CN103667106A - Lactobacillus plantarum, bacteriocins and culture, separation and purification method thereof - Google Patents

Abstract

The invention discloses a Lactobacillus plantarum, bacteriocins and a culture, separation and purification method thereof. The collection number of the Lactobacillus plantarum 163 strain is CGMCC No.8224. The Lactobacillus plantarum is cultured in an MRS liquid culture medium or a celery cabbage/tomato culture medium, and the bacteriocins can be generated through fermentation, wherein the generated bacteriocins are two novel bacteriocins having a primary structure composed of two amino acid sequences and can be obtained by performing four times of separation, purification and identification. Determination results show that the bacteriocins have wide antibacterial spectrum and favorable heat stability and pH value stability, and can be widely used in the fields of foods and medicine.

Description

A kind of plant lactobacillus, its bacteriocin and cultivation and separation purification method

Technical field

The present invention relates to a kind of plant lactobacillus and bacteriocin thereof, particularly a kind of plant lactobacillus, its bacteriocin and cultivation and separation purification method.

Background technology

In foodstuffs industry, the anti-corrosive fresh-keeping of related food is a major issue urgently to be resolved hurrily all the time.According to FDA statistics, it is various putrid and deteriorated that the whole world approximately has the food of 10%-20% to be lost in every year, and in the process of food spoilage, microorganism plays conclusive effect.For the reason of food spoilage, take different processing means to control the microorganism in food, but some method for disinfection can produce detrimentally affect to the quality of food, local flavor and nutrition.Food preservatives is easy to use effectively because of it, and the commodity value that can preserve well food, remains one of important measures of food antiseptic at present.

Milk-acid bacteria and the tunning mankind have had the use of thousands of years historical, are not only applied in now food mfg, but also be applied in cure the disease, health care aspect.The utilization fine quality historical and that have no side effect of thousands of years makes milk-acid bacteria be considered to GRAS(generally recognized as safe generally recognized as safe) bacterium.Its tunning is regarded as GRAS material.Wherein the milk-acid bacteria of some kind can be by Ribosome biogenesis micromolecule polypeptide----bacteriocin in metabolic process, and parts of fine rhzomorph can suppress the growth and breeding of pathogenic micro-organism, putridness bacterium, has anticorrosion application prospect.At present, research both at home and abroad the most deeply, the most thorough nisin--Nisin, more than 80 countries and regions, be used widely (being also unique a kind of commercial applications approval that obtains).China also listed Nisin in 1992 the amendments in the GB GB2660-86 implementing on October 1st, 1992, can be used in the preservation of tinned pre-, vegetable protein beverage, milk-product and meat product.Yet Nisin activity under alkaline environment is lower; Only Glan positive bacteria is had to effect, to the negative pathogenic bacterium of Glan, yeast, mould, viral unrestraint effect; Activity is subject to biological enzyme to affect larger grade and has limited its use.Thereby the new high-efficiency broad spectrum bacteriocin lab of continuation screening becomes worldwide internal medicine scholar's a popular research direction in recent years.

In 32 kinds of sanitass that at present China's foodstuff additive standard (GB2760) allows to use, take Chemical Preservative as main, natural antiseptic agent is two kinds of nisin (Nisin) and tennecetins (Natamycin) only.The common cost of Chemical Preservative is lower, stable chemical performance and antimicrobial spectrum are wide, favorable anti-corrosion effect, but research at present shows, to human body, there is in various degree certain toxicity in Chemical Preservative, some of them have and lure carcinous and teratogenecity, easily cause food poisoning use in the situation that exceeding standard.

Along with the raising of living standards of the people and the enhancing of Consciousness of food security, people are to the safety and sanitation of food and self health status growing interest, not containing the whole food of Chemical Preservative and green food more easily by consumer acceptance and acceptance.Therefore, the nontoxic natural antiseptic agent of research and development highly effective and safe has become the main direction of current international food Development of Additives.

China allows the antimicrobial substance Nisin and the Natamycin that use to be microbial source natural antiseptic agent.Nisin is the synthetic small peptide molecule of streptococcus acidi lactici, and to gram-positive microorganism, particularly to clostridium botulinum and spore thereof, stearothermophilus gemma and spore thereof, streptococcus aureus, listeria bacteria etc. have remarkable antagonistic activity.Nisin is current generally recognized as safe and the widest antiseptics for natural food of range of application, but also there is some limitation, its antimicrobial spectrum is narrower, only gram-positive microorganism is shown to remarkable restraining effect, Gram-negative bacteria and fungi are not had to activity, under neutral and alkaline condition to thermally labile, thereby make its application in food be subject to certain limitation.Natamycin comes from actinomycetes Natal mould (Streptomyces natalensis), is polyene hydrocarbon Macrolide fungistat, effectively mould fungus inhibition, yeast and filamentous fungus, but invalid to bacterium and virus.

Although as more in bacteriocin lab, antibacterial peptide research to the microorganism natural antiseptic agent of different sources both at home and abroad, but practical application seldom in foodstuffs industry, this is mainly because some of them natural antiseptic agent antimicrobial spectrum is narrow, fungistatic effect is not ideal enough, anti-microbial property is unstable, especially on the high side, productive rate is low and security can not determined, is also difficult at present effect and the status of substituted chemistry sanitas.Therefore the antiseptics for natural food of, developing wide spectrum, efficient, stable, safety, the high and low cost of productive rate has potential using value and the huge market requirement.

Summary of the invention

The object of the invention is to provides a kind of plant lactobacillus, its bacteriocin and cultivation and separation purification method in order to overcome above the deficiencies in the prior art.

The present invention is realized by following technique means:

A kind of plant lactobacillus (Lactobacillus plantarum) 163, culture presevation number is CGMCC NO.8224.

Above-mentioned plant lactobacillus (Lactobacillus plantarum) 163, on September 20th, 2013, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (be called for short CGMCC, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preservation registration number is CGMCC NO.8224.

A bacteriocin, above-described plant lactobacillus (Lactobacillus plantarum) 163 fermentations obtain.

Described bacteriocin, can have the aminoacid sequence shown in SEQ ID NO.1.

Described bacteriocin, can have the aminoacid sequence shown in SEQ ID NO.2.

A preparation method for described bacteriocin, is obtained by following step cultivation and fermentation by plant lactobacillus (Lactobacillus plantarum) 163:

(1) plant lactobacillus (Lactobacillus plantarum) 163 seed liquor preparations: by the bacterial classification of preservation, be inoculated in MRS liquid nutrient medium 37 ℃ of standing cultivation 12-16h;

(2) plant lactobacillus (Lactobacillus plantarum) 163 fermentations: by the seed liquor of activation, by volume per-cent is 1% to be inoculated in MRS liquid nutrient medium or Chinese cabbage tomato substratum, after MRS liquid nutrient medium standing cultivation 24h or the standing cultivation of Chinese cabbage tomato substratum 30h, the centrifugal fermented liquid obtaining containing bacteriocin of 5000rpm.

The preparation method of above-described bacteriocin, wherein the Chinese cabbage tomato culture medium prescription described in step (2) can be glucose 10g, cabbage juice 200mL, tomato 50mL, distilled water 750mL, pH6.5.

The separation purification method of the bacteriocin described in more than one, first carries out cultivation and fermentation by plant lactobacillus (Lactobacillus plantarum) 163, then takes following steps to carry out:

(1) ammonium sulfate precipitation purifying: first that plant lactobacillus MRS fermented liquid is concentrated, concentrated solution adds Powdered ammonium sulfate to saturation ratio 60%, stir, centrifugally at 4 ℃ get supernatant liquor after completely and continue to add Powdered ammonium sulfate to saturation ratio 85%, stir, centrifugal complete at 4 ℃, obtain precipitation, precipitation is dissolved with distilled water;

(2) Sephadex G25 purifying: the sample that ammonium sulfate precipitation is got off concentrates, draws sample, opens the valve below the post that installs SephadexG25 column material, open constant flow pump, with the abundant wash-out of pure water, regulate flow velocity, utilize automatic collector to collect cut;

(3) Sephadex LH-20 purifying: Sephadex LH-20 fills post, adds distilled water to dissolve, loading after the active fractions freeze-drying out of G25 purifying, use 80% methanol-eluted fractions, flow velocity 1.5mL/9min, every cut meets 1.5mL, collect cut, after purifying, obtain two active elution peaks;

(4) HPLC purifying: first active elution peak, utilizes RP-HPLC look to boil method purifying, purification condition: C18 post, A: water+0.1%TFA, B: acetonitrile+0.1%TFA, Time:60min, A:95%-5%, B:5%-95% gradient elution, wavelength: 267nm, flow velocity 0.4mL/min; Second active elution peak, purification condition: 95%A+5%B isocratic elution, A: water+0.1%TFA, B: acetonitrile+0.1%TFA, time: 40min, pillar: C18 post, flow velocity 3mL/min, detects wavelength 267nm.

Above-described bacteriocin, can be applied in food antiseptic or preparation germ resistance medicine.

Beneficial effect

The present invention is purified into two kinds of bacteriocins (SEQ ID NO.1 and SEQ ID NO.2) with broad-spectrum antibacterial action from plant lactobacillus, and its beneficial effect is as follows:

(1) source safety, plant lactobacillus separation, from Guizhou traditional fermented food fermentation pickled radiss, has time-honored edible history, therefore from safety, the tunning of plant lactobacillus (two kinds of bacteriocins, SEQ ID NO.1 and SEQ ID NO.2) is direct-edible.

(2) plant lactobacillus (Lactobacillus plantarum) 163 acid producing abilities are strong, itself produces again the bacteriocin of broad-spectrum antibacterial action, therefore can be used as the bacterium that sets out of industrial food fermentation, and can reduce the use of even stopping its other sanitass of leavened food, extend the shelf-lives of leavened food.

(3) bacteriocin SEQ ID NO.1 and the SEQ ID NO.2 of the broad-spectrum antibacterial action that plant lactobacillus (Lactobacillus plantarum) 163 produces, has a broad antifungal spectrum, can suppress gram positive organism, gram-negative bacteria, can add in food as natural food-preservative, extend the shelf-lives of food.

(4) SEQ ID NO.1 and SEQ ID NO.2 character are comparatively stable, and pH, temperature are little to its activity influence, and in the scope of pH3-6 and temperature below 80 ℃, its activity can retain substantially completely, is more conducive to the application in food.

Accompanying drawing explanation

Fig. 1 is Sephadex LH-20 active elution curve after purifying for the third time.

Fig. 2 is the HPLC figure of SEQ ID NO1.

Fig. 3 is MALDI-TOF-MS and the MALDI-TOF-MS/MS figure of SEQ ID NO1, and wherein a is MALDI-TOF-MS, and b is MALDI-TOF-MS/MS.

Fig. 4 is the amino acid composition analysis figure of SEQ ID NO1.

Fig. 5 is the HPLC figure of SEQ ID NO2.

Fig. 6 is the MALDI-TOF-MS figure of SEQ ID NO2.

Fig. 7 is the MALDI-TOF-MS/MS figure of SEQ ID NO2.

Embodiment

The present invention's separation and purification from the traditional fermented food pickled radiss of Guizhou goes out milk-acid bacteria 430 strains, therefrom filters out the plant lactobacillus (Lactobacillus plantarum) 163 that a strain can produce broad-spectrum antibacterial action bacteriocin.Main biological property: on MRS solid medium, be white in color or oyster white point, without gemma gram-positive microorganism, rod-short, atrichia, catalase is negative, can tolerate 6.5%(mass ratio, lower same) following NaCl, and produce acid, do not produce CO ₂, H ₂s, NH ₃fermentable fibres element, polychrom, fructose, semi-lactosi, glucose, lactose, gluconic acid, Sorbitol Powder, maltose, N.F,USP MANNITOL, seminose, melibiose, sucrose, raffinose, saligenol, trehalose, wood sugar, pectinose, ribose but unfermentable pectinose and rhamnosyl.In defined medium, fermentation culture can produce the bacteriocin with broad spectrum antibiotic activity.

Plant lactobacillus (Lactobacillus plantarum) 163 can adopt MRS substratum or Chinese cabbage tomato substratum, 37 ℃ of standing cultivations.

MRS substratum described in following examples and Chinese cabbage tomato substratum all adopt following formula:

MRS culture medium prescription: peptone 10g, beef leaching thing 10g, yeast extract 5g, glucose 5g, sodium acetate 5g, citric acid diamines 2g, tween 80 1mL, dipotassium hydrogen phosphate 2g, magnesium sulfate heptahydrate 0.2g, seven water manganous sulfate 0.05g, calcium carbonate 20g, distilled water 1L, pH6.4.

Chinese cabbage tomato culture medium prescription: glucose 10g, cabbage juice 200mL, tomato 50mL, distilled water 750mL, pH6.5.

Embodiment 1

1, produce the screening of broad-spectrum antimicrobial bacteriocin milk-acid bacteria

(1) milk-acid bacteria purifying: ten times of dilutions of collected specimens, obtain 10 ^-4, 10 ^-5, 10 ^-6three concentration, get each concentration 0.1mL, and the MRS culture medium flat plate of calcium carbonate is added in coating, at 37 ℃, cultivate 48 hours, there is the bacterium colony of molten calcium circle in random picking, adds the MRS substratum line separation and purification of calcium carbonate, obtains molten calcium circle obvious, single bacterium colony that colonial morphology is single, picking list bacterium colony, further carries out catalase test and Gram staining dyeing after separation and purification, catalase test is negative, the bacterium colony of the gram's staining positive, ℃ preservation of glycerine pipe-70.

(2) the milk-acid bacteria primary dcreening operation of bacteriocinogeny:

1) preparation MRS solid medium, good dull and stereotyped, after solidifying, transfering loop or toothpick inoculating lactic acid bacteria strain, on MRS substratum, are cultivated 2 days at 37 ℃, treat that it grows bacterium colony.

2) indicator is inoculated in 10mL beef extract-peptone liquid nutrient medium, 37 ℃ of shaking tables are cultivated 10 hours, by 1%(v/v) be inoculated in 50 ℃, the extractum carnis egg of agar concentration 0.8% is poured over the beef extract-peptone with indicator on the MRS substratum that grows milk-acid bacteria bacterium colony.

3) 37 ℃ of cultivations, observe it and whether occur inhibition zone (inhibition zone is greater than 5mm and thinks there is fungistatic effect) after one day.

(3) milk-acid bacteria of bacteriocinogeny is sieved again: the bacterial strain that primary dcreening operation is positive, inoculation activates after MRS substratum, standing cultivation 24h, and 5000g is centrifugal, obtain supernatant liquor, the bacterial strain that still shows bacteriostatic activity after supernatant liquor eliminating acid inhibition and hydrogen peroxide suppress is considered as positive bacteria.Experiment obtains 5 strain positive bacterias, and wherein plant lactobacillus (Lactobacillus plantarum) 163 fungistatic effects are best.

2, the evaluation of bacterial strain

Physiology and biochemistry is identified: according to the < < lactic-acid-bacterium classification evaluation of < < uncle Jie Shi Bacteria Identification handbook > > and Ling Daiwen and experimental technique > >, the bacterial strain screening is carried out to gram's staining and physio-biochemical characteristics evaluation.

Result shows, bacterial strain is without gemma gram-positive microorganism, rod-short, atrichia, catalase is negative, can tolerate 6.5%(mass ratio, lower with) following NaCl, produce sourly, do not produce CO ₂, H ₂s, NH ₃fermentable fibres element, polychrom, fructose, semi-lactosi, glucose, lactose, gluconic acid, Sorbitol Powder, maltose, N.F,USP MANNITOL, seminose, melibiose, sucrose, raffinose, saligenol, trehalose, wood sugar, pectinose, ribose but unfermentable pectinose and rhamnosyl.Concrete outcome is in Table 1.

Table 1 bacterial strain Physiology and biochemistry qualification result

Identify column	Bacterial strain reaction	Identify column	Bacterial strain reaction	Identify column	Bacterial strain reaction
						Pectinose	-	Maltose	+	Trehalose	+
Cellobiose	+	N.F,USP MANNITOL	+	Wood sugar	+
						Polychrom	+	Seminose	+	6.5%NaCL	-
Fructose	+	Melibiose	+	18%NaCl	-
						Semi-lactosi	+	Raffinose	+	Aerogenesis	-
Glucose	+	Rhamnosyl	-	Arginine produces ammonia	-
						Gluconate	+	Ribose	+	Motion	-
Lactose	+	Saligenin	+	Produce hydrogen sulfide	-
						Sorbyl alcohol	+	Sucrose	+

16S rDNA identifies: primer: P15 ＇-AGAGTTTGATCCTGGCTCAG-3 ＇, (SEQ ID NO.3) P25 ＇-CTACGGCTACCTTGTTACGA-3 ＇, (SEQ ID NO.4) pcr amplification goes out after DNA, send (the Jin Sirui of order-checking company, Nanjing) order-checking, sequencing result is right Databases of the GenBank of NCBI.

Result shows, pcr amplification checks order after going out sequence empirical tests, obtain the base pair (GenBank accession number JX524228) of 1440bp, right Databases of the GenBank of NCBI, find that there is 4 lactobacillus plantarums therewith the similarity of bacterial strain reach 100%, in conjunction with previous Physiology and biochemistry qualification result, determine that this bacterial strain is plant lactobacillus.

3, the cultivation and fermentation of plant lactobacillus (Lactobacillus plantarum) 163

(1) plant lactobacillus (Lactobacillus plantarum) 163 seed liquor preparations: by the bacterial classification of preservation, be inoculated in MRS liquid nutrient medium 37 ℃ of standing cultivation 12-16h;

(2) plant lactobacillus (Lactobacillus plantarum) 163 fermentations: by the seed liquor of activation, by volume per-cent is 1% to be inoculated in MRS liquid nutrient medium or Chinese cabbage tomato substratum, after MRS liquid nutrient medium standing cultivation 24h or the standing cultivation of Chinese cabbage tomato substratum 30h, the centrifugal fermented liquid obtaining containing bacteriocin of 5000rpm.

4, SEQ ID NO1, separation and purification and the evaluation of SEQ ID NO2

(1) ammonium sulfate precipitation preliminary purification

Concentrated 2 times of plant lactobacillus MRS fermented liquid, concentrated solution slowly adds Powdered ammonium sulfate to saturation ratio 60%, stirs after half an hour 8000rpm, centrifugal 15min at 4 ℃, obtain supernatant liquor (precipitation abandons), supernatant liquor continues slowly to add Powdered ammonium sulfate to saturation ratio 85%, stirs after half an hour, 8000rpm, at 4 ℃, centrifugal 15min, obtains precipitation, and precipitation is dissolved with 10mL distilled water.

(2) Sephadex G25 purifying for the second time

The sample that ammonium sulfate precipitation gets off,, draws 1mL sample with suction pipe by concentrated 2 times, open the valve below the post that installs Sephadex G25 column material, open constant flow pump, with the abundant wash-out of pure water, regulate flow velocity (1.8mL/5min), utilize automatic collector to collect cut.After fraction collection, take bacillus pumilus as indicator, detect its bacteriostatic activity.

(3) Sephadex LH-20 purifying for the third time

Sephadex LH-20 pre-treatment fills well post, adds 1mL distilled water to dissolve, loading after the active fractions 15mL freeze-drying out of G25 purifying, use 80% methanol-eluted fractions, flow velocity 1.5mL/9min, every cut connects after 1.5mL. fraction collection, take bacillus pumilus as indicator, detect its bacteriostatic activity.

Sephadex LH-20 after purifying, obtains active elution peak for the third time, and wherein first active elution peak is for SEQ IDNO1, and second active elution peak is shown in Fig. 1 for SEQ ID NO2().

(4) the 4th purifying of HPLC

1) for first active elution peak, finally utilize RP-HPLC look to boil method final purification, purification condition: C18 post, A: water+0.1%TFA, B: acetonitrile+0.1%TFA, Time:60min, A:95%-5%, B:5%-95% gradient elution, wavelength: 267nm, flow velocity 0.4mL/min, preferably obtains retention time and is 22.277 peak, is SEQ ID NO.1(and sees Fig. 2).

2) evaluation of SEQ ID NO.1: utilize MALDI-TOF-MS to draw its molecular weight, the ms fragment that utilizes MALDI-TOF-MS/MS to obtain SEQ ID NO.1 (is shown in Fig. 3, a is MALDI-TOF-MS figure, b is MALDI-TOF-MS/MS figure), utilize n terminal amino acid order-checking to obtain 10 aminoacid sequences before SEQ ID NO.1, utilize amino acid composition analysis to show that the amino acid of SEQ ID NO1 forms (see figure 4), finally infers the aminoacid sequence structure of SEQ ID NO.1.

3) for second active elution peak, through HPLC purifying, purification condition: 95%A+5%B isocratic elution, A: water+TFA(0.1%), B: acetonitrile+TFA(0.1), time:40min, pillar: C18 post (4.6*250mm) flow velocity 3mL/min, detects wavelength 267nm.Collect Peak Activity (see figure 5), MALDI-TOF-MS mass spectrometric detection SEQ ID NO2 molecular weight (see figure 6), MALDI-TOF-MS/MS detects the ion fragment peak (see figure 7) of SEQ ID NO.2, finally infers the aminoacid sequence structure of SEQ ID NO.2.

5, the antimicrobial spectrum of SEQ ID NO.1, SEQ ID NO.2

The SEQ ID NO.1 that purifying obtains and SEQ ID NO.2, the bacterium described in following table 2 is indicator respectively, the bacteriostatic activity of test SEQ ID NO.1 and SEQ ID NO.2.Result shows: SEQ ID NO.1 and SEQ ID NO.2 have inhibition to 7 kinds of gram positive organisms, 3 kinds of gram-negative bacterias, wherein the inhibition of SEQ ID NO.2 is better than the inhibition of SEQ ID NO.1 far away, but SEQ ID NO.1 and SEQ ID NO.2 are to 3 kinds of equal unrestraint effects of fungi.

The antimicrobial spectrum of table 2SEQ ID NO.1 and SEQ ID NO.2

6, the proteolytic enzyme susceptibility of SEQ ID NO.1 and SEQ ID NO.2.

Purifying SEQ ID NO.1 and SEQ ID NO.2 out, described according to the form below 3 remarks, process, testing 4 kinds of proteolytic enzyme acts on it, 4 kinds of proteolytic enzyme are: trypsin Trypsin) (2500u/mg), Proteinase K (Protease K) (30u/mg), (3k-3.5k/mg), alpha-chymotrypsin (α-chymotrypsin) (1000u/mg) for stomach en-(Pepsin).

The effect of table 3 proteolytic enzyme to SEQ ID NO.1 and SEQ ID NO.2

As can be seen from the above table, SEQ ID NO.1 bacteriocin can be by all protease hydrolysiss, therefore lose anti-microbial activity, and the anti-microbial activity of SEQ ID NO.2 bacteriocin can be suppressed by trypsinase and alpha-chymotrypsin, by stomach en-and Proteinase K hydrolysis, be lost anti-microbial activity completely.

7, SEQ ID NO.1 thermostability and pH stability.

Purifying SEQ ID NO.1 out, temperature and time shown according to the form below 4 is processed water-bath out rear (121 ℃ is high-pressure sterilizing pot), take bacillus pumilus as indicator, detect its residual activity (aperture 5mm, loading 40 μ L), for pH treatment process, be: the SEQ ID NO.1 solution that respectively takes a morsel, with HCl or NaOH, be adjusted to corresponding pH, 37 ℃ of insulations are recalled to original pH(pH4 after 3h again) test its bacteriostatic activity.

Thermostability and the pH stability of table 4 SEQ ID NO.1

Process	Antibacterial circle diameter (mm)
		CK	11.20±0.17
60℃10min	11.29±0.05
		60℃30min	11.02±0.07
80℃10min	10.93±0.15
		80℃30min	11.02±0.12
100℃10min	9.37±0.06
		100℃30min	8.36±0.08
121℃20min	8.19±0.24
		pH2	6.63±0.17
pH3	10.55±0.06
		pH4（CK）	10.88±0.05
pH5	10.21±0.05
		pH6	8.46±0.12
pH7	8.28±0.03
		pH8	8.54±0.34
pH9	7.81±0.14
		pH10	5.72±0.08

As can be seen from the above table, the heat tolerance of SEQ ID NO.1 is better, below 80 ℃, and its active basic reservation, but more than 100 ℃, its active loss is larger.In pH tolerance, pH4-pH6, its active basic reservation, but when pH2 or pH3, its active loss is larger.Loss surpasses 40% activity, pH7-pH10, and its active loss also approaches 30%.From heat tolerance and pH tolerance, SEQ ID NO.1 can be applicable to Partial Food anticorrosion on.

8, SEQ ID NO.2 thermostability and pH stability.

Purifying SEQ ID NO.2 out, temperature and time shown according to the form below is processed water-bath out rear (121 ℃ is high-pressure sterilizing pot), take bacillus pumilus as indicator, detect its residual activity (aperture 5mm, loading 40 μ L), for pH treatment process, be: the SEQ ID NO.2 solution that respectively takes a morsel, with HCL or NAOH, be adjusted to corresponding pH, 37 ℃ of insulations are recalled to original pH(pH4 after 3h again) test its bacteriostatic activity.

Process	Average diameter of inhibition zone (mm)
		CK	13.89±0.1
60℃10min	13.71±0.16
		60℃30min	13.69±0.12
80℃10min	13.22±0.2
		80℃30min	13.61±0.32
100℃10min	13.26±0.29
		100℃30min	13.44±0.04
121℃20min	12.88±0.1
		pH2	10.58±0.1
pH3	13.78±0.1
		pH4（CK）	13.64±0.08
pH5	13.71±0.05
		pH6	13.59±0.04
pH7	12.71±0.2
		pH8	12.39±0.04
pH9	10.71±0.14
		pH10	9.6±0.1

As can be seen from the above table, SEQ ID NO.2 heat tolerance is better, and at 121 ℃, its activity also retains more than 90%.PH tolerance is also better, and at pH3-pH6, its activity is not lost substantially, but at extreme pH(pH2, pH10) under condition, its active loss is larger.From heat tolerance and pH tolerance, SEQ ID NO.2, as foodstuff additive, can meet a big chunk food for the requirement of heat and pH, and application potential is very high.

From above-described content, can find out, plant lactobacillus provided by the invention, bacteriocin that its fermentation produces has good fungistatic effect, and natural, safety simultaneously completely can be through being processed into foodstuff additive or germ resistance pharmaceutical preparation is applied in food or field of medicaments.

Sequence table

<110> Agricultural University Of Nanjing

<120> plant lactobacillus, its bacteriocin and cultivation and separation purification method

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<170> PatentIn version 3.3

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Claims (9)

1. A plant lactobacillus ( lactobacillus plantarum) 163, culture presevation number is CGMCC NO.8224.
2. 2. a bacteriocin, is characterized in that, by plant lactobacillus claimed in claim 1 ( lactobacillus plantarum) 163 fermentation obtain.
3. 3. bacteriocin according to claim 2, is characterized in that, has the aminoacid sequence shown in SEQ ID NO.1.
4. 4. bacteriocin according to claim 2, is characterized in that, has the aminoacid sequence shown in SEQ ID NO.2.
5. 5. a preparation method for bacteriocin claimed in claim 2, is characterized in that, by plant lactobacillus ( lactobacillus plantarum) 163 by following step cultivation and fermentation, obtain:

   (1) plant lactobacillus ( lactobacillus plantarum) 163 seed liquor preparations: by the bacterial classification of preservation, be inoculated in MRS liquid nutrient medium 37 ℃ of standing cultivation 12-16 h;

   (2) plant lactobacillus ( lactobacillus plantarum) 163 fermentations: by the seed liquor of activation, by volume per-cent is that 1 % is inoculated in MRS liquid nutrient medium or Chinese cabbage tomato substratum, after standing cultivation 24 h of MRS liquid nutrient medium or standing cultivation 30 h of Chinese cabbage tomato substratum, the centrifugal fermented liquid obtaining containing bacteriocin of 5000 rpm.
6. 6. the preparation method of bacteriocin according to claim 5, is characterized in that, the Chinese cabbage tomato culture medium prescription described in step (2) is glucose 10 g, cabbage juice 200 mL, and tomato 50 mL, distilled water 750 mL, pH 6.5.
7. 7. a separation purification method for the bacteriocin described in claim 2 or 3 or 4, is characterized in that, first by plant lactobacillus ( lactobacillus plantarum) 163 carry out cultivation and fermentation, then take following steps to carry out:

   (1) ammonium sulfate precipitation purifying: first that plant lactobacillus MRS fermented liquid is concentrated, concentrated solution adds Powdered ammonium sulfate to saturation ratio 60%, stir, centrifugally at 4 ℃ get supernatant liquor after completely and continue to add Powdered ammonium sulfate to saturation ratio 85%, stir, centrifugal complete at 4 ℃, obtain precipitation, precipitation is dissolved with distilled water;

   (2) Sephadex G25 purifying: the sample that ammonium sulfate precipitation is got off concentrates, draws sample, opens the valve below the post that installs Sephadex G25 column material, open constant flow pump, with the abundant wash-out of pure water, regulate flow velocity, utilize automatic collector to collect cut;

   (3) Sephadex LH-20 purifying: Sephadex LH-20 fills post, adds distilled water to dissolve, loading after the active fractions freeze-drying out of G25 purifying, by 80 % methanol-eluted fractions, flow velocity 1.5 mL/9 min, every cut meets 1.5 mL, collect cut, after purifying, obtain two active elution peaks;

   (4) HPLC purifying: first active elution peak, utilize RP-HPLC look to boil method purifying, purification condition: C18 post, A: water+0.1 %TFA, B: acetonitrile+0.1 %TFA, Time:60 min, A:95 %-5 %, B:5 %-95 % gradient elution, wavelength: 267 nm, flow velocity 0.4 mL/min; Second active elution peak, purification condition: 95%A+5%B isocratic elution, A: water+0.1%TFA, B: acetonitrile+0.1%TFA, time: 40min, pillar: C18 post, flow velocity 3mL/min, detects wavelength 267nm.
8. 8. the application of the bacteriocin described in claim 2 or 3 or 4 in food antiseptic.
9. 9. the application of the bacteriocin described in claim 2 or 3 or 4 in preparation germ resistance medicine.

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