in 20 visuals field are taken, the bacterial population that cell is adhered to, the bacterial population being averagely attached on each cell are calculated
As adhesion index, each salinity do three repetitions, and experiment carries out data analysis after being repeated 3 times.Lactobacillus plantarum TH103 is viscous
The measurement result of attached intestinal epithelial cell HT-29 performance is listed in table 4.
According to lactobacillus plantarum TH103 is acidproof, bile tolerance, resistance to NaCl and adherency intestinal epithelial cell HT-29 performance are surveyed
Determine the identical mode of method and carried out check experiment using lactobacillus plantarum ST-III, test result is also found in table 2,3 and of table
Table 4.
Table 2: the acid-resistant property measurement result of lactobacillus plantarum TH103
Table 3: the bile tolerance characteristic measurement result of lactobacillus plantarum TH103
Table 4: the resistance to NaCl of lactobacillus plantarum TH103 and adhesion characteristics measurement result
The result of table 2 to table 4 clearly illustrates that lactobacillus plantarum TH103 has acid resistance, can grow at pH2.0；
With bile tolerance ability, the gallbladder salinity that is resistant to is up to 0.4%；With high resistance to NaCl ability, the concentration for being resistant to NaCl reaches
10%；Ability with preferable adherency intestinal epithelial cell.
5, lactobacillus plantarum TH103 inhibits salmonella typhimurium to grow in egg and milk
By the lactobacillus plantarum TH103 of freezen protective according in terms of MRS culture medium weight 2~4% inoculum concentrations be inoculated in MRS
It in culture medium, is cultivated at 37 DEG C for 24 hours, in the same manner using the switching of MRS culture solution culture 2-3 times, then takes activation bacteria suspension
1mL is added in 50mL centrifuge tube, and 4500rpm centrifugation 5min is resuspended after collecting thallus with PBS buffer solution.It handles in the same way
Salmonella typhimurium ATCC14028.Then, salmonella typhimurium ATCC14028 bacterium solution is smeared in product egg surface, dried in the air
It after dry, then smear and lactobacillus plantarum TH103 bacterium solution is resuspended, counted after natural air drying 5h using dressing plate colony counting method.It is right
It is carried out according to test according to the same manner, replaces lactobacillus plantarum TH103 to smear egg table using sterile MRS fluid nutrient medium
Face.These test results are listed in Table 5 below.
Table 5 is in egg surface cream lactobacillus plantarum TH103 to the inhibiting effect of salmonella typhimurium
Test result shows that lactobacillus plantarum TH103 is able to suppress the growth of salmonella typhimurium in egg surface.
Lactobacillus plantarum TH103 and salmonella typhimurium ATCC14028 are handled according to mode same as mentioned above,
Then by they according in terms of cow's milk weight 0.5% inoculum concentration be respectively connected to sterilizing cow's milk in, every 2h use dressing plate
Colony counting method is counted, these measurement results are listed in Fig. 1.Test result shows lactobacillus plantarum TH103 in cow's milk
Equally it is able to suppress the growth of salmonella typhimurium.
6, there is protective effect to Salmonella Typhimurium Infection Intestinal epithelium cell HT-29 cell.
Determine that its inhibition is invasive by following test methods:
(Chinese Academy of Sciences's allusion quotation is purchased from using RPMI1640 culture solution (Gibco Products) culture enterocyte HT-29
Type culture collection committee cell bank).HT-29 cell is in 5%CO2With under the gas condition of 95% air in 37 DEG C of incubators
In cultivated, every culture just replacement culture solution 1 time for 24 hours, continuous culture to degrees of fusion reaches 80%.Use 0.2% tryptose
Enzyme solutions digest HT-29 cell at room temperature, and cell concentration is then adjusted to 2 × 10 with RPMI1640 culture solution5A/
ML, then be dispensed into 6 porocyte culture plates, every hole 2mL.Replacement cell culture fluid daily, until forming cell monolayer.
By lactobacillus plantarum TH103 in MRS fluid nutrient medium 37 DEG C of culture 12h.4500rpm is centrifuged 5min and collects bacterium
Body, PBS buffer solution washing thalline 3 times are simultaneously resuspended, and adjustment bacterial concentration is 108cfu/mL.To salmonella typhimurium
ATCC14028 is handled according to above-mentioned same method.
Test is divided into four groups and carries out, and first group is inhibition adherency group, and second group is to inhibit intrusion group, and third group is adherency
Control group, the 4th group is intrusion control group.Cell culture fluid is sucked out from above-mentioned 6 porocyte culture plates, is cleaned with PBS buffer solution
Lactobacillus plantarum TH103 suspension 1mL and RPMI1640 culture solution is added in HT-29 cell 3 times, first group and second group every holes
1mL is mixed, and infection multiplicity is about 200:1.Third group and addition 2mL RPMI1640 culture solution in the 4th group of every hole, 37 DEG C,
5%CO2It is cleaned 3 times with being incubated under the gas condition of 95% air after 1h with PBS buffer solution, mouse typhus sand is added in all holes
Door Salmonella suspension 1mL and RPMI1640 culture solution 1mL, mixes.37 DEG C, 5%CO2Continue to incubate under the gas condition of 95% air
1h is educated, obtained cell monolayer is washed 3 times with PBS buffer solution, and first group is added 0.5mL 0.5%Triton X- with third group
100 solution cultivate 8min, and 0.5mLPBS buffer is added, then carries out gradient dilution；Second group be added after the 4th group of washing
RPMI1640 culture solution of the 1mL containing gentamicin (100 μ g/mL), 37 DEG C, 5%CO2Continue under the gas condition of 95% air
It is incubated for 1h, is washed 3 times after incubation with PBS buffer solution, 0.5mL 0.5%Triton X-100 solution is added and cultivates 8min,
0.5mLPBS buffer is added and then carries out gradient dilution.It is husky to the porose mouse typhus of institute using salmonella typhimurium culture medium
Door Salmonella is counted using dressing plate colony counting method, and each test group carries out 3 parallel tests, is repeated 3 times.Suppression
Adhesion rate processed=third group count results/the first group count results；Inhibit invasion rate=(the 4th group of count results-third group meter
Number result)/(second group of the-the first group of count results count results), these test results are listed in table 6.
6 lactobacillus plantarum TH103 of table infects the inhibiting effect of HT-29 cell to salmonella typhimurium
It can be seen that, lactobacillus plantarum TH103 adheres to salmonella typhimurium and invades colon cancer cell from table 6
It significantly inhibits, therefore, there is protection to make Salmonella Typhimurium Infection Intestinal epithelium cell HT-29 cell
With.
It is clearly illustrated by above-mentioned test, the lactobacillus plantarum TH103 has the following properties:
(1) there is acid resistance, still can survive, grow at pH2.0；
(2) there is bile tolerance ability, the gallbladder salinity being resistant to is up to 0.4% (w/v, similarly hereinafter)；
(3) have salt resistance ability, be resistant to NaCl at concentrations up to 10%；
(4) there is very high adhesive capacity to intestinal epithelial cell HT-29；
(5) have to pathogenic bacteria salmonella typhimurium (Salmonella typhimurium ATCC14028) significant
Inhibiting effect；
(6) adherency of the salmonella typhimurium to enterocyte HT-29 can obviously be inhibited；
(7) invasion of the salmonella typhimurium to intestinal epithelial cell HT-29 can be substantially reduced.
Embodiment 1: fermented vegetable
In the present invention, the fermented vegetable is prepared using following step:
Firstly, vegetables are cleaned drain after by the vegetables peeling, cutting, be added according to the vegetables poidometer 1.0%
~1.2% sucrose and 6~8% salt, stir evenly in the pre- marinated 36~48h of room temperature, drain away the water, obtain a kind of salting down in advance
Vegetables processed.
Then, weigh edible spice, use by it is described using spice weight in terms of 8-10 times of water at 75-80 DEG C of temperature
4~6h is extracted, the white wine for using spice poidometer 10% is added after cooling, is uniformly mixed so as to obtain a kind of spice juice.
Then, lactobacillus plantarum TH103 in terms of MRS fluid nutrient medium volume 1~3% is inoculated in MRS fluid nutrient medium
In, 10~15min of centrifugation is carried out under conditions of 37 DEG C of cultures 20~28h, 4500rpm, abandons supernatant, the bacterium mud nothing collected
Bacterium water is resuspended, so that the viable bacteria concentration of lactobacillus plantarum TH103 reaches 108Cfu/mL or more, to obtain a kind of bacteria suspension；
Finally, the spice juice that the pre- Pickle poidometer 10-12% will be added in obtained pre- Pickle
And 2~5% bacteria suspension, mix tinning, after sealing at room temperature fermentation until fermentation liquid pH value up to 3.0~4.0, obtain institute
State fermented vegetable.
Heretofore described vegetables are one or more selected from Chinese cabbage, asparagus lettuce, cowpea, carrot, white radishes, asparagus lettuce, volume
The vegetables of heart dish, pimento, onion or celery；The spice be it is one or more selected from capsicum, Chinese prickly ash, ginger, garlic, illiciumverum,
Fennel, orange peel, spiceleaf spice.
Embodiment 2: working stock culture of the invention is prepared
The working stock culture is prepared as steps described below:
According to 1~2% inoculum concentration in terms of MRS fluid nutrient medium weight, lactobacillus plantarum TH103 original strain is inoculated in
In MRS fluid nutrient medium, 20~28h is cultivated under conditions of 37 DEG C and is activated, continuously carries out activation 2-3 in the same manner
Secondary, obtaining lactobacillus plantarum TH103 viable count in activation culture object is 108Cfu/mL or more, then 2~6 DEG C of temperature with
10~15min of centrifugation is carried out under conditions of revolving speed 4500rpm, abandons supernatant, the PBS buffer solution of the bacterium mud pH7.2 collected
After rinsing 3-4 times, freeze drying protectant suspension is added, adjustment bacterial concentration is 109Cfu/mL carries out vacuum freeze drying after mixing
The lactobacillus plantarum TH103 leavening is obtained, and is saved at 4 DEG C.Wherein, the composition of freeze drying protectant suspension is with weight
Meter sucrose 2%, skimmed milk 15%, lactose 5%, glycerol 1% and 75% water.
Embodiment 3: ensilage is prepared using working stock culture of the invention
Ensilage preparation step is as follows:
Firstly, ensilage raw material is rubbed be switched to 2~5cm after it is stand-by.
Then, 50~60kg ensilage raw material is weighed, 0.5~0.6g is prepared according to claim 5
Lactobacillus plantarum TH103 working stock culture is uniformly mixed with 200~250mL sterile water, is sprayed onto ensilage raw material, is turned
It is fitted into ensilage bucket and is pressed and sealed after mixing, obtain after 1-2 months are stored under normal temperature condition containing lactobacillus plantarum
The ensilage of TH103.
Heretofore described ensilage raw material is one or more selected from straw, corn stover, broomcorn straw, sweet potato
Seedling, native grass, tame forage grass, weeds in field, water plant or spray leaf ensilage.
After measured, which contains 106Cfu/g or more lactobacillus plantarum TH103, with utilization 3 phase of embodiment
It is made with technique, but TH103 is not added and compares, can obviously inhibit growth and survival of the salmonella typhimurium in feed.
Embodiment 4: ferment sausage is prepared using working stock culture of the invention
Ferment sausage is prepared as steps described below:
Meat selected from pork, beef or mutton is shredded, after carrying out 24~36h of pre-freeze in 5 DEG C~8 DEG C of refrigerating chamber, with
Salt, white granulated sugar, sodium nitrite are sufficiently mixed according to weight ratio 100:4:1.5:0.015.Mixture is placed at 0~4 DEG C of temperature
Marinated 24~48h, is added according to 5~10% spice of meat poidometer, is stirred, is then added according to claim 8
The working stock culture of method preparation, additional amount are fermented after mixing work is contained 0.5~0.8% in terms of meat weight
The meat of agent is filled into casing, the fermentation 25~30 under conditions of 14 DEG C~15 DEG C of temperature with relative humidity 80% of obtained bowel lavage
It；It is toasted at 65-70 DEG C of temperature after fermentation, the ferment sausage is obtained after cooling.The spice is
One or more spices for being selected from capsicum, Chinese prickly ash, ginger, garlic, illiciumverum, fennel, orange peel, spiceleaf.
Preparation-obtained ferment sausage tart flavour is soft, gives off a strong fragrance, of good quality, and product slice property is good, real with utilizing
The production of 4 same process of example is applied, but compared with the ferment sausage that lactobacillus plantarum TH103 is not added, therefrom detects Salmonella typhimurium
The probability of bacterium and other harmful bacterias is substantially reduced.
It should be noted that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although referring to preferable
Embodiment describes the invention in detail, those skilled in the art should understand that, it can be to technology of the invention
Scheme is modified or replaced equivalently, and without departing from the spirit and scope of the technical solution of the present invention, should all be covered in this hair
In bright scope of the claims.
Claims (8)
1. a kind of lactobacillus plantarum TH103, it is characterised in that: the bacterial strain is on April 23rd, 2015 in China Microbiological bacterium
Kind preservation administration committee General Microbiological Culture collection preservation, the number that preservation is registered on the books is CGMCC10739.
2. a kind of lactobacillus plantarum TH103 according to claim 1 is in the application in fermented vegetable field, it is characterised in that:
Including,
Vegetables are cleaned drain after by the vegetables peeling, cutting, be added according to the vegetables poidometer 1.0%~1.2%
Sucrose and 6%~8% salt, stir evenly in the pre- marinated 36~48h of room temperature, drain away the water, obtain a kind of pre- Pickle；
Edible spice is weighed, use uses 8~10 times of water in terms of spice weight to extract 4~6h at 75~80 DEG C of temperature by described,
The white wine that edible spice poidometer 10% is added after cooling, is uniformly mixed so as to obtain a kind of spice juice；
Lactobacillus plantarum TH103 in terms of fluid nutrient medium volume 1~3% is inoculated in fluid nutrient medium,
10~15min of centrifugation is carried out under conditions of 37 DEG C of cultures 20~28h, 4500rpm, abandons supernatant, and the bacterium mud collected is used
Sterile water is resuspended, so that the viable bacteria concentration of lactobacillus plantarum TH103 reaches 108Cfu/mL or more, so that it is outstanding to obtain a kind of bacterium
Liquid；
The spice juice and 2 of addition in terms of the pre- Pickle weight 10~12% in the pre- Pickle
~5% bacteria suspension, mix tinning, after sealing at room temperature fermentation until fermentation liquid pH value up to 3.0~4.0, sent out
Ferment vegetables.
3. lactobacillus plantarum TH103 according to claim 2 is in the application in fermented vegetable field, it is characterised in that: described
Vegetables be for one or more of Chinese cabbage, asparagus lettuce, cowpea, carrot, white radishes, cabbage, pimento, onion or celery.
4. lactobacillus plantarum TH103 according to claim 2 is in the application in fermented vegetable field, it is characterised in that: institute
Stating spice is one or more of capsicum, Chinese prickly ash, ginger, garlic, illiciumverum, fennel, orange peel, spiceleaf.
5. a kind of application of lactobacillus plantarum TH103 according to claim 1 in working stock culture field, feature
Be: the concentration of lactobacillus plantarum TH103 is 10 in the working stock culture9Cfu/g or more.
6. lactobacillus plantarum TH103 according to claim 5 is in the application in working stock culture domain, it is characterised in that: packet
It includes,
According to 1~2% inoculum concentration in terms of culture medium weight, lactobacillus plantarum TH103 original strain is inoculated in culture medium,
20~28h is cultivated under conditions of 37 DEG C to be activated, and is continuously carried out activation 2~3 times, is obtained in activation culture object in the same manner
Lactobacillus plantarum TH103 viable count is not less than 108cfu/mL；
10~15min of centrifugation is carried out under conditions of 2~6 DEG C of temperature with revolving speed 4500rpm, abandons supernatant, the bacterium mud collected
It is rinsed 3~4 times with the buffer of pH7.2, then, freeze drying protectant suspension is added into the bacterium mud, adjusts bacterial concentration
For not less than 109Cfu/mL carries out vacuum freeze drying and obtains the lactobacillus plantarum TH103 leavening after mixing, wherein
The composition of the freeze drying protectant suspension is sucrose 2%, skimmed milk 15%, lactose 5%, glycerol 1% and 75% by weight
Water.
7. a kind of lactobacillus plantarum TH103 according to claim 1 exists in the use in fermentation ensilage field, feature
In: including ensilage raw material being rubbed stand-by after cutting section；50~60kg ensilage raw material is weighed, by 0.5~0.6g as weighed
Benefit require 6 described in lactobacillus plantarum TH103 leavening be uniformly mixed with 200~250mL sterile water, be sprayed onto ensilage
It in raw material, turns to be fitted into after mixing in ensilage bucket and is pressed and sealed, obtained after being stored 1~2 month under normal temperature condition
The ensilage of the TH103 containing lactobacillus plantarum.
8. a kind of lactobacillus plantarum TH103 according to claim 1 exists in the application in ferment sausage field, feature
In: including,
Meat selected from pork, beef or mutton is shredded, after carrying out 24~36h of pre-freeze in 5 DEG C~8 DEG C of refrigerating chamber, with food
Salt, white granulated sugar, sodium nitrite are sufficiently mixed according to weight ratio 100:4:1.5:0.015；
Mixture is placed at 0~4 DEG C of temperature and pickles 24~48h, is added according to 5~10% spice of meat poidometer, stirring is mixed
It closes, the lactobacillus plantarum TH103 leavening according to claim 7 is then added, additional amount is in terms of meat weight
0.5~0.8%, the meat containing working stock culture is filled into casing after mixing, obtained bowel lavage 14 DEG C of temperature~
15 DEG C with relative humidity 80% under conditions of ferment 25~30 days；
It is toasted at 65~70 DEG C of temperature after fermentation, the ferment sausage is obtained after cooling.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510385613.6A CN104928219B (en) | 2015-06-30 | 2015-06-30 | A kind of lactobacillus plantarum TH103 and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510385613.6A CN104928219B (en) | 2015-06-30 | 2015-06-30 | A kind of lactobacillus plantarum TH103 and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104928219A CN104928219A (en) | 2015-09-23 |
| CN104928219B true CN104928219B (en) | 2018-12-18 |
Family
ID=54115647
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510385613.6A Active CN104928219B (en) | 2015-06-30 | 2015-06-30 | A kind of lactobacillus plantarum TH103 and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104928219B (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107467586B (en) * | 2017-08-31 | 2021-02-26 | 四川民福记食品有限公司 | Sichuan-flavor pickle making method |
| CN107988094A (en) * | 2017-11-14 | 2018-05-04 | 北京好实沃生物技术有限公司 | One plant of lactobacillus fermenti HEW-A846 and its application |
| CN109234181B (en) * | 2017-12-22 | 2020-06-09 | 浙江大学 | Lactobacillus plantarum ZJUF HN9 and application thereof |
| CN110106112B (en) * | 2019-05-08 | 2021-06-15 | 北京农学院 | Mixed probiotics for fermented vegetables, fermented vegetables and preparation method thereof |
| CN110835620B (en) * | 2019-12-16 | 2022-08-09 | 吉林农业大学 | Lactobacillus plantarum and application thereof |
| CN111165651A (en) * | 2020-01-14 | 2020-05-19 | 南昌大学 | Additive premixed feed and application thereof |
| CN111616290A (en) * | 2020-04-26 | 2020-09-04 | 河北工程大学 | Lactic acid bacteria fermented red date beverage and preparation method thereof |
| CN111616289A (en) * | 2020-04-26 | 2020-09-04 | 河北工程大学 | Lactic acid bacteria fermented red date and sea buckthorn beverage and preparation method thereof |
| CN111513219A (en) * | 2020-04-26 | 2020-08-11 | 河北工程大学 | Lactobacillus fermented sea-buckthorn beverage and preparation method thereof |
| CN113564068B (en) * | 2021-05-25 | 2023-05-30 | 南昌大学 | Lactobacillus plantarum 1201 and application thereof in preparation of salmonella typhimurium inhibition products |
| CN115044498B (en) * | 2022-05-13 | 2023-10-03 | 江南大学 | Lactobacillus plantarum strain suitable for fermenting red jujube beverage and application thereof |
| CN114908017B (en) * | 2022-05-31 | 2023-03-14 | 四川农业大学 | Lactobacillus plantarum and application thereof in silage |
| CN115250872B (en) * | 2022-08-29 | 2023-06-16 | 承德市艺风园林绿化工程有限公司 | Organic matrix containing probiotics for planting and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103014166A (en) * | 2012-12-28 | 2013-04-03 | 江南大学 | Method for screening lactobacillus plantarum ST-III salt-tolerance gene |
| CN103275921A (en) * | 2013-05-16 | 2013-09-04 | 江南大学 | Method for improving cholate tolerance of lactobacillus |
| CN103275920A (en) * | 2013-05-16 | 2013-09-04 | 江南大学 | Method for improving capacity of lactobacillus adhered to epithelial cells of intestinal tract |
-
2015
- 2015-06-30 CN CN201510385613.6A patent/CN104928219B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103014166A (en) * | 2012-12-28 | 2013-04-03 | 江南大学 | Method for screening lactobacillus plantarum ST-III salt-tolerance gene |
| CN103275921A (en) * | 2013-05-16 | 2013-09-04 | 江南大学 | Method for improving cholate tolerance of lactobacillus |
| CN103275920A (en) * | 2013-05-16 | 2013-09-04 | 江南大学 | Method for improving capacity of lactobacillus adhered to epithelial cells of intestinal tract |
Non-Patent Citations (3)
| Title |
|---|
| 曲菲.长白山地区发酵食品来源益生菌的分离、筛选及应用研究.《中国优秀硕士学位论文数据库，工程科技辑》.2015,正文第2.2.1节-第3.2.6节，第3.4节. * |
| 植物乳杆菌的生理功能及应用;王水泉 等;《中国农业科技导报》;20101231;第12卷(第4期);摘要，第2.1-2.2节 * |
| 长白山地区发酵食品来源益生菌的分离、筛选及应用研究;曲菲;《中国优秀硕士学位论文数据库，工程科技辑》;20150215;正文第2.2.1节-第3.2.6节，第3.4节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104928219A (en) | 2015-09-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104928219B (en) | A kind of lactobacillus plantarum TH103 and application thereof | |
| US9862955B2 (en) | Plant endophytic bacteria Bacillus oryzicola isolated from rice rizhosphere, and development of agent for natural plant protection and plant enhancement using the same | |
| Gupta et al. | Probiotic potential of Lactobacillus plantarum LD1 isolated from batter of Dosa, a South Indian fermented food | |
| CN105557756B (en) | Plant bacteriostasis method adopting bacillus methylotrophicus strain NKG-1 | |
| CN109536415A (en) | A kind of Lactobacillus rhamnosus and its application | |
| CN102559563B (en) | Pediococcus acidilactici CCFM7902 and application thereof | |
| CN108841760A (en) | One plant can improve Growth of Grass Carps Ctenopharyngodon Idellus performance, immunocompetence and the bacillus licheniformis FA6 and its application that adjust intestinal microflora | |
| CN112795496B (en) | Paenibacillus polymyxa and application thereof in preventing and treating stem basal rot of Chinese cabbage | |
| CN108384735A (en) | Lactobacillus plantarum CCFM1019, its fermented food and its application in medicine preparation | |
| CN107177522A (en) | One plant height activity forage plant lactobacillus and its cultural method and application | |
| CN116083289B (en) | Bacillus subtilis strain 4-4 and application, product and method thereof | |
| Andriani et al. | Characterization of Bacillus subtilis and B. licheniformis potentials as probiotic bacteria in Vanamei shrimp feed (Litopenaeus vannamei Boone, 1931) | |
| CN109161498A (en) | Bacillus subtilis M406 and its preparing the application in bacteriocin and cellulase | |
| CN114231473A (en) | Probiotic lactobacillus plantarum and application thereof in preparation of low-salt fermented meat food | |
| CN110408577B (en) | Lactobacillus casei for controlling vegetable bacterial soft rot and application thereof | |
| CN108641989A (en) | One plant of Methylotrophic bacillus and its application | |
| CN104877940B (en) | One plant of streptococcus thermophilus | |
| CN107699526B (en) | Actinomycete strain for preventing and treating gray mold and application thereof | |
| CN101892170B (en) | Entomopathogenic nematode symbiotic bacteria and application thereof | |
| CN105018395B (en) | One bacillus pumilus and its application in alternaria leaf spot of apple is prevented | |
| CN116536207A (en) | Bacillus atrophaeus WLKYSY-4, biological microbial inoculum and application thereof | |
| Ortega-Morales et al. | Antagonism of Bacillus spp. isolated from marine biofilms against terrestrial phytopathogenic fungi | |
| Huang | Mathematical modeling and numerical analysis of the growth of non-O157 Shiga toxin-producing Escherichia coli in spinach leaves | |
| CN109402023A (en) | A kind of mould proof Bacillus strain and its application in foodstuff preservation | |
| JP6261664B2 (en) | Lyophilized product containing Lactobacillus plantarum LLP5193, which is excellent in acid resistance, bile resistance and cell adhesion ability, as an active ingredient |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20211119 Address after: 610213 No. 3510, floor 35, building 1, No. 500, middle section of Tianfu Avenue, Chengdu hi tech Zone, China (Sichuan) pilot Free Trade Zone, Chengdu, Sichuan Patentee after: Sichuan qianyiding Technology Co.,Ltd. Address before: South Street, Hanshan District bright 056038 Hebei province Handan City No. 199 Patentee before: HEBEI University OF ENGINEERING |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20211209 Address after: 214400 No. 173, Chengjiang Middle Road, Jiangyin City, Wuxi City, Jiangsu Province (business place: building C2, No. 6, Dongsheng West Road, Jiangyin City) Patentee after: JIANGSU NEW-BIO BIOTECHNOLOGY CO.,LTD. Address before: 610213 No. 3510, floor 35, building 1, No. 500, middle section of Tianfu Avenue, Chengdu hi tech Zone, China (Sichuan) pilot Free Trade Zone, Chengdu, Sichuan Patentee before: Sichuan qianyiding Technology Co.,Ltd. |