CN103667106B - A kind of plant lactobacillus, its bacteriocin and cultivation and separation purification method - Google Patents

Abstract

The invention discloses a kind of plant lactobacillus, its bacteriocin and cultivation and separation purification method, wherein plant lactobacillus (<i>Lactobacillus? do are plantarum</i>) 163 culture presevation number CGMCC? NO.8224.This plant lactobacillus is cultivated in MRS liquid nutrient medium or Chinese cabbage tomato substratum, can be fermented generation bacteriocin, wherein produced bacteriocin is that bacteriocin is obtained by four separation and purification and qualification by two kinds of novel fine rhzomorphs of two seed amino acid Sequence composition primary structures.Through measuring, this bacteriocin has wider antimicrobial spectrum and preferably thermostability and pH stability, can be widely used in food and field of medicaments.

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 the food 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 about 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 the microorganism in different process means control food, but some method for disinfection can produce detrimentally affect to the quality of food, local flavor and nutrition.Food preservatives is effectively easy to use because of it, can preserve the commodity value of food well, one of important measures remaining food antiseptic at present simultaneously.

Milk-acid bacteria and the tunning mankind have had the use history of thousands of years, are not only applied in food mfg now, but also be applied in cure the disease, health care aspect.Utilize history and the fine quality that has no side effect of thousands of years make 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 by Ribosome biogenesis micromolecule polypeptide----bacteriocin in metabolic process, and part bacteriocin 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, be used widely more than 80 countries and regions (being also that unique one obtains commercial applications approval).Nisin was also listed in the amendments in the GB GB2660-86 of enforcement on October 1st, 1992 in 1992 by China, can be used in the preservation of tinned pre-, vegetable protein beverage, milk-product and meat product.But Nisin activity under alkaline environment is lower; Only there is effect to Glan positive bacteria, to Glan negative pathogens, yeast, mould, viral unrestraint effect; Active affect the use limiting it such as larger by biological enzyme.Thus the popular research direction that the new high-efficiency broad spectrum bacteriocin lab of screening becomes worldwide internal medicine scholar is in recent years continued.

Based on Chemical Preservative in 32 kinds of sanitass that the permission of current China's foodstuff additive standard (GB2760) uses, natural antiseptic agent is nisin (Nisin) and tennecetin (Natamycin) two kinds only.The usual cost of Chemical Preservative is lower, stable chemical performance and antimicrobial spectrum are comparatively wide, favorable anti-corrosion effect, but research at present shows, certain toxicity is there is in various degree in Chemical Preservative to human body, some of them have and lure carcinous and teratogenecity, easily cause food poisoning when exceeding standard and using.Along with the raising of living standards of the people and the enhancing of Consciousness of food security, people 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 Main way that the nontoxic natural antiseptic agent of highly effective and safe has become current international food Development of Additives is is researched and developed.

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

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 is little in the food 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 to the function and position of substituted chemistry sanitas at present.Therefore, develop wide spectrum, antiseptics for natural food that is efficient, stable, the high and low cost of safety, productive rate has potential using value and the huge market requirement.

Summary of the invention

The object of the invention is to provide a kind of plant lactobacillus, its bacteriocin and cultivation and separation purification method 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, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 20th, 2013 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 kind of bacteriocin, above-described plant lactobacillus (Lactobacillus plantarum) 163 fermentation obtains.

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 preparation: by the bacterial classification of preservation, be inoculated in MRS liquid nutrient medium, 37 DEG C of quiescent culture 12-16h;

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

The preparation method of above-described bacteriocin, the Chinese cabbage tomato culture medium prescription wherein 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 plant lactobacillus MRS fermented liquid is concentrated, concentrated solution adds Powdered ammonium sulfate to saturation ratio 60%, stir, at 4 DEG C centrifugal completely after get supernatant liquor and continue to add Powdered ammonium sulfate to saturation ratio 85%, stir, completely centrifugal at 4 DEG C, obtain precipitation, precipitation distilled water dissolves;

(2) Sephadex G25 purifying: the sample got off by ammonium sulfate precipitation concentrates, pipette samples, opens the valve below the post installing 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 and dissolves, loading after the active fractions freeze-drying that G25 is purified, 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, determined wavelength 267nm.

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

Beneficial effect

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

(1) safe source, plant lactobacillus is separated from Guizhou traditional fermented food fermentation pickled radiss, has time-honored edible history, therefore from safety, the tunning (two kinds of bacteriocins, SEQ ID NO.1 and SEQ ID NO.2) of plant lactobacillus 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 the use of even stopping its other sanitass of leavened food can be reduced, extend the shelf-lives of leavened food.

(3) the bacteriocin SEQ IDNO.1 of broad-spectrum antibacterial action that produces of plant lactobacillus (Lactobacillus plantarum) 163 and SEQ ID NO.2, has a broad antifungal spectrum, gram positive organism can be suppressed, 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 comparatively stable, pH, temperature are little to its activity influence, and in the scope of pH3-6 and temperature less than 80 DEG C, its activity can retain substantially completely, is more conducive to the application in food.

Accompanying drawing explanation

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

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, 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 strain, therefrom filters out the plant lactobacillus (Lactobacillus plantarum) 163 that a strain can produce broad-spectrum antibacterial action bacteriocin.Main biological property: in white or oyster white point on MRS solid medium, without gemma gram-positive microorganism, rod-short, atrichia, negative catalase, can tolerate 6.5%(mass ratio, lower same) following NaCl, 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 DEG C of quiescent culture.

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, the screening of broad-spectrum antimicrobial bacteriocin milk-acid bacteria is produced

(1) milk-acid bacteria purifying: collected specimens ten times dilution, obtains 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, cultivate 48 hours at 37 DEG C, there is the bacterium colony of molten calcium circle in random picking, adds the MRS substratum line separation and purification of calcium carbonate, obtain molten calcium circle obvious, single bacterium colony that colonial morphology is single, picking list bacterium colony, carry out catalase test and Gram staining dyeing after further separation and purification, catalase test is negative, the bacterium colony of the gram's staining positive ,-70 DEG C of preservations of glycerine pipe.

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

1) prepare MRS solid medium, good dull and stereotyped, after to be solidified, transfering loop or toothpick inoculating lactic acid bacteria strain, on MRS substratum, are cultivated 2 days at 37 DEG C, are treated that it grows bacterium colony.

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

3) 37 DEG C 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, quiescent culture 24h, and 5000g is centrifugal, obtain supernatant liquor, supernatant liquor still shows bacteriostatic activity bacterial strain after getting rid of acid suppression and hydrogen peroxide suppression is considered as positive bacteria.Experiment obtains 5 strain positive bacterias, and wherein plant lactobacillus (Lactobacillus plantarum) 163 fungistatic effects are best.

2, the qualification of bacterial strain

Physiology and biochemistry is identified: carry out gram's staining and physio-biochemical characteristics qualification according to " uncle Jie Shi Bacteria Identification handbook " and " lactic-acid-bacterium taxonomic identification and the experimental technique " of Ling Daiwen to the bacterial strain screened.

Result shows, bacterial strain is without gemma gram-positive microorganism, rod-short, atrichia, and negative catalase can tolerate 6.5%(mass ratio, lower same) following NaCl, produces acid, does 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

Qualification column	Bacterial strain reacts	Qualification column	Bacterial strain reacts	Qualification column	Bacterial strain reacts
						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 ＇, after (SEQ ID NO.4) pcr amplification goes out DNA, send order-checking company (Jin Sirui, Nanjing) order-checking, sequencing result is right GenBank Databases of NCBI.

Result shows, pcr amplification checks order after going out sequence empirical tests, obtain the base pair (GenBank accession number JX524228) of 1440bp, GenBank Databases at NCBI right, the similarity finding that there is 4 lactobacillus plantarums bacterial strain therewith reaches 100%, in conjunction with before 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 preparation: by the bacterial classification of preservation, be inoculated in MRS liquid nutrient medium, 37 DEG C of quiescent culture 12-16h;

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

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

(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%, after stirring half an hour, and 8000rpm, centrifugal 15min at 4 DEG C, obtain supernatant liquor (precipitation abandons), supernatant liquor continues slowly to add Powdered ammonium sulfate to saturation ratio 85%, after stirring half an hour, 8000rpm, at 4 DEG C, centrifugal 15min, obtains precipitation, and precipitation 10mL distilled water dissolves.

(2) Sephadex G25 second time purifying

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

(3) Sephadex LH-20 third time purifying

Sephadex LH-20 pre-treatment fills post well, add 1 mL distilled water after the active fractions 15 mL freeze-drying that G25 is purified to dissolve, loading, by 80 % methanol-eluted fractions, flow velocity 1.5 mL/9 min, after every cut connects 1.5 mL. fraction collections, be indicator with bacillus pumilus, detect its bacteriostatic activity.

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

(4) HPLC the 4th purifying

1) for first active elution peak, RP-HPLC look is finally utilized to boil method final purification, 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, preferably obtains the peak that retention time is 22.277, is SEQ ID NO.1(and sees Fig. 2).

2) qualification of SEQ ID NO.1: utilize MALDI-TOF-MS to draw its molecular weight, MALDI-TOF-MS/MS is utilized to obtain the ms fragment of SEQ ID NO.1 (see Fig. 3, a is MALDI-TOF-MS figure, b is MALDI-TOF-MS/MS figure), utilize n terminal amino acid to check order and obtain 10 aminoacid sequences before SEQ ID NO.1, utilize amino acid composition analysis to draw the amino acid composition (see figure 4) of SEQ ID NO.1, finally infer the amino acid sequence structure of SEQ ID NO.1.

3) for second active elution peak, through HPLC purifying, purification condition: 95 %A+5 %B isocratic elutions, A: water+TFA(0.1 %), B: acetonitrile+TFA(0.1), time:40 min, pillar: C18 post (4.6*250mm) flow velocity 3 mL/min, determined wavelength 267 nm.Collect Peak Activity (see figure 5), MALDI-TOF-MS mass spectrometric detection SEQ IDNO.2 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 amino acid 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, respectively with the bacterium described in following table 2 for indicator, test SEQID NO.1 and SEQ ID NO.2 bacteriostatic activity.Result shows: SEQ ID NO.1 and SEQ ID NO.2 has 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 is to 3 kinds of equal unrestraint effects of fungi.

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

Indicator

Indicator is originated

Antibacterial circle diameter (mm)

		SEQ ID NO.1	SEQ ID NO.2
				Bacillus pumilus	ATCC25923	9.31±0.54	16.28±0.47
Bacillus cereus	ATCC19114	11.82±0.39	16.30±1.36
				Staphylococcus aureus	CMCC63202	11.27±0.32	19.38±0.68
Micrococcus luteus	AS1.1846	7.10±0.52	13.07±0.27
				Listeria bacteria	CMCC28001	9.96±0.84	16.25±0.58
Thermophilic lacto-bacilli	This Laboratories Accession	7.33±0.75	9.55±0.52
				Lactobacillus rhamnosus	This Laboratories Accession	6.84±0.28	9.6±0.54
Intestinal bacteria	ATCC25922	10.10±1.14	15.35±0.67
				Pseudomonas aeruginosa	AS1.2620	8.62±0.74	9.91±1.3
Pseudomonas fluorescens	AS3.6452	6.79±0.98	12.92±1.46
				Point mould	AS3.4356	0	0
Aspergillus niger	AS3.6459	0	0
				Rhizopus stolonifer	AS3.822	0	0

6, the protease sensitive of SEQ ID NO.1 and SEQ ID NO.2.

The SEQ ID NO.1 be purified and SEQ ID NO.2, processes described according to the form below 3 remarks, and test 4 kinds of proteolytic enzyme and act on it, 4 kinds of proteolytic enzyme are: trypsin Tr _ypsin) (2500u/mg), Proteinase K (Protease K) (30u/mg), stomach en-(Pepsin) (3k-3.5k/mg), alpha-chymotrypsin (α-chymotrypsin) (1000u/mg).

Table 3 proteolytic enzyme is to the effect of SEQ ID NO.1 and SEQ ID NO.2

As can be seen from the above table, SEQ ID NO.1 bacteriocin can 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, lost anti-microbial activity by stomach en-and Proteinase K hydrolysis completely.

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

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

The thermostability of table 4SEQ ID NO.1 and pH stability

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, less than 80 DEG C, its active basic reservation, but more than 100 DEG C, its active loss is larger.In pH tolerance, pH4-pH6, its active basic reservation, but during pH2 or pH3, its active loss is larger.The activity of loss more than 40%, pH7-pH10, its active loss is also close to 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.

The SEQ ID NO.2 be purified, after the process of temperature and time shown according to the form below water-bath out (121 DEG C is high-pressure sterilizing pot), take bacillus pumilus as indicator, detect its residual activity (aperture 5mm, loading 40 μ L), be: respectively take a morsel SEQ ID NO.2 solution, is adjusted to corresponding pH with HCL or NaOH recall to original pH(pH4 again after 37 DEG C of insulation 3h for pH treatment process) 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 DEG C, 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 is as foodstuff additive, and can meet a big chunk food for requirement that is hot and pH, application potential is very high.

As can be seen from above-described content, plant lactobacillus provided by the invention, bacteriocin that its fermentation produces has good fungistatic effect, simultaneously natural, safety, completely can 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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Tyr Val Cys Ala Ser Pro Trp

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Val Phe His Ala Tyr Ser Ala Arg Gly Asn Tyr Tyr Gly Asn Cys Pro

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Ala Asn Trp Pro Ser Cys Arg Asn Asn Tyr Lys Ser Ala Gly Gly Lys

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Claims (8)

1. a plant lactobacillus ( lactobacillus plantarum) 163, culture presevation number is CGMCC NO.8224.

2. a bacteriocin, is characterized in that, by plant lactobacillus according to claim 1 ( lactobacillus plantarum) 163 fermentation obtain, be made up of the aminoacid sequence shown in SEQ ID NO.1.

3. a bacteriocin, is characterized in that, by plant lactobacillus according to claim 1 ( lactobacillus plantarum) 163 fermentation obtain, be made up of the aminoacid sequence shown in SEQ ID NO.2.

4. a preparation method for the bacteriocin described in Claims 2 or 3, is characterized in that, by plant lactobacillus ( lactobacillus plantarum) 163 to be obtained by following step cultivation and fermentation:

(1) plant lactobacillus ( lactobacillus plantarum) 163 seed liquor preparations: by the bacterial classification of preservation, be inoculated in MRS liquid nutrient medium, 37 DEG C of quiescent culture 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 MRS liquid nutrient medium quiescent culture 24 h or Chinese cabbage tomato substratum quiescent culture 30 h, the centrifugal fermented liquid namely obtained containing bacteriocin of 5000 rpm.

5. the preparation method of bacteriocin according to claim 4, is characterized in that, the Chinese cabbage tomato culture medium prescription described in step (2) is glucose 10 g, cabbage juice 200 mL, tomato 50 mL, distilled water 750 mL, pH 6.5.

6. a separation purification method for the bacteriocin described in Claims 2 or 3, 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 plant lactobacillus MRS fermented liquid is concentrated, concentrated solution adds Powdered ammonium sulfate to saturation ratio 60%, stir, at 4 DEG C centrifugal completely after get supernatant liquor and continue to add Powdered ammonium sulfate to saturation ratio 85%, stir, completely centrifugal at 4 DEG C, obtain precipitation, precipitation distilled water dissolves;

(2) Sephadex G25 purifying: the sample got off by ammonium sulfate precipitation concentrates, pipette samples, opens the valve below the post installing 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 and dissolves, loading after the active fractions freeze-drying that G25 is purified, 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, RP-HPLC look is utilized 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, determined wavelength 267nm.

7. the application of the bacteriocin described in Claims 2 or 3 in food antiseptic.

8. the application of the bacteriocin described in Claims 2 or 3 in preparation germ resistance medicine.