CN101333507B - Enterococcus faecium Grx28 with flame resistance and its use - Google Patents
Enterococcus faecium Grx28 with flame resistance and its use Download PDFInfo
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- CN101333507B CN101333507B CN2008100212813A CN200810021281A CN101333507B CN 101333507 B CN101333507 B CN 101333507B CN 2008100212813 A CN2008100212813 A CN 2008100212813A CN 200810021281 A CN200810021281 A CN 200810021281A CN 101333507 B CN101333507 B CN 101333507B
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Abstract
Disclosed is an enterococcus faecium Grx28 having the heat-resistant quality as well as a method for the production thereof and purposes, relating to the biotechnology field, in particular to an enterococcus faecium isolated strain with good heat-resistant quality separated from the traditional dairy products of the Yunnan nationality as well as applications thereof. The preparation method includes the following steps of: separating out lactobacillus from the traditional dairy products in the Dali or DengChuan area in Yunnan Province; and screening lactobacillus having the 60DEG C/10min- or 65DEG C/5min-resistant thermal handling capacity, the number of live bacteria exceeds 10<7> or 10<5>cfu/mL, namely, the live bacteria are enterococcus aecia Grx28. The method is simple, easy in operation and high in preparation rate, and can be used for making dairy fan, activity lactobacillus beverage, yoghurt powder and fermentation food, and the number of live bacteria produced by the method is 100 times of that produced by the traditional alkekengy, so the products are applicable for normal temperature sales, and the guarantee period reaches 15 to 30 days.
Description
Technical field
The present invention relates to biological technical field, particularly from Yunnan's Ethnic tradition milk-product, separate the faecium strain isolated that acquisition has good heat-resistant quality, and use.
Technical background
But milk-acid bacteria is a class fermentable carbohydrates, and mainly generates the general name of the bacterium of lactic acid.Milk-acid bacteria has very high using value in industry, agricultural, food and health care etc. and the closely-related field of the mankind, now be widely used in many fields such as food-processing, medicines and health protection, feed fermentation and livestock and poultry disease control and prevention.
In the preparation of starter, leavened food such as cheese, lactic acid fermented beverage, and the aspects such as production of viable lactic acid bacteria preparation all need product to keep higher number of viable after processing.A lot of research work are devoted to improve the wherein quantity of milk-acid bacteria, perhaps add special nutrition composition, efficient degeneration-resistant protective material etc. as the method that reduces milk-acid bacteria death.Their production needs to bear the milk-acid bacteria of general processing conditions but the most basic is, only control milk-acid bacteria loss in the treating processes of process for processing, the vigor and the number of viable that could in storage, be kept milk-acid bacteria, in the hope of when entering the consumption link, still having the viable bacteria of some amount, therefore have the screening of the milk-acid bacteria of certain tolerance, the importance that domestication becomes food microorganisms research.
Summary of the invention
The present invention's first purpose is to provide a kind of milk-acid bacteria with temperature capacity.
Be faecium Grx28, on June 24th, 2008 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, preserving number CGMCC NO:2555.
Through evidence, faecium Grx28 has high-temperature tolerance, under the treatment condition of 60 ℃/10min, number of viable before the thermal treatment 8.4 * 10
8Cfu/mL only drops to 2.4 * 10 after the processing
7Cfu/mL under the treatment condition of 65 ℃/5min, drops to 2.1 * 10 after the processing
5Cfu/mL.
Second purpose of the present invention is to provide the preparation method of above-mentioned faecium Grx28:
May further comprise the steps:
1, from Dali or the geographic traditional milk-product of Deng Chuan, isolates milk-acid bacteria;
2, screening has the milk-acid bacteria of 60 ℃/10min of tolerance or 65 ℃/5min heat treatment capacity, is faecium Grx28.
Present method is simple, easy handling, produce the rate height, filter out safe milk-acid bacteria,, and carry out Analysis and Identification by the French Mei Aili API of company series indentifying substance bar and 16SrRNA again by ne ar, physiology and cultural characteristic with excellent temperature capacity.The result has confirmed that bacterial strain of the present invention is novel manure enterococcin strain.
The 3rd purpose of the present invention is to invent the purposes of faecium Grx28:
Can be used for making the breast fan.Viable count reaches 5.2 * 10 in the breast fan finished product
6Cfu/g is higher 100 times than the product viable count of producing with traditional wintercherry.
Also can be used for making lactic acid fermented beverage.Product can be suitable for normal temperature to be sold, and the quality guaranteed period reaches 15-30 days.
Also can be used for making yogurt powder.Because bacterial strain of the present invention has good heat-resisting property, therefore the product living lactic acid bacteria quantity of producing is more than 100 times of ordinary lactic acid bacteria.
Being used to make other leavened food also is a kind of good selection.
Description of drawings
Fig. 1 is the fluorescence photo figure of faecium Grx28 genomic dna.
The fluorescence photo figure of the 16S rDNA-PCR product of Fig. 2 faecium Grx28.
Embodiment
Faecium Grx28 of the present invention on June 24th, 2008 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, preserving number CGMCC NO:2555.
Following embodiment has set forth practice of the present invention and preferred embodiment at present.
1, the source of sample and collection
The Yunnan multiple microorganism growth that suits, the milk-acid bacteria aboundresources, and the biological characteristics of milk-acid bacteria has obtained good preservation.Living all the year round based on the herdsman who herds in the pastoral area, Yunnan, they continue to use the traditional zymotic milk-product with national characters of the unique flavor of traditional way production, and are nutritious, have good sense organ and functional performance.Because traditional milk-product are by making by hand over the past thousands of years, make the milk-acid bacteria of the many good characters in traditional milk-product be existed and biological characteristics has obtained good preservation.
Originating in lactic acid bacterium of the present invention is national milk-product in the Dali---the breast fan.The breast fan is that Yunnan Province of China is rich in one of traditional milk-product of characteristic, is a kind of tension type cheese of acid cure that adopts the natural fermented wintercherry of milk-acid bacteria to make that adds with cow's milk.Breast fan sample is collected in area, Chinese yunnan Bai Autonomous Prefecture of Dali Eryuan respectively.Sample is deposited in the sterilization sampling bottle, places under 4 ℃ of conditions and preserves.
2, from the breast fan, isolate milk-acid bacteria
Get the 1.0g solid sample, aseptic pulverizing places sterilized water, and thorough mixing is respectively got the substratum that 1mL is inoculated in table 1 respectively, according to the array mode enrichment culture of substratum and culture condition.
To be collected in Dali national milk-product breast fan sample in the M17 liquid nutrient medium, cultivate enrichment in 24 hours for 42 ℃.This culture is coated on the M17 agar plate, cultivated 48 hours at 42 ℃ then.After the cultivation, be collected in the bacterium colony that occurs on the nutrient agar, and be further purified, eliminate non-lactic bacterium strains.
The bacterial strain that will separate obtain is with the broth culture that contains 11% skimming milk, cultivation, freeze-drying, is kept at-18 ℃, is used for the test of various bacterial detection features then.
3, the screening of thermotolerant lactic acid bacteria
The heat-resistant quality that to screen 108 strains of lactic acid bacteria that obtain from Yunnan compares.Different milk-acid bacterias are inoculated in the degreasing milk medium by 4% inoculum size respectively, are cultured to curdled milk, when measuring curdled milk respectively and through 60 ℃/5min, 60 ℃/10min, 65 ℃/5min, the number of viable after the 65 ℃/10min thermal treatment, the relatively heat-resistant quality of different strains.Select the best bacterial strain of temperature capacity.
By the comparison to screening bacterial strain temperature capacity, as can be seen, there is very big-difference in the different strains heat-resistant quality.In 108 strain bacterial strains, there are 49 strain bacterium not tolerate the thermal treatment intensity of 60 ℃/5min, account for more than 45% of test strain quantity, but have 27 strain bacterial strains can tolerate the thermal treatment intensity of 60 ℃/10min, number of viable still can reach 10 after the bacterial strain thermal treatment wherein of the present invention
7Cfu/mL is for the highest.Have 92 strain bacterium not tolerate the thermal treatment intensity of 65 ℃/5min, account for more than 85% of test strain quantity, in the 16 strain bacterial strains that the viable bacteria survival is arranged, bacterial strain number of viable of the present invention can reach 10
5Cfu/mL is for the highest.
4, lactic bacterium strains gramstaining
Adopt gram staining method to detect the feature of bacterial strain.Bacterial strain of the present invention is gram-positive microorganism, and is spherical for no gemma, and size is 1.0-1.5um.
Then, study this milk-acid bacteria, to illustrate its cultural characteristic.The result is, this bacterial classification grows up to the white colony that diameter is 2-3mm on the M17 agar plate, and optimum culturing temperature is 42 ℃.
5, identification of strains
(1) use API20 STREP to the bacterial strain analysis
By API20 STREP the invention bacterial strain is identified, shown that 99.3% is faecium (E.faecium).
The API20 STREP identification systems reaction result of table 1 bacterial strain of the present invention
| VP | HIP | ESC | PYRA | αGAL | βGUR | βGAL | PAL | LAP | ADH | RIB |
| + | - | + | + | + | - | + | - | + | + | + |
| ARA | MAN | SOR | LAC | TRE | INU | RAE | AMD | GLGY | HEM | |
| + | + | - | + | + | - | - | + | - | - |
(2) bacterial strain 16SrDNA Sequence Identification
● the extraction of milk-acid bacteria DNA
Adopt bacterial genomes DNA extraction agent box in a small amount, operate according to explanation, the genome dna electrophoresis band of the faecium Grx28 that is extracted is clear, as shown in Figure 1, illustrate that the genomic dna integrity better can be used for the 16SrDNA amplification, wherein, swimming lane M is: LambdaDNA/EcoRI+HindIII Marker, 3; Other 6 swimming lanes are the genome of invention milk-acid bacteria.
● 16SrDNA amplification, clone and order-checking
With the genomic dna is template, and milk-acid bacteria of the present invention is carried out pcr amplification, and amplified production is done electrophoresis detection.Amplified production compares with Marker, clear band (as shown in Figure 2) occurs at 1375～1584bp place.Wherein, swimming lane M is: LambdaDNA/EcoRI+HindIII Marker, 3; Other 6 swimming lanes are represented the amplified production of milk-acid bacteria of the present invention respectively.
Sequencing is finished by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, adopts two-way order-checking.
The length that faecium Grx2816SrDNA measures sequence is 1472bp.
● the online BLAST result of bacterial strain 16SrDNA sequence of the present invention
With the online BLAST comparison of 16SrDNA sequence of each bacterial strain of measuring, the 16SrDNA gene order of bacterial strain of the present invention as a result and E.faecium NRIC 0114 only differs 2 bases, and homology is up to 99.9%.From online comparison result as can be seen, bacterial strain of the present invention and the bacterial strain of delivering in the world have higher homology, and there is some difference but partial sequence is still arranged, and show that bacterial strain of the present invention belongs to faecium, but with the faecium of having reported difference is also arranged, belong to new bacterial strain.
Application example
Embodiment 1: the breast fan
(1) breast fan manufacture craft flow process
Fresh cow milk, sterilization, be cooled to 60 ℃, add wintercherry, stirring, rubbing are agglomerating, washing, blanching stretch, dry, the newborn fan of finished product.
(2) concrete operations step
● fermentation wintercherry: adopt faecium Grx28 and L.helveticusNRg11, S.thermophilusST
1With the starter of 1: 1: 1 mixed as the fermentation wintercherry of producing the breast fan, make the breast fan with the wintercherry of producing, the wintercherry of finally fermenting pH is 3.30.
● sterilization: fresh cow milk adopts the processing condition sterilization of 72 ℃/15～20s.
● select slurry: the cow's milk after the sterilization is cooled to 60 ℃, adds the pH3.30 wintercherry and make the protein gelling, and whey pH4.6 after the slurry curdled milk of reference mark.
● washing, stretch: protein gel is crumpled agglomerating, simultaneously with the wintercherry washing of pH4.6,65 ℃ of control wash temperatures, washing time 4min, the nodule that makes under these processing condition carry out blanching and stretch.
(3) the various physical and chemical indexs of breast fan
The breast fan that makes under above-mentioned processing condition is carried out drying treatment, obtain finished product breast fan, recording its protein content is 38.44%, lipid content is 39.13%, and ash content is 4.64%, proofreaies and correct yield rate and reaches 6.46%, the total protein utilization ratio is 72.61%, and the fat utilization rate is 71.74%.Viable count reaches 5.2 * 10 in the breast fan finished product
6Cfu/g is higher 100 times than the product viable count of producing with traditional wintercherry.
Embodiment 2: lactic acid fermented beverage
The 1000g raw milk is preheating to 65 ℃, add 70g sugar, dissolve the back under 20MPa pressure homogeneous once, at 95 ℃ of following thermal treatment 10min, be cooled to 45 ℃ then, add hybrid bacterial strain fermentation (faecium Grx28: L.helveticusNRg11: S.thermophilus ST
1It is 1: 1: 1).Behind 42 ℃ of fermentation 7h, add through sterilization and refrigerative 1200g water, reach an amount of sucrose, stablizer, essence etc., after stirring, health or sterile filling are adopted in sterilization under the condition of 65-75 ℃/5-30S behind the homogeneous, and the product number of viable reaches 10
6-10
8Cfu/mL, product can be suitable for normal temperature and sell, and the quality guaranteed period reaches 15-30 days.
Embodiment 3: yogurt powder
After 90-95 ℃/5-10min of the 1000g raw milk process thermal treatment, inoculation hybrid bacterial strain fermentation (faecium Grx28: L.helveticusNRg11: S.thermophilus ST
1Be 1: 1: 1) at 42 ℃ of fermentation 7h, then immediately at 53 ℃, 16 * 10
3Low pressure concentrates and obtains concentrating breast under the vacuum of handkerchief, and the concentration of controlling 50 ℃ is 12～16
0Be ', milk solids content are 45%～50%.With other auxiliary material through 85 ℃/20s sterilization after, be cooled to 45 ℃ then, mix with concentrated fermented-milk, homogeneous carries out spraying drying at last, the spraying drying inlet temperature is 170 ℃, 75 ℃ of tower temperature, temperature of outgoing air is 70～80 ℃.Selling point: because bacterial strain of the present invention has good heat-resisting property, therefore the product living lactic acid bacteria quantity of producing is more than 100 times of ordinary lactic acid bacteria.
Embodiment 4: leavened foods such as cheese
Because many leavened foods are in the course of processing, after the product fermentation, often need it is carried out certain thermal treatment, in cheese processing, after curdled milk, need carry out hyperthermic treatment to granular curd, improve the elasticity and the intensity of granular curd, accelerate wheys draining, but in the after ripening process, also need certain milk-acid bacteria to participate in the after ripening process simultaneously, therefore utilize bacterial strain of the present invention to produce leavened foods such as cheese, just have a good application prospect.
As be applied to the production of natural cheese, concrete technical process is as follows:
With sterilization after the raw dairy stdn (71 ℃～75 ℃/15s), through 30 ℃～32 ℃ coolings, add starter (Lactococcus lactis breast subspecies: lactococcus lactis subsp: dimethyl diketone suis: faecium Grx28=1: 1: 1: 1, inoculum size is 1%-2%), after adjusting acidity, adding calcium chloride, add pigment, add milk coagulant, the grumeleuse cutting is again through stirring, heat, discharge whey, after moulding squeezing, salt marsh, the maturation, wax is hung in colouring.
＜110〉Yangzhou University
＜120〉have faecium Grx28 and preparation method thereof, the purposes of heat-resistant quality
<160>1
<210>1
<211>1472
<212>DNA
＜213〉milk-acid bacteria
<220>
＜223〉from Dali or the geographic traditional milk-product of Deng Chuan, separate
<400>1
gcgattggcg atcatgttat acatgcaagt cgtacgcttc tttttccacc ggagcttgct 60
ccaccggaaa aagaagagtg gcgaacgggt gagtaacacg tgggtaacct gcccatcaga 120
aggggataac acttggaaac aggtgctaat accgtataac aatcgaaacc gcatggtttt 180
gatttgaaag gcgctttcgg gtgtcgctga tggatggacc cgcggtgcat tagctagttg 240
gtgaggtaac ggctcaccaa ggccacgatg catagccgac ctgagagggt gatcggccac 300
attgggactg agacacggcc caaactccta cgggaggcag cagtagggaa tcttcggcaa 360
tggacgaaag tctgaccgag caacgccgcg tgagtgaaga aggttttcgg atcgtaaaac 420
tctgttgtta gagaagaaca aggatgagag taactgttca tcccttgacg gtatctaacc 480
agaaagccac ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt 540
ccggatttat tgggcgtaaa gcgagcgcag gcggtttctt aagtctgatg tgaaagcccc 600
cggctcaacc ggggagggtc attggaaact gggagacttg agtgcagaag aggagagtgg 660
aattccatgt gtagcggtga aatgcgtaga tatatggagg aacaccagtg gcgaaggcgg 720
ctctctggtc tgtaactgac gctgaggctc gaaagcgtgg ggagcaaaca ggattagata 780
ccctggtagt ccacgccgta aacgatgagt gctaagtgtt ggagggtttc cgcccttcag 840
tgctgcagct aacgcattaa gcactccgcc tggggagtac gaccgcaagg ttgaaactca 900
aaggaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg 960
aagaacctta ccaggtcttg acatcctttg accactctag agatagagct tccccttcgg 1020
gggcaaagtg acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa 1080
gtcccgcaac gagcgcaacc cttattgtta gttgccatca ttcagttggg cactctagca 1140
agactgccgg tgacaaaccg gaggaaggtg gggatgacgt caaatcatca tgccccttat 1200
gacctgggct acacacgtgc tacaatggga agtacaacga gttgcgaagt cgcgaggcta 1260
agctaatctc ttaaagcttc tctcagttcg gattgcaggc tgcaactcgc ctgcatgaag 1320
ccggaatcgc tagtaatcgc ggatcagcac gccgcggtga atacgttccc gggccttgta 1380
cacaccgccc gtcacaccac gagagtttgt aacacccgaa gtcggtgagg taaccttttt 1440
ggagccagcc gcctaaggtg gaaagatgct gg
Claims (5)
1. the faecium Grx28 that has heat-resistant quality, its feature in, preserving number is CGMCCNO.2555.
2. the faecium Grx28 that has heat-resistant quality according to claim 1 is used to make the breast fan.
3. the faecium Grx28 that has heat-resistant quality according to claim 1 is used to make lactic acid fermented beverage.
4. the faecium Grx28 that has heat-resistant quality according to claim 1 is used to make yogurt powder.
5. the faecium Grx28 that has heat-resistant quality according to claim 1 is used to make leavened food.
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| CN101333507B true CN101333507B (en) | 2010-07-21 |
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| CN101629155B (en) * | 2009-04-30 | 2012-07-25 | 邵峰 | Enterococcus faecium |
| CN101701202B (en) * | 2009-11-27 | 2012-06-06 | 南京农业大学 | Enterococcus faecalis and application thereof |
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Owner name: YANGZHOU YANGDA KANGYUAN DAIRY INDUSTRY CO., LTD. Free format text: FORMER OWNER: YANGZHOU UNIVERSITY Effective date: 20141226 |
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Effective date of registration: 20141226 Address after: 225000 No. 88 Dingxing Road, Guangling District, Jiangsu, Yangzhou Patentee after: Yangzhou Yangda Kuangyuang Dairy Industry Co., Ltd. Address before: 225009 Yangzhou University, Jiangsu, South Road, No. 88 Patentee before: Yangzhou University |