CN1670184A - Peptides natural microbial antiseptic agent producing strain, its use and preparation method for antiseptic agent - Google Patents
Peptides natural microbial antiseptic agent producing strain, its use and preparation method for antiseptic agent Download PDFInfo
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Abstract
This invention relates to an antiseptic producing bacteria, its metabolite application and preparing natural microbios antiseptic process using the metabolite as material. Naming the invention as Lactobacillus paracaseiHD1.7, preservation number being CCTCC: M205015. Its metabolite is a kind of peptide antiseptic, being capable of inhibiting colibacillus, Staphylococcus aureus, micrococcus flavus, bacillus subtilis, also salmonella, bacillus megaterium, Bacillus cereus, Shigella, proteus and such like these pathogenic bacteria; in condition of 120Deg.C, 60min still sustaining its bacterial inhibition active, with strong heat stability; being irresponsive to several proteinases, working well in neutral or acidic conditions.
Description
Technical field:
The present invention relates to that a kind of peptide class sanitas produces the isolation identification of bacterium and application that this sanitas produces the bacterium meta-bolites and be the method for feedstock production natural microbial sanitas with the meta-bolites that this sanitas produces bacterium.
Background technology:
Sanitas is divided into two kinds of Chemical Preservative and natural microbial sanitass.At present both at home and abroad a large amount of sanitass that use are still artificial chemical process synthetic in foodstuffs industry, and the raw material of Shi Yonging, auxiliary material and additive itself or the impurity that wherein contains all may damage human health in process of production; Chemical Preservative much is not the material that nature exists naturally, and whether it exists potential chronic toxicity or three to cause effect to HUMAN HEALTH also imperfectly understands.The natural microbial sanitas that China's approval is at present used have only tennecetin and Nisin etc. a few, kind is very single, and the microorganism natural antiseptic agent has the defective of peculiar smell and aspect such as variegated.
Be that Chemical Preservative or natural antiseptic agent all exist the narrow problem of antimicrobial spectrum, only gram-positive microorganism had restraining effect and to Gram-negative fungus unrestraint effect as the Nisin that on market, occupies at present critical role.The antibacterial of most sanitass all requires suitable environment, or acid or neutral, only under acidic conditions bacteriostatic action arranged as Nisin.But many milk-acid bacteria metabolism produce peptide matters, studying clearly at present, Nisin is that the streptococcus acidi lactici metabolism produces, many scholars have been separated to the milk-acid bacteria of multiple generation sanitas, but about the character of product, molecular weight size, produce bacterium kind etc. at present many places among research.
In the foreign study, Richter reports this bacterium and is used to produce lactic acid, and lactic acid yield reaches 94%; Mercenier, report Lactobacillus paracasei such as Shaw have very strong immunostimulatory potency; Philippe report Lactobacillus paracasei can be used for diseases such as treatment of allergic rhinitis, also has many reports that have good treatment intestinal dysfunction about this bacterium in addition; The H.MINOR-PEREZ of Mexico has reported Lactobacillus paracasei can produce bacteriocin.Disclose a kind of " new strains of lactobacillus paracasei " among the CN1556851A, this bacterial strain is used to the cheese that ferments.A kind of " the antibacterium note of novel anti-helicobacter pylori and the lactobacillus paracasei class cheese subspecies bacterial strain of intestinal bacteria 157:H7 " disclosed among the CN1406274A, it is the strains of lactobacillus paracasei that obtains from the ight soil of pig, has the characteristic of anti-helicobacter pylori and intestinal bacteria 0517.
Summary of the invention:
The purpose of this invention is to provide the preparation method that a kind of peptides natural microbial sanitas produces bacterium and application and sanitas, the meta-bolites of this generation bacterium can be used as sanitas, peptide class sanitas is to adopt the metabolism of microorganism pure-blood ferment to produce, and product is the peptide class, and security is very high.
Peptide class sanitas of the present invention produces bacterium in (address: China, China typical culture collection center, Wuhan, Luo Jiashan, postcode: 430072) preservation, called after lactobacillus paraceasi HD1.7 (Lactobacillus paracasei HD1.7), deposit number is CCTCC NO:M205015, and preservation date is on February 5th, 2005.
Generation bacterium of the present invention is to adopt conventional plate streaking partition method, uses the milk-acid bacteria ball to separate acquisition with milk-acid bacteria bar two class isolation mediums from the rich derived food L-of the company limited milk-acid bacteria pickling sauerkraut fermented liquid of Qiqihar.Determine that according to thalli morphology and size Lactobacillus paracasei HD1.7 is a bacterium, determine that according to the definition of lactic-acid-bacterium this bacterium is a milk-acid bacteria, determine that according to the physiological and biochemical property of " the lactic-acid-bacterium classification is identified and experimental technique " lactobacillus this bacterium is a lactobacillus.16SrDNA sequences Design primer according to the Bacterium lacticum close with this bacterium Physiology and biochemistry character, identify, extract plasmid, order-checking, comparison by pcr amplification, connection T carrier, conversion, PCR, high homology according to many strains dna sequence dna in itself and the lactobacillus paraceasi is accredited as Lactobacillus paracasei (lactobacillus paraceasi); According to called after Lactobacillus paracasei HD1.7 strains such as the separation of this bacterial strain source and new product features, its biological characteristics is as follows again:
A, Lactobacillus paracasei HD1.7 are gram-positive microorganism, and shaft-like, bacterium colony is little, and is rounded; Under 15～45 ℃ of conditions, cultivate all growths; Heterofermentation can utilize multiple sugar and alcohol, as seminose, lactose, glucose, fructose, melizitose, cellobiose, maltose, wood sugar, saligenin, Vitamin C2, semi-lactosi, melibiose etc.; Catalase test is negative, does not produce H2S, and substratum is to grow under the condition of pH4.5, and liquefy gelatin does not produce indoles, benzidine reaction feminine gender, nitrate reduction test feminine gender;
B, Lactobacillus paracasei HD1.7 cultivate and to enter the logarithmic growth after date, begin to produce relative molecular weight and be 3000～30000 peptide class; Stationary phase, output peaked, and follow-up production has by a small margin to be increased; The peptide matters that Lactobacillus paracasei HD1.7 produces all can detect inside and outside thalline;
C, begin to producing a large amount of lactic acid stationary phase from cultivation, later lactic acid production has a small amount of increase; This bacterium can be used as lactic acid and produces bacterium, is used for the production and the applicable industry of lactic acid;
D, this bacterium have multiple therapeutic efficiencies such as the gastrointestinal function of adjustment, treatment of allergic rhinitis.
The peptide class sanitas that this bacterium produces is a kind of novel natural microbial sanitas, and this sanitas is natural, safety, scope of restraining fungi is wider, the scope of application is wideer; The sanitas that this bacterium produces is the peptide matters of molecular weight 3000～30000; Through Fermentation Conditions, determined the culture condition of Lactobacillus paracasei HD1.7; Through medium component optimization research, determine seed culture medium and fermentation culture based component.The meta-bolites of this bacterium can be produced the peptides natural microbial sanitas.
The preparation method of the peptide class sanitas that Lactobacillus paracasei HD1.7 produces comprises the steps: a, the fermentation of peptide class sanitas: with the preservation inclined-plane streak inoculation of Lactobacillus paracasei HD1.7 to fermention medium, after cultivating 36～48h, 35～40 ℃ of inversions are inoculated in the seed fermentation liquid, static cultivation 24～36h under 35～40 ℃, be connected in the enlarged culturing fermented liquid by 1～3% inoculum size again, static cultivation 24～36h under 35～40 ℃, be connected in the secondary enlarged culturing fermented liquid with 1～3% inoculum size again, static cultivation 24～36h under 35～40 ℃, continuation is inoculated in the fermented liquid by 1～3% inoculum size, at 35～40 ℃ of static cultivation 30～50h; The separation of b, peptide class sanitas: with Lactobacillus paracasei HD1.7 bacterial strain fermentation liquor centrifugal 10min under the condition of 4500r/min, in supernatant liquor, add n-propyl alcohol and NaCl, extract repeatedly, saltout to supersaturation; The acetone precipitation peptide class sanitas that adds-20 ℃ then is dissolved in throw out among the HCl again, adds KH after spending the night
2PO
4Precipitation of peptides class sanitas is dissolved in throw out among the HCl again; Add butanols and acetic acid, and transfer the pH value to 3.5 of system, add cold acetone in the upper strata, the centrifugal acquisition peptide of throw out class sanitas dry powder with ammoniacal liquor.Owing to contain salt in the sample that makes, adopting relative molecular weight to hold back scope is 3500～7000 dialysis tubing dialysis; Use CM Sephadex C-25 gel chromatography again, receive and the merging activeconstituents, use vacuum freeze drier to carry out lyophilize, make peptide class sanitas dry powder.After further separation and purification, can solve the problem that the microorganism natural antiseptic agent has peculiar smell and aspect such as variegated through peptide class sanitas that Lactobacillus paracasei HD1.7 fermentation makes well.
The present invention has been separated to the bacterial strain of the new product natural antiseptic agent of a strain, through Physiology and biochemistry and 16SrDNA order-checking, comparison, is accredited as lactobacillus paraceasi (Lactobacillus paracasei HD1.7).The peptides natural sanitas that meta-bolites is a kind of safety, scope of restraining fungi is wider, the scope of application is wideer of lactobacillus paraceasi (Lactobacillus paracasei HD1.7).The peptide class sanitas antimicrobial spectrum broad that Lactobacillus paracasei HD1.7 produces had not only had bacteriostatic action but also Gram-negative bacteria had been had restraining effect gram-positive microorganism, and various pathogens is also had bacteriostatic action.The peptide class sanitas that Lactobacillus paracaseiHD1.7 fermentation obtains all has bacteriostatic action in acid and neutral slant acidity environment.
It has following characteristics:
(1) this meta-bolites is a peptide matters: meta-bolites is after protease treatment, and bacteriostatic activity disappears, and illustrates that product is protein or peptide matters; Meta-bolites carries out electrophoresis after organic solvent extraction is slightly carried, through determining to have three kinds of peptide matters at least in the product with the Marker comparison, molecular weight is between 3000～30000;
(2) antimicrobial spectrum of peptide matters and bacteriostasis: small-molecular peptides all has restraining effect to intestinal bacteria, streptococcus aureus, micrococcus flavus, subtilis etc., promptly gram-positive microorganism and negative bacterium is all had restraining effect; Pathogenic bacterium such as Salmonellas, bacillus megaterium, Bacillus cereus, Shigellae, listeria bacteria, Bacillus proteus all there is restraining effect;
(3) character of peptide class sanitas:
A, to the susceptibility of temperature: the peptide class sanitas that Lactobacillus paracasei HD1.7 produces is handled 60min under 120 ℃ of conditions, its bacteriostatic activity is unaffected substantially, illustrates to have very strong thermostability;
B, to the susceptibility of enzyme: insensitive to stomach en-, papoid, K-proteolytic enzyme;
C, under condition of different pH fungistatic effect: at pH is under 2,3,4,5,6,7 conditions bacteriostatic activity to be arranged all, and bacteriostatic activity is the strongest during pH=2.
The present invention has following characteristics:
1, has been separated to the bacterial strain---Lactobacillus paracaseiHD1.7 (lactobacillus paraceasi HD1.7) of the new generation natural antiseptic agent of a strain, this bacterial strain can not only produce wide spectrum, efficient peptide class sanitas, can also produce a large amount of lactic acid, therefore not only can be used for producing sanitas but also can be used for producing lactic acid; This bacterium itself has health care, therapeutic efficiency, can be used for regulating gastrointestinal function, is applied to baby formula milk powder, also can be used for disease of immune system such as treatment of allergic rhinitis;
2, Lactobacillus paracasei HD1.7 metabolism has produced a kind of novel, natural peptide quasi-microorganism sanitas, this sanitas scope of restraining fungi is wider, not only gram-positive microorganism there was restraining effect, but also Gram-negative bacteria was had restraining effect, various pathogens was also had restraining effect; The scope of application is wideer, under acid and neutral slant acidity condition bacteriostatic action is arranged all, is applicable to the environment of various food such as fruits and vegetables, breast, meat, egg; Fungistatic effect is stronger, obviously is better than at home and abroad market and occupies the like product Nisin of critical role; Enrich natural microbial sanitas market, can promote greatly only to allow to add the green food of natural antiseptic agent, the development of protective foods, also further protected human beings'health;
3, the present invention studies the fermentation condition of having known Lactobacillus paracasei HD1.7, has determined inoculum size, culture temperature, incubation time, training method; Study through medium component, determined the influence of different N source, different C source, different metal ion, different concns phosphorus source to peptide class sanitas output and thalline biomass, and optimize and to have obtained can obtaining the seed culture medium of high thalline biomass and can obtaining peptide class sanitas high yield and low-cost fermention medium, for the operation of this project is laid a good foundation.
Description of drawings:
Fig. 1 is the colonial morphology figure of Lactobacillus paracasei HD1.7 of the present invention.
Embodiment:
Embodiment one: present embodiment is such isolation identification lactobacillus paraceasi HD1.7:
1. adopt the isolating method of conventional plate streaking isolated strains from L-milk-acid bacteria pickling sauerkraut fermented liquid.
2. can produce the screening of antibacterial substance bacterial strain: each single bacterium colony is after dull and stereotyped enlarged culturing, insert fermented liquid and cultivate 48h for 37 ℃, the centrifugal 12min of fermented liquid 3500r/min, draw 100 μ l supernatant liquors, the bilayer of clicking and entering 12 kinds of indicators such as Bacillus subtillis, streptococcus aureus, intestinal bacteria, Salmonellas respectively detects in the flat board, cultivate 12～24h, observe fungistatic effect, keep the bacterial strain that antimicrobial spectrum is wide, bacteriostasis is strong.
3. a strain energy metabolism that obtains produces the bacterial strain of peptide class sanitas, checks order, compares through Physiology and biochemistry and 16SrDNA, is accredited as lactobacillus paraceasi (Lactobacillus paracasei HD1.7).
Embodiment is as follows: (1) adopts conventional cultural characters, physio-biochemical characteristics to identify that it is lactobacillus that peptide class sanitas produces bacterium; (2) adopt the methods analyst program of the base sequence analysis of 16SrDNA oligonucleotide to be: according to the conserved sequence design PCR primer at the 16SrDNA gene two ends of many kinds in the lactobacillus, adopt N,O-Diacetylmuramidase-SDS-Potassium ethanoate method to extract DNA, pass through pcr amplification, electrophoresis, reclaim the PCR product, with the product cloning of PCR to plasmid vector, after amplification in thalline with the carrier that has the 16SrDNA gene as template, with M13F or M13R is the primer order-checking, carry out the 16SrDNA sequential analysis, identify that it is Lactobacillus paracaseiHD1.7 (lactobacillus paraceasi HD1.7) that peptide class sanitas produces bacterium.
Embodiment two: the peptides natural microbial sanitas preparation method of present embodiment comprises the steps: a, the fermentation of peptide class sanitas: with the preservation inclined-plane of Lactobacillus paracasei HD1.7, streak inoculation is to fermention medium, after cultivating 36～48h, 35～40 ℃ of inversions are inoculated in the seed fermentation liquid, static cultivation 24～36h under 35～40 ℃, be connected in the enlarged culturing fermented liquid by 1～3% inoculum size again, static cultivation 24～36h under 35～40 ℃, be connected in the secondary enlarged culturing fermented liquid with 1～3% inoculum size again, static cultivation 24～36h under 35～40 ℃, continuation is inoculated in the fermented liquid by 1～3% inoculum size, at 35～40 ℃ of static cultivation 30～50h; The separation of b, peptide class sanitas is purified: with Lactobacillus paracasei HD1.7 bacterial strain fermentation liquor centrifugal 10min under the condition of 4500r/min, add n-propyl alcohol and NaCl to supersaturation in supernatant liquor, extract repeatedly, saltout; The acetone precipitation peptide class sanitas that adds-20 ℃ then is dissolved in throw out among the HCl again, adds KH after spending the night
2PO
4Precipitation of peptides class sanitas is dissolved in throw out among the HCl again; Add butanols and acetic acid, and transfer the pH value to 3.5 of system, add cold acetone in the upper strata, the centrifugal acquisition peptide of throw out class sanitas dry powder with ammoniacal liquor.
Fermentation parameter is:
(1) substratum:
1, seed fermentation liquid, enlarged culturing fermented liquid and secondary enlarged culturing fermented liquid are formed by following compositions: carbon source: 1～3g/L, nitrogenous source: 1～5g/L, metal ion: 0.0001～0.001mol/L, tween: 0.1～1.0ml, potassium primary phosphate or dipotassium hydrogen phosphate: 0.01～0.02g/L.
A, carbon source can be one or more in glucose, sucrose, the lactose, and optimum carbon source is a glucose, and concentration is 1～3g/L;
In the organic nitrogen sources such as b, yeast extract paste, extractum carnis, peptone, tryptone, poly-peptone one or more, urea, soya-bean milk, (NH
4)
2SO
4, NH
4Cl, NH
4NO
3, (NH
4)
2CO
3, NaNO
3All can be used as nitrogenous source etc. in the inorganic nitrogen-sourced bacterium one or more, optimum nitrogen source is yeast extract paste and/or extractum carnis, and concentration is yeast extract paste 1～5g/L, extractum carnis 1～5g/L;
C, Mg
2+, Ca
2+, Mn
2+, Zn
2+, Co
2+, Fe
2+Deng in one or more metal ions all have and promote this bacteria growing and promote the effect that peptide matters produces that the best metal ion is Ca
2+And/or Mn
2+, concentration is Ca
2+: 0.001～0.01mol/L, Mn
2+: 0.0001～0.001mol/L;
2, fermention medium is made up of following compositions: the bean sprouts: 50g～500g/L, tomato: 100～300g/L, Chinese cabbage: 100～700g/L, sucrose: 10～30g/L or glucose: 5～20g/L, vitamin complex: 10～50ug/L, peptone: 1～5g/L or extractum carnis: 1～5g/L or yeast extract paste: 1～5g/L, NaCl:5～30g/L.
(2) culture condition:
A, at aerobic, anaerobic, amphimicrobian, fill nitrogen, fill CO
2Lactobacillus paracaseiHD1.7 all can grow, produce peptide matters under the condition;
B, Lactobacillus paracasei HD1.7 all can grow, produce peptide matters under 15～45 ℃ of conditions of culture temperature;
C, Lactobacillus paracasei HD1.7 all can grow, produce peptide matters under fermented liquid pH value 4.0～7.5 conditions;
D, Lactobacillus paracasei HD1.7 all can produce peptide matters under inoculum size 1～10% condition;
E, Lactobacillus paracasei HD1.7 produce peptide class sanitas peak period for cultivating 20～30 hours, are to make the bacterium of incomplete synchronous growth all fully produce peptide class sanitas during production, and fermentation time is decided to be 30～50 hours;
Lactobacillus paracasei HD1.7 is cultivated in f, static cultivation and concussion all can grow, produce peptide matters.
Embodiment three: what present embodiment and embodiment two were different is, it also comprises the c step, the c step is the purifying of peptide class sanitas: owing to contain salt in the sample that makes, adopting relative molecular weight to hold back scope is 3500～7000 dialysis tubing dialysis; Use CM Sephadex C-25 gel chromatography again, receive and the merging activeconstituents, use vacuum freeze drier to carry out lyophilize, make peptide class sanitas dry powder.After further separation and purification, can solve the problem that the microorganism natural antiseptic agent has peculiar smell and aspect such as variegated through peptide class sanitas that Lactobacillus paracasei HD1.7 fermentation makes well.
Sequence table
＜110〉Heilongjiang University
＜120〉the peptides natural microbial sanitas produces the fermentation process and the application of bacterium and meta-bolites thereof
<160>1
<170>Patent-In?3.1
<210>1
<211>1109
<212>DNA
＜213〉the 16SrDNA sequence of lactobacillus paraceasi HD1.7 (Lactobacillus paracasei HD1.7)
<400>1
ccggggatcc?tctagagatt?acggctacct?tgttacgact?tcaccctaat?catttgtccc 60
accttagacg?gctcgctccc?taaaagggtt?acgccaccgg?cttcgggtgt?tacaaactct 120
catggtgtga?cgggcggtgt?gtacaaggcc?cgggaacgta?ttcaccgcgg?cgtgctgatc 180
cgcgattact?agcgattccg?acttcgtgta?ggcgagttgc?agcctacagt?ccgaactgag 240
aatggcttta?agagattagc?ttgacctcgc?ggtctcgcaa?ctcgttgtac?catccattgt 300
agcacgtgtg?tagcccaggt?cataaggggc?atgatgattt?gacgtcatcc?ccaccttcct 360
ccggtttgtc?accggcagtc?ttactagagt?gcccaactaa?atgctggcaa?ctagtcataa 420
gggttgcgct?cgttgcggga?cttaacccaa?catctcacga?cacgagctga?cgacaaccat 480
gcaccacctg?tcattttgcc?cccgaagggg?aaacctgatc?tctcaggtga?tcaaaagatg 540
tcaagacctg?gtaaggttct?tcgcgttgct?tcgaattaaa?ccacatgctc?caccgcttgt 600
gcgggccccc?gtcaattcct?ttgagtttca?accttgcggt?cgtactcccc?aggcggaatg 660
cttaatgcgt?tagctgcggc?actgaagggc?ggaaaccctc?caacacctag?cattcatcgt 720
ttacggcatg?gactaccagg?gtatctaatc?ctgttcgcta?cccatgcttt?cgagcctcag 780
cgtcagttac?agaccagaca?gccgccttcg?ccactgtgtt?tcttccatat?atctacgcat 840
tcaccgctac?acatggagtt?ccactgtcct?cttctgcact?caggttccag?ttccgatgcg 900
cttctcggtt?aggcggaggg?cttcacatcc?gactaaaaac?cgctgccctc?gcttacgcca 960
taaatccgaa?aaccttgcac?ctacgtaata?cgcggctgct?gcacgtatta?gcctggcttc 1020
cggttggaat?ccgttcagcc?ggaacgttac?tttgccgaac?aatcttttct?caccaccgag 1080
ttatccaccc?cgaaggcctt?ctttactca 1109
Claims (10)
1, the peptides natural microbial sanitas produces bacterium, called after lactobacillus paraceasi HD1.7 (Lactobacillus paracasei HD1.7), and deposit number is CCTCC NO:M205015.
2, peptides natural microbial sanitas according to claim 1 produces bacterium, it is characterized in that the 16SrDNA sequence of described lactobacillus paraceasi HD1.7 is:
CCGGGGATCCTCTAGAGATTACGGCTACCTTGTTACGACTTCACCCTAATCATTTGTCCCACCTTAGACGGCTCGCTCCCTAAAAGGGTTACGCCACCGGCTTCGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAATGGCTTTAAGAGATTAGCTTGACCTCGCGGTCTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTACTAGAGTGCCCAACTAAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTTTGCCCCCGAAGGGGAAACCTGATCTCTCAGGTGATCAAAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGTGTTTCTTCCATATATCTACGCATTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAGGTTCCAGTTCCGATGCGCTTCTCGGTTAGGCGGAGGGCTTCACATCCGACTAAAAACCGCTGCCCTCGCTTACGCCATAAATCCGAAAACCTTGCACCTACGTAATACGCGGCTGCTGCACGTATTAGCCTGGCTTCCGGTTGGAATCCGTTCAGCCGGAACGTTACTTTGCCGAACAATCTTTTCTCACCACCGAGTTATCCACCCCGAAGGCCTTCTTTACTCA。
3, the peptides natural microbial sanitas produces the application of bacterium, it is characterized in that the meta-bolites of peptide class sanitas generation bacterium is used to produce the peptides natural microbial sanitas.
4, the preparation method of peptides natural microbial sanitas, it is characterized in that it comprises the steps: a, the fermentation of peptide class sanitas: with Lactobacillus paracasei HD1.7 slant preservation, streak inoculation is to fermention medium, after cultivating 36～48h, 35～40 ℃ of inversions are inoculated into seed fermentation liquid, static cultivation 24～36h under 35～40 ℃, be connected in the enlarged culturing fermented liquid by 1～3% inoculum size again, static cultivation 24～36h under 35～40 ℃, be connected in the secondary enlarged culturing fermented liquid with 1～3% inoculum size again, static cultivation 24～36h under 35～40 ℃, continuation is inoculated in the fermented liquid by 1～3% inoculum size, at 35～40 ℃ of static cultivation 30～50h; The separation of c, peptide class sanitas: with Lactobacillus paracasei HD1.7 bacterial strain fermentation liquor centrifugal 10min under the condition of 4500r/min, in supernatant liquor, add n-propyl alcohol and NaCl, extract repeatedly, saltout to supersaturation; The acetone precipitation peptide class sanitas that adds-20 ℃ then is dissolved in throw out among the HCl again, adds KH after spending the night
2PO
4Precipitation of peptides class sanitas is dissolved in throw out among the HCl again; Add butanols and acetic acid, and transfer the pH value to 3.5 of system, add cold acetone in the upper strata, the centrifugal acquisition peptide of throw out class sanitas dry powder with ammoniacal liquor.
5, the preparation method of peptides natural microbial sanitas according to claim 4, it is characterized in that it also comprises the d step, the d step is the purifying of peptide class sanitas: adopting relative molecular weight to hold back scope is that 3500～7000 dialysis tubing is dialysed to the sanitas that the c step obtains; Receive with CMSephadex C-25 gel chromatography again and merge activeconstituents, use vacuum freeze drier to carry out lyophilize, make the peptide class sanitas dry powder behind the purifying.
6, the preparation method of peptides natural microbial sanitas according to claim 4 is characterized in that described fermention medium is made up of following compositions: bean sprouts: 50g～500g/L, tomato: 100～300g/L, Chinese cabbage: 100～700g/L, sucrose: 10～30g/L, sucrose or glucose: 5～20g/L, vitamin complex: 10～50ug/L, peptone: 1～5g/L, extractum carnis: 1～5g/L, yeast extract paste: 1～5g/L, NaCl:5～30g/L.
7, the preparation method of peptides natural microbial sanitas according to claim 4 is characterized in that seed fermentation liquid, enlarged culturing fermented liquid and secondary enlarged culturing fermented liquid form by following compositions: carbon source: 1～3g/L, nitrogenous source: 1～5g/L, metal ion: 0.0001～0.001mol/L, tween: 0.1～1.0ml, potassium primary phosphate or dipotassium hydrogen phosphate: 0.01～0.02g/L.
8, the preparation method of peptides natural microbial sanitas according to claim 7 is characterized in that described carbon source is one or more the mixture in glucose, sucrose, the lactose; Described nitrogenous source is yeast extract paste, extractum carnis, peptone, tryptone, poly-peptone, urea, soya-bean milk, (NH
4)
2SO
4, NH
4Cl, NH
4NO
3, (NH
4)
2CO
3, NaNO
3In one or more mixture; Described metal ion is Mg
2+, Ca
2+, Mn
2+, Zn
2+, Co
2+, Fe
2+In one or more mixture.
9, the preparation method of peptides natural microbial sanitas according to claim 8 is characterized in that described carbon source is a glucose; Described nitrogenous source is yeast extract paste and/or extractum carnis; Described metal ion is Ca
2+And/or Mn
2+
10, the preparation method of peptides natural microbial sanitas according to claim 7 is characterized in that the pH value of seed fermentation liquid, enlarged culturing fermented liquid and secondary enlarged culturing fermented liquid is 4.0～7.5.
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| CN1285722C CN1285722C (en) | 2006-11-22 |
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| CN 200510009788 Expired - Fee Related CN1285722C (en) | 2005-03-07 | 2005-03-07 | Peptides natural microbial antiseptic agent producing strain, its use and preparation method for antiseptic agent |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181393A (en) * | 2011-04-11 | 2011-09-14 | 中国农业科学院农产品加工研究所 | High-autolysis-degree lactic acid bacteria and application thereof to cheese |
| CN101768559B (en) * | 2008-12-31 | 2012-03-07 | 生展生物科技股份有限公司 | Lactobacillus, paracasei subsp. paracasei strain, bacteriostatic composition and applications thereof |
| CN105695498A (en) * | 2016-03-18 | 2016-06-22 | 蔡河齐 | Construction method of gene engineering strain of lactobacillus paracasei with high yield of bacteriocin |
| CN105755031A (en) * | 2016-03-18 | 2016-07-13 | 黑龙江大学 | Construction method of gene engineering strain of Lactobacillus paracasei with high bacteriocin yield |
| CN108004182A (en) * | 2017-12-31 | 2018-05-08 | 武汉市天辰生物科技有限公司 | One plant of lactobacillus paracasei and its application in aquaculture |
| CN115039885A (en) * | 2021-05-14 | 2022-09-13 | 艾兰得生物科技研究泰州有限公司 | Lactobacillus paracasei with function of inhibiting growth of proteus mirabilis, and probiotic composition, fermentation liquid and application thereof |
| CN116458516A (en) * | 2023-04-23 | 2023-07-21 | 南京市畜牧兽医站(南京市动物疫病预防控制中心) | Antibacterial composition for improving livestock and poultry environment and preparation method and application thereof |
| CN117070413A (en) * | 2023-08-21 | 2023-11-17 | 广东海洋大学 | Lactobacillus paracasei BY5 and application thereof |
| CN117070413B (en) * | 2023-08-21 | 2024-05-31 | 广东海洋大学 | Lactobacillus paracasei BY5 and application thereof |
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2005
- 2005-03-07 CN CN 200510009788 patent/CN1285722C/en not_active Expired - Fee Related
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101768559B (en) * | 2008-12-31 | 2012-03-07 | 生展生物科技股份有限公司 | Lactobacillus, paracasei subsp. paracasei strain, bacteriostatic composition and applications thereof |
| CN102181393A (en) * | 2011-04-11 | 2011-09-14 | 中国农业科学院农产品加工研究所 | High-autolysis-degree lactic acid bacteria and application thereof to cheese |
| CN102181393B (en) * | 2011-04-11 | 2012-11-21 | 中国农业科学院农产品加工研究所 | High-autolysis-degree lactic acid bacteria and application thereof to cheese |
| CN105695498A (en) * | 2016-03-18 | 2016-06-22 | 蔡河齐 | Construction method of gene engineering strain of lactobacillus paracasei with high yield of bacteriocin |
| CN105755031A (en) * | 2016-03-18 | 2016-07-13 | 黑龙江大学 | Construction method of gene engineering strain of Lactobacillus paracasei with high bacteriocin yield |
| CN108004182A (en) * | 2017-12-31 | 2018-05-08 | 武汉市天辰生物科技有限公司 | One plant of lactobacillus paracasei and its application in aquaculture |
| CN115039885A (en) * | 2021-05-14 | 2022-09-13 | 艾兰得生物科技研究泰州有限公司 | Lactobacillus paracasei with function of inhibiting growth of proteus mirabilis, and probiotic composition, fermentation liquid and application thereof |
| CN115039885B (en) * | 2021-05-14 | 2023-11-03 | 江苏艾兰得营养品有限公司 | Lactobacillus paracasei with function of inhibiting growth of Proteus mirabilis, and probiotic composition, fermentation liquor and application thereof |
| CN116458516A (en) * | 2023-04-23 | 2023-07-21 | 南京市畜牧兽医站(南京市动物疫病预防控制中心) | Antibacterial composition for improving livestock and poultry environment and preparation method and application thereof |
| CN116458516B (en) * | 2023-04-23 | 2024-01-30 | 南京市畜牧兽医站(南京市动物疫病预防控制中心) | Antibacterial composition for improving livestock and poultry environment and preparation method and application thereof |
| CN117070413A (en) * | 2023-08-21 | 2023-11-17 | 广东海洋大学 | Lactobacillus paracasei BY5 and application thereof |
| CN117070413B (en) * | 2023-08-21 | 2024-05-31 | 广东海洋大学 | Lactobacillus paracasei BY5 and application thereof |
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| CN1285722C (en) | 2006-11-22 |
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