CN102021127B

CN102021127B - Lactobacillus paracasei and application thereof

Info

Publication number: CN102021127B
Application number: CN2010102386713A
Authority: CN
Inventors: 陈坚; 张娟; 堵国成; 王淼; 张茜; 吴重德; 王松; 毕洁; 顾磊
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2010-07-28
Filing date: 2010-07-28
Publication date: 2012-05-30
Anticipated expiration: 2030-07-28
Also published as: CN102021127A

Abstract

The invention discloses Lactobacillus paracasei BE10-215, which has been preserved in China center for type culture collection with the preservation number of CCTCC M 2010169 on 7th July, 2010. The strain has higher clearing efficiency of algal toxins, is applied to the field of health care food and has the efficacies of improving the human immunity, assisting the nutrition and digestion and the like.

Description

One strain lactobacillus paraceasi and application thereof

Technical field

The invention belongs to technical field of bioengineering, relate to a strain lactobacillus paraceasi and an application method thereof.

Background technology

World nutrition educational circles is for the definition of " probiotic bacterium ": refer to the intestinal physiology bacterium of one type of useful human health, also often be described to the bacterium of " optimum " or " health ".It comprises bacteriums such as lactobicillus bulgaricus, thermophilus streptococcus, bifidus bacillus, Lactobacterium acidophilum, lactobacillus salivarius, lactobacillus fermentum.Be applied to the milk-product, meat product, fruit and vegetable product of the probiotic products profitable probliotics fermentation of human body etc., the medicines and health protection industry.Milk-acid bacteria is main a type in the probiotic bacterium, have antitumor, alleviate physiological actions such as lactose intolerance, strengthening immunity, reducing cholesterol, adjustment intestinal microflora.Milk-acid bacteria is mainly used in the production of leavened foods such as fermented meat prods, fermentation aquatic product article; Suppress the growth of spoilage organism and pathogenic bacterium and the generation of toxin, the preservation period of leavened food is prolonged greatly, helped storage and circulation; Edible safety is better, more abundant in nutrient.

Microcystin (MCs) is a kind of ring-type seven peptide materials that produced by blue-green algaes such as the microcystic aeruginosa in the eutrophication water, wawter bloom anabena, beads algaes.Microcystin is the effect target organ with the animal livers, thereby the arrestin phosphatase activity brings out a series of pathologies such as cancer specifically.Because strong toxicity, in food chain, to involve scope wide and have distinct regional feature, Microcystin becomes the healthy important killer of harm humans day by day.At present, the means of intervention MCs can be divided into physical control, chemical prevention and biological control.Wherein, the part probiotic bacterium really can be implemented in because of its security (GRAS) becomes the intervention effect of MCs and remove the biocontrol agent that contacts with water source, food " zero distance " in the MCs process in the microorganism treatment.Simultaneously and since probiotic bacterium grow surely behind the human intestinal suppress the harmful microorganism growth, improve body immunity, assist digestion and dietetic alimentation etc. many aspect the long-term efficacy of performance, make probiotic bacterium more outstanding as the value of MCs in the body " street cleaner ".

Summary of the invention

The invention provides a strain lactobacillus paraceasi ( Lactobacillus paracaseiBBE10-215).

Lactobacillus paraceasi BBE10-215 provided by the present invention ( Lactobacillus paracasei), being preserved in Chinese typical culture collection center on July 7th, 2010, the depositary institution address is Chinese Wuhan, Wuhan University, deposit number is CCTCC NO:M 2010169, taxonomy called after lactobacillus paraceasi ( Lactobacillus paracasei); Its 16SrDNA classifies basic phylogenetic tree as shown in Figure of description 1 with its 16SrDNA total order shown in SEQ ID NO:1 in the nucleotides sequence tabulation.

The invention provides a kind of application of said lactobacillus paraceasi, is the removing that cultured lactobacillus paraceasi is used for Microcystin.The ratio that cultured lactobacillus paraceasi adjustment bacterium is dense is seeded in the algae toxin soiutions to optimum range, and static cultivation or not timing are stirred solution is mixed under the room temperature.

Said lactobacterium casei is for to screen from traditional fermented food such as pickles, sausage; Its storage conditions is following: get 2-3 ring probiotic bacterium from well-grown flat board and move into the MRS substratum; 37 ℃ leave standstill cultivation 20 h; Get 0.7 mL immigration and fill in the glycerine pipe of the aseptic glycerine of 0.3 mL, put-70 ℃ of refrigerators and preserve.

Said MRS substratum is: glucose 20 g L ^-1, peptone 10 g L ^-1, Carnis Bovis seu Bubali cream 10 g L ^-1, Yeast diffusion juice 5 g L ^-1, anhydrous sodium acetate 5 g L ^-1, potassium primary phosphate 2 g L ^-1, Triammonium citrate 2 g L ^-1, MgSO ₄7H ₂O 0.2 g L ^-1, MnSO ₄H ₂O 0.05 g L ^-1, Tween-80 1 mL L ^-1, pH 5.5.

Said lactobacterium casei culture condition is: the probiotic bacterium of glycerine being guaranteed the Tibetan is inserted the MRS liquid nutrient medium, leaves standstill at 37 ℃ and cultivates 20 h to the logarithm middle and later periods, measures-70 ℃ of glycerine stock solutions with 2% inoculation and is inoculated in the MRS substratum, and 37 ℃ leave standstill cultivation 20 h.

At the algae toxin concentration is 1000 μ g L ^-1Condition under, 10 ⁹CFU mL ^-1Thalline cultivate 24 h altogether with it after, degradation rate is up to 12% (Fig. 2).At the algae toxin concentration is 100 μ g L ^-1Condition under, bacterium is dense to be 10 ⁹CFU mL ^-1Thalline cultivate 24 h altogether with it after, degradation rate is up to 76%.At algae toxin initial concentration is 500 μ g L ^-1Condition under, bacterium is dense to be 10 ⁹CFU mL ^-1Thalline cultivate 24 h altogether with it after, degradation rate is up to 39%.

The method of performance liquid is adopted in the detection of Microcystin:

Chromatographic column: Agilent ZORBAX Eclipse XDB-C18 (4.6 * 150 mm, 5.0 μ m)

Moving phase: acetonitrile: H ₂O (0.05% TFA)=40:60 (v/v)

Sample size: 20 μ l; Flow velocity: 1 mL/min; Column temperature: 40 ℃

Detector: DAD 238 nm; MCs RT: 3.48 min

The present invention has the algae toxin and removes good effectiveness, and lactobacillus paraceasi is particularly suitable for being applied to field of food as a kind of probiotic bacterium, makes that the algae toxin obtains fast, safety and removing efficiently.

Description of drawings

Fig. 1: probiotic lactobacillus phylogenetic tree

Embodiment

Embodiment 1: the lactobacillus paraceasi screening method

From traditional fermented food such as pickles, sausage, get certain sample and join enrichment culture in the 30 mL MRS substratum, cultivate 20 h for 37 ℃.With the nutrient solution gradient dilution, it is dull and stereotyped to coat the MRS that adds 0.02% purpurum bromocresolis, cultivates 48 h in 37 ℃ of incubators.Picking makes the tangible single bacterium colony of purpurum bromocresolis variable color circle.Carry out plate streaking repeatedly and separate, triplicate obtains single bacterium colony of purifying, supplies next step to identify and uses.

Identify and mainly to pass through: 1) colony characteristics is observed: direct viewing with the naked eye, choosing colony diameter between 1 ~ 3 mm, rounded protuberance, smooth surface or coarse slightly, milky white, greyish white or dark yellow etc. meet the bacterial strain of milk-acid bacteria colony characteristics.2) individual morphology is observed: observe with the opticmicroscope gramstaining.Select Gram-positive, no gemma bacterial strain.The bacterial strain that satisfies the evaluation condition is chosen glycerine guarantee the Tibetan.

Measure-70 ℃ of glycerine stock solutions with 2% inoculation and be inoculated in the MRS substratum, 37 ℃ leave standstill cultivation.Get 37 ℃ and leave standstill the fermented liquid of cultivating 20 h (logarithm middle and later periods), centrifugal 10 min (3200 * g, 4 ℃) collect thalline, use centrifugal 2 times of phosphate buffered saline buffer (pH 7.0) washing again, and the adjustment bacterium is dense 10 ⁹CFU mL ^-1About.The thalline that obtains is resuspended in to contain algae toxin (MC-LR) concentration be 1000 μ g L ^-1Phosphate buffered saline buffer (pH 7.0) in, place 37 ℃ of shaking tables (150 r min ^-1) the lucifuge cultivation.Take a sample after cultivating 24 h, centrifugal 10 min (12000 * g, 4 ℃) get supernatant and detect the remaining concentration of MC-LR with HPLC.

Detect through HPLC; Screen a strain and have the bacterial strain that strong MC-LR removes ability; Can know that through 16SrDNA sequential analysis (see in the nucleotides sequence tabulation shown in the SEQ ID NO:1) this bacterial strain is a lactobacillus paraceasi; Be preserved in DSMZ on July 7th, 2010, deposit number is CCTCC M 2010169.

Embodiment 2:

The MC-LR starting point concentration is 100 μ g L ^-1

Measure-70 ℃ of glycerine stock solutions with 2% inoculation and be inoculated in the MRS substratum, 37 ℃ leave standstill cultivation.Get 37 ℃ and leave standstill the fermented liquid of cultivating 20 h (logarithm middle and later periods), centrifugal 10 min (3200 * g, 4 ℃) collect thalline, use centrifugal 2 times of phosphate buffered saline buffer (pH 7.0) washing again, and the adjustment bacterium is dense 10 ⁹CFU mL ^-1About.The thalline that obtains is resuspended in to contain MC-LR concentration be 100 μ g L ^-1Phosphate buffered saline buffer (pH 7.0) in, place 37 ℃ of shaking tables (150 r min ^-1) the lucifuge cultivation.Take a sample after cultivating 24 h, centrifugal 10 min (12000 * g, 4 ℃) get supernatant and detect the remaining concentration of MC-LR with HPLC.Calculating acquisition bacterial strain is 76% to the clearance rate of MC-LR.

Embodiment 3:

The MC-LR starting point concentration is 500 μ g L ^-1

Measure-70 ℃ of glycerine stock solutions with 2% inoculation and be inoculated in the MRS substratum, 37 ℃ leave standstill cultivation.Get 37 ℃ and leave standstill the fermented liquid of cultivating 20 h (logarithm middle and later periods), centrifugal 10 min (3200 * g, 4 ℃) collect thalline, use centrifugal 2 times of phosphate buffered saline buffer (pH 7.0) washing again, and the adjustment bacterium is dense 10 ⁹CFU mL ^-1About.The thalline that obtains is resuspended in to contain MC-LR concentration be 500 μ g L ^-1Phosphate buffered saline buffer (pH 7.0) in, place 37 ℃ of shaking tables (150 r min ^-1) the lucifuge cultivation.Take a sample after cultivating 24 h, centrifugal 10 min (12000 * g, 4 ℃) get supernatant and detect the remaining concentration of MC-LR with HPLC.Calculating acquisition bacterial strain is 39% to the clearance rate of MC-LR.

Embodiment 4:

The MC-LR starting point concentration is 1000 μ g L ^-1

Measure-70 ℃ of glycerine stock solutions with 2% inoculation and be inoculated in the MRS substratum, 37 ℃ leave standstill cultivation.Get 37 ℃ and leave standstill the fermented liquid of cultivating 20 h (logarithm middle and later periods), centrifugal 10 min (3200 * g, 4 ℃) collect thalline, use centrifugal 2 times of phosphate buffered saline buffer (pH 7.0) washing again, and the adjustment bacterium is dense 10 ⁹CFU mL ^-1About.The thalline that obtains is resuspended in to contain MC-LR concentration be 1000 μ g L ^-1Phosphate buffered saline buffer (pH 7.0) in, place 37 ℃ of shaking tables (150 r min ^-1) the lucifuge cultivation.Take a sample after cultivating 24 h, centrifugal 10 min (12000 * g, 4 ℃) get supernatant and detect the remaining concentration of MC-LR with HPLC.Calculating acquisition bacterial strain is 12% to the clearance rate of MC-LR.

It is understandable that, concerning those of ordinary skills, can be equal to replacement or change according to technical scheme of the present invention and inventive concept thereof, and all these changes or replacement all should belong to the protection domain of the appended claim of the present invention.

The nucleotides sequence tabulation

< 110>Southern Yangtze University

< 120>one strain lactobacillus paraceasi and application thereof

<160> 1

<170> PatentIn?version?3.3

<210> 1

<211> 1462

<212> DNA

<213> Lactobacillus?paracasei?BBE10-215

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gtgctataca?tgcagtcgaa?cgagttctcg?ttgatgatcg?gtgcttgcac?cgagattcaa 60

catggaacga?gtggcggacg?ggtgagtaac?acgtgggtaa?cctgccctta?agtgggggat 120

aacatttgga?aacagatgct?aataccgcat?agatccaaga?accgcatggt?tcttggctga 180

aagatggcgt?aagctatcgc?ttttggatgg?acccgcggcg?tattagctag?ttggtgaggt 240

aatggctcac?caaggcgatg?atacgtagcc?gaactgagag?gttgatcggc?cacattggga 300

ctgagacacg?gcccaaactc?ctacgggagg?cagcagtagg?gaatcttcca?caatggacgc 360

aagtctgatg?gagcaacgcc?gcgtgagtga?agaaggcttt?cgggtcgtaa?aactctgttg 420

ttggagaaga?atggtcggca?gagtaactgt?tgtcggcgtg?acggtatcca?accagaaagc 480

cacggctaac?tacgtgccag?cagccgcggt?aatacgtagg?tggcaagcgt?tatccggatt 540

tattgggcgt?aaagcgagcg?caggcggttt?tttaagtctg?atgtgaaagc?cctcggctta 600

accgaggaag?cgcatcggaa?actgggaaac?ttgagtgcag?aagaggacag?tggaactcca 660

tgtgtagcgg?tgaaatgcgt?agatatatgg?aagaacacca?gtggcgaagg?cggctgtctg 720

gtctgtaact?gacgctgagg?ctcgaaagca?tgggtagcga?acaggattag?ataccctggt 780

agtccatgcc?gtaaacgatg?aatgctaggt?gttggagggt?ttccgccctt?cagtgccgca 840

gctaacgcat?taagcattcc?gcctggggag?tacgaccgca?aggttgaaac?tcaaaggaat 900

tgacgggggc?ccgcacaagc?ggtggagcat?gtggtttaat?tcgaagcaac?gcgaagaacc 960

ttaccaggtc?ttgacatctt?ttgatcacct?gagagatcag?gtttcccctt?cgggggcaaa 1020

atgacaggtg?gtgcatggtt?gtcgtcagct?cgtgtcgtga?gatgttgggt?taagtcccgc 1080

aacgagcgca?acccttatga?ctagttgcca?gcatttagtt?gggcactcta?gtaagactgc 1140

cggtgacaaa?ccggaggaag?gtggggatga?cgtcaaatca?tcatgcccct?tatgacctgg 1200

gctacacacg?tgctacaatg?gatggtacaa?cgagttgcga?gaccgcgagg?tcaagctaat 1260

ctcttaaagc?cattctcagt?tcggactgta?ggctgcaact?cgcctacacg?aagtcggaat 1320

cgctagtaat?cgcggatcag?cacgccgcgg?tgaatacgtt?cccgggcctt?gtacacaccg 1380

cccgtcacac?catgagagtt?tgtaacaccc?gaagccggtg?gcgtaaccct?ttagggagcg 1440

agccgtctaa?gggacaaaag?gt 1462

Claims

One strain lactobacillus paraceasi ( Lactobacillus paracasei) BBE10-215, deposit number CCTCC NO:M 2010169.
2. a method of cultivating the said lactobacillus paraceasi of claim 1 is in the MRS substratum, to leave standstill the cultivation lactobacillus paraceasi.
3. cultural method as claimed in claim 2 is characterized in that pH is 5.5; Culture temperature is 37 ℃.
4. the application of the said lactobacillus paraceasi of claim 1 is the Microcystin that cultured lactobacillus paraceasi is used for removing water body.