CN102559561B - Food-sourced lactic acid bacterium and application thereof - Google Patents
Food-sourced lactic acid bacterium and application thereof Download PDFInfo
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- CN102559561B CN102559561B CN201210044449.9A CN201210044449A CN102559561B CN 102559561 B CN102559561 B CN 102559561B CN 201210044449 A CN201210044449 A CN 201210044449A CN 102559561 B CN102559561 B CN 102559561B
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- food
- milk
- acid bacteria
- lactic acid
- acid
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Abstract
The invention discloses a novel food-sourced lactic acid bacterial strain Lactobacillus curieae CCTCC NO: M2011381. The lactic acid bacterium has a good antimicrobial effect on Gram-positive bacterium Gram-negative bacterium can be added into food as a food preservative.
Description
Technical field
The invention belongs to microorganism field, specifically, is about a kind of food source property milk-acid bacteria and application thereof.
Background technology
Milk-acid bacteria (Lactic acid bacteria, LAB) refers to that a class can produce the bacterium of lactic acid by homofermentation or heterofermentation, is generally Gram-positive, and anaerobism is without gemma, acidproof.Be not the section in phylogenetic systematics or a genus on stricti jurise, this class bacterium of having found at present at least comprises 8 genus on systematic bacteriology, as: the minority kind in lactobacillus genus, genus bifidobacterium, streptococcus, bright beading Coccus, enterococcus spp, lactococcus, carnivorous Bacillaceae, unusual Pseudomonas, Pediococcus, Aerococcus, roaming Coccus, Listeria, Sporolactobacillus, bacillus, circumfili mattress genus, erysipelothrix, twin Pseudomonas and sugared Coccus etc.The overwhelming majority is anaerobism or amphimicrobian chemoheterotrophic bacteria, and growth and breeding is in anaerobism or the abundant sour environment of micro-oxygen consumption, mineral substance and organic nutrient substance.In the digestive tube of animal, content is higher, account for greatly normal microflora 10%, be the integral part of humans and animals normal microflora, usually used as the probiotic agent of safety, add in food and animal-feed, and the ecology of milk-acid bacteria and the ecology of normal microflora closely related.
Special physiological activity and the trophic function of milk-acid bacteria, cause people's attention just day by day in recent years.Milk-acid bacteria can be given tart flavour and the fragrance that food is soft, improves quality and the nutrition of food, have antibacterial, reduce cholesterol, maintain micro-ecological environment, the important biomolecule such as antitumor, anti-mutagen and strengthening immunity learns function, in food, be widely used.The food of lactobacillus-fermented is acknowledged as functional foodstuff.Milk-acid bacteria is considered to optimal natural food-preservative, its anti-microbial activity showing also can prevent microbial contamination, it can produce some natural antiseptic-germicides, comprises organic acid (lactic acid, acetic acid, formic acid, phenyllactic acid, caproic acid), carbonic acid gas, hydrogen peroxide, di-acetyl, ethanol and bacteriocin etc.
Summary of the invention
Present inventor is in the research process of the microbial diversity in bean curd with odor saltwater brine, and separation has obtained a kind of food source property milk-acid bacteria, through being accredited as a kind of new milk-acid bacteria.
Therefore, primary and foremost purpose of the present invention is to provide a kind of new food source property milk-acid bacteria.
Second object of the present invention is to provide the application of described food source property milk-acid bacteria.
The 16S rRNA sequence of food of the present invention source property milk-acid bacteria is as shown in SEQ NO:1.
According to a preferred embodiment of the present invention, the preserving number of described milk-acid bacteria is CCTCC NO:M2011381.
According to a preferred embodiment of the present invention, described milk-acid bacteria can be used for food antiseptic.
The present invention not only provides a kind of new food source property milk-acid bacteria, and this milk-acid bacteria has bacteriostatic action, therefore can add to and in food, be used as food preservatives.
Accompanying drawing explanation
Fig. 1 is the transmission electron microscope picture of the food source property milk-acid bacteria that obtains of the present invention.
Fig. 2 is food source property milk-acid bacteria and the evolutionary tree of other milk-acid bacterias based on 16S rRNA that the present invention obtains.
Embodiment
Below, by specific embodiment, the present invention is described in further details.Should be understood that following examples are only for the present invention is described but not for limiting scope of the present invention.
The present invention's bacterial classification used is milk-acid bacteria Lactobacillus curieae S1L19, on September 29th, 2011, is preserved in Chinese Typical Representative culture collection center, and preservation address is Wuhan, China Wuhan University, and preserving number is CCTCC NO:M2011381.
The formula of the MRS substratum using in following examples is: trypticase 1%, extractum carnis powder 0.5%, yeast powder 0.4%, glucose 2%, sodium acetate 0.5%, triammonium citrate 0.2%, tween 80 0.1%, dipotassium hydrogen phosphate 0.2%, magnesium sulfate 0.02%, manganous sulfate 0.005%, pH6.0.
Separated and the evaluation of embodiment 1, food source property milk-acid bacteria
1.1, growth conditions
Present inventor is when research bean curd with odor saltwater brine microbial diversity, separation obtains a strain food source property bacterial strain, its growth conditions is measured, result shows that it is at de Man-Rogosa-Sharpe(MRS) well-grown on substratum, growth temperature range is 15～45 ℃, optimum growth temperature is 30 ℃, and growth pH scope is 4.0～8.5.
1.2, strain morphology
Observe the form of the bacterial strain obtaining in 1.1, under the condition of 30 ℃, cultivate 48h on MRS substratum after, this bacterial strain is carried out to transmission electron microscope observing (magnification is 10000), result as shown in Figure 1, cell size is (1.2～1.3) * (0.6～0.8) μ m, and growth pattern is single, paired or bunchiness growth.
1.3, utilization of carbon source
Use API50CHL system (BioM é rieux, France) to measure the utilization of carbon source situation of the bacterial strain obtaining in 1.1, concrete operation method is according to working instructions.
Result shows that this bacterial strain can utilize following carbon source: L-arabinose, D-ribose, D-semi-lactosi, D-Glucose, D-Fructose, D-Maltose, D-melibiose, D-sucrose, D-melizitose, D-raffinose, D-turanose, methyl-α-D-glucopyranoside, D-MANNOSE (faint utilization), D-lactose (faint utilization), gluconic acid (faint utilization).
This bacterial strain can not utilize following carbon source: glycerine, erythritol, D-R, L-wood sugar, D-Pentitol, methyl-β-D-xylopyranoside, L-sorbose, L-rhamnosyl, melampyrin, inositol, PEARLITOL 25C, D-glucitol, methyl-β-D-mannopyranose alcohol, N-acetyl-glucosamine, arbutin, saligenin, D-cellobiose, D-trehalose, inulin, glycogen, Xylitol, amygdaloside, β-gentiobiose, D-lyxose, D-Tag, D-Fucose, L-fucose, D-R alcohol, L-arabinose alcohol, 5-ketone group Potassium Gluconate and 2-ketone group Potassium Gluconate.
1.4,16S rRNA order-checking
The bacterial strain obtaining in 1.1 is carried out to 16S rRNA order-checking, its sequence is compared to the 16SrRNA of relevant kind, adopt ortho position phase connection (neighbor-joining) to draw evolutionary tree, result as shown in Figure 2.Wherein, the homology of the 16S rRNA of bacterial strain of the present invention and other milk-acid bacterias is respectively: L.senioris JCM17472(96.5%), L.diolivorans JCM12183(96.5%), L.parafarraginis JCM14109(96.4%), L.hilgardii ATCC8290(96.3%), L.rapi DSM19907(96.3%).Therefore, bacterial strain and other milk-acid bacterias that the present invention obtains have very high affinity, but not identical with known milk-acid bacteria.
1.5, Product Identification
Take glucose as substrate, use D-/L-Plasma lactate test kit (R-Biopharm AG, Germany) to measure the degraded product of the bacterial strain obtaining in 1.1, concrete operation method is according to working instructions.Result shows, comprises D type and L-type lactic acid in its product.
In sum, can identify that the bacterial strain that the present invention obtains is the new milk-acid bacteria of a strain, by its called after Lactobacillus curieae S1L19, be preserved in Chinese Typical Representative culture collection center, preserving number is CCTCC NO:M2011381.
The biological activity of embodiment 2, food source property milk-acid bacteria
2.1, fermentation
The mono-bacterium colony of picking food source property milk-acid bacteria Lactobacillus curieae CCTCC NO:M2011381, is seeded to 3ml de Man-Rogosa-Sharpe(MRS) substratum, at 30 ℃, under the condition of 200rpm, shaking table is cultivated 22 hours.By fermented liquid, in 4 ℃, centrifugal 10 minutes of 12000rpm, gets supernatant liquor, with aseptic 0.22 μ m filter filtration sterilization, is positioned in-20 ℃ of refrigerators stand-by.
2.2, biological activity determination
The biological activity determination of food of the present invention source property milk-acid bacteria Lactobacillus curieae CCTCC NO:M2011381 adopts double-deck agar diffusion method, and concrete measuring method is as follows:
Get indicator streptococcus aureus (gram-positive microorganism) and each 50 μ l of dust Xi Shi intestinal bacteria (Gram-negative bacteria) of the preservation of glycerine pipe, be seeded to 3ml Luria-Bertani(LB) substratum, at 37 ℃, under the condition of 200rpm, shaking table is cultivated 16 hours.Aseptic blue rifle head is inverted, is then placed on equably on 1.5% the agar solidifying.Get the LB substratum that 20ml melts and is cooled to approximately 55 ℃, add streptococcus aureus or the colibacillary bacterium liquid of dust Xi Shi of 50 μ l incubated overnight, after mixing, be added on 1.5% the agar that is placed with rifle head, treat that it is cooling, extract rifle head.In each hole, add the food source property milk-acid bacteria Lactobacillus curieae CCTCC NO:M2011381 fermented liquid obtaining in 200 μ l3.1, in 37 ℃ cultivate 10 hours after observations.
Result shows, the fermented liquid of food of the present invention source property milk-acid bacteria Lactobacillus curieae CCTCC NO:M2011381 can suppress streptococcus aureus and the colibacillary growth of dust Xi Shi, to two kinds of indicators, can produce the inhibition zone of 2.0～2.5 centimetres.
Food of the present invention source property milk-acid bacteria Lactobacillus curieae CCTCC NO:M2011381 has good fungistatic effect to gram-positive microorganism and Gram-negative bacteria, and this milk-acid bacteria separation is from food, therefore can add in food and be used as food preservatives, this is apparent for a person skilled in the art.
Sequence table
<110> East China University of Science
<120> food source property milk-acid bacteria and application thereof
<130>P4-12110151
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<170>PatentIn version3.3
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<213>Lactobacillus curieae CCTCC NO：M2011381
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agagtttgat catggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgaac 60
gcgtcctcgc tgatgaagtt gaagtgcttg cacggattct gattcaacat ttggacgagt 120
ggcgaactgg tgagtaacac gtgggtaacc tgccctcaag caggggataa cacttggaaa 180
caggtgctaa taccgtataa caatgaaaac cgcatggttt tcatttgaaa gatggtttcg 240
gctatcactt gaggatggac ccgcggcgta ttagctagtt ggtgaggtaa cggctcacca 300
aggcaatgat acgtagccga cctgagaggg taatcggcca cattgggact gagacacggc 360
ccaaactcct acgggaggca gcagtaggga atcttccaca atggacgaaa gtctgatgga 420
gcaacgccgc gtgagtgaag aagggtttcg gctcgtaaaa ctctgttgtt aaagaagaac 480
aggtgtaaga gtaactgttt acaccgtgac ggtatttaac cagaaagcca cggctaacta 540
cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggattta ttgggcgtaa 600
agcgagcgca ggcggtcttt taagtctgat gtgaaagcct tcggcttaac cggagaagtg 660
catcggaaac tgggagactt gagtgcagaa gaggacagtg gaactccatg tgtagcggtg 720
aaatgcgtag atatatggaa gaacaccagt ggcgaaggcg gctgtctggt ctgtaactga 780
cgctgaggct cgaaagcatg ggtagcgaac aggattagat accctggtag tccatgccgt 840
aaacgatgag tgctaagtgt tggagggttt ccgcccttca gtgctgcagc taacgcatta 900
agcactccgc ctggggagta cgaccgcaag gttgaaactc aaaggaattg acgggggccc 960
gcacaagcgg tggagcatgt ggtttaattc gatgctacgc gaagaacctt accaggtctt 1020
gacatcttct gctaacccaa gagattgggc gttcccttcg gggacggaat gacaggtggt 1080
gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1140
ccttattgtt agttgccagc atttagttgg gcactctagc aagactgccg gtgacaaacc 1200
ggaggaaggt ggggacgacg ccaaatcatc atgcccctta tgacctgggc tacacacgtg 1260
ctacaatgga cggtacaacg agtcgcgaaa ccgcgaggtc aagctaatct cttaaagccg 1320
ttctcagttc ggattgcagg ctgcaactcg cctgcatgaa gttggaatcg ctagtaatcg 1380
tggatcagca tgccacggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca 1440
tgagagtttg taacacccaa agtcggtgag gtaacctttt ggagccagcc gcctaaggtg 1500
ggacagatga ttagggtgaa gtcgtaacaa ggtaaccgta 1540
Claims (2)
1. a food source property milk-acid bacteria, is characterized in that, the preserving number of described milk-acid bacteria is CCTCC NO:M2011381.
2. the application of milk-acid bacteria as claimed in claim 1, is characterized in that, for food antiseptic.
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