CN105274023A - Lactobacillus plantarum L. plantarum HM6055 and use thereof - Google Patents
Lactobacillus plantarum L. plantarum HM6055 and use thereof Download PDFInfo
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- CN105274023A CN105274023A CN201510635793.9A CN201510635793A CN105274023A CN 105274023 A CN105274023 A CN 105274023A CN 201510635793 A CN201510635793 A CN 201510635793A CN 105274023 A CN105274023 A CN 105274023A
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- bread
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Abstract
The invention discloses a Lactobacillus plantarum L. plantarum HM6055 and a use thereof. The Lactobacillus plantarum HM6055 is a separated brand new Lactobacillus plantarum, and the preservation number of the Lactobacillus plantarum is CGMCC No.11213. The above strain can generate high content of phenyllactic acid, and can be used for the fields of micro-ecologic preparations and foods.
Description
Technical field
The present invention relates to microorganism field, particularly relate to plant lactobacillus
l.plantarumHM6055and uses thereof.
Background technology
Along with the raising of people's living standard and the reinforcement of environmental consciousness, people pay special attention to the health of oneself life, and be concerned about that so special chemical is on the impact of health, thus food preservatives is also faced with new selection.The feature that microbiological antiseptic is safe with it, natural, healthy and receiving much concern, various countries drop into manpower and materials research one after another.Along with China is to the pay attention to day by day of food safety, replace Chemical Preservative to become food with biological preservative
preservationthe development trend of technology.
Phenyllactic acid has very strong anti-corrosive fresh-keeping effect, can be used on fruit wine, beverage, meat, food, cake makes, vegetables (olive, gherkin, pearl onion) are pickled and can processing, grain processing, fruit storage, have adjust ph, antibacterial, extend the shelf life, seasoning, maintenance food color, the effect such as to improve the quality of products;
The consumption of China in Recent Years to baking bread based food increases year by year, also day by day improves the requirement of its quality, has occurred much utilizing plant lactobacillus and the fermentation of thermophilus streptococcus auxiliary yeast, to improve bread nutritive value, to extend the novel method of its shelf-lives.
Summary of the invention
The object of the present invention is to provide a kind of new strains plant lactobacillus
l.plantarumHM6055.
For achieving the above object, the invention provides plant lactobacillus
l.plantarumHM6055, it is characterized in that,
preservationbe numbered CGMCCNo.11213.
On the other hand, the present invention also provides described plant lactobacillus
l.plantarumHM6055purposes in probiotics, food.
Further, described purposes is plant lactobacillus
l.plantarumHM6055for generation of the purposes of the phenyllactic acid of high level.
On the other hand, the present invention also provides a kind of probiotics, and described probiotics comprises described plant lactobacillus
l.plantarumHM6055.
On the other hand, the present invention also provides a kind of food, and described food contains described plant lactobacillus
l.plantarumHM6055or adopt described plant lactobacillus
l.plantarumHM6055prepare.
Further, described food is foodstuff additive.
Further, described food is dough.
Further, described food is bread.
The screening method of plant lactobacillus of the present invention is the pickled vegetable fermentation liquor gathering spontaneous fermentation, gets 1ml and be diluted to 10 in 9ml stroke-physiological saline solution
-3-10
-7, draw 0.2ml and coat on improvement M17 plate culture medium, 37 DEG C of aerobic cultivation 24-48h.Select the further separation and purification of bacterium colony of Gram-positive, negative catalase, until obtain pure bacterial strain.The bacterial strain obtained by primary dcreening operation is carried out bread application experiment and is measured the output of its phenyllactic acid by HPLC, obtains bacterial strain through multiple sieve.
l.plantarumHM6055(plant lactobacillus
lactobacillusplantarumHM6055) be the probiotics be separated in spontaneous fermentation sauerkraut sample, plant lactobacillus of the present invention (
l.plantarumHM6055)
preservation.
preservationinformation:
Strain name: plant lactobacillus
lactobacillusplantarumHM6055
preservationdate: on August 10th, 2015,
preservationunit: in
state northno. 3, No. 1, North Star West Road, Jing Shi Chaoyang District institute, Chinese microorganism strain
preservationmanagement
the councilcommon micro-organisms center (CGMCC),
preservationnumbering: CGMCCNo.11213.
The present invention, according to the diet formula of Chinese Consumer's and food habits, utilizes plant lactobacillus
l.plantarumHM6055development lactobacillus-fermented bread, while its shelf preservation period that is intended to delay, improves bread structure, gives bread flavour, expand the range of application of milk-acid bacteria in food particularly bread.
Use
l.plantarumHM6055the standby bread of fermentation can extend bread shelf-lives, improves bread flavor, improve bread appearance features.
l.plantarumHM6055as a kind of probiotic bacterium, be with a wide range of applications in food (baked goods) field.
Probiotics (Porbioties), also active bacteria formulation (Bigone) or bacteria-promoting agent is, refer to and use microecology principle, utilize the growth-promoting substance to the useful and harmless probiotic bacterium of host or probiotic bacterium, through the preparation that special process is made.Current probiotics has been applied in each fields such as feed, agricultural, medicines and health protection and food.
As can be seen from embodiments of the invention, by
l.plantarumHM6055bacterium powder fermentation does not stimulate for the bread sour of gained is moderate, and ferment strongly fragrant pleasant, wheat fragrance is dense, and overall fragrance is pure and fresh; Surface is in golden yellow, and uniform color is consistent, glossy, and bread be circular, and outwardly rouse convex, tangent plane pore is evenly fine and closely woven, and without macroscopic void, the pure white and high resilience of endoplasm, organizes fluffy spongy, the nothing life heart; It can detect phenyllactic acid content apparently higher than control strain, thus can effective Shelf-life.
Accompanying drawing explanation
fig. 1it is the outward appearance of embodiment 2 blank group bread.
fig. 2embodiment 2 by
l.plantarumHM6055bacterium powder fermentation is for the outward appearance of the bread of gained.
Embodiment
Be described below in detail embodiments of the invention, the example of described embodiment exists
in accompanying drawingillustrate, wherein same or similar label represents same or similar element or has element that is identical or similar functions from start to finish.Below by reference
accompanying drawingthe embodiment described is exemplary, is intended to for explaining the present invention, and can not be interpreted as limitation of the present invention.Unreceipted concrete technology or condition person in embodiment, according to the technology described by the document in this area or condition or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
embodiment 1:plant lactobacillus
l.plantarumHM6055screening and Identification
1, screen
Gather Sichuan farmers' self-control pickles (30 degree of fermentation 240h), get the pickled vegetable fermentation liquor of 1ml spontaneous fermentation in 9ml stroke-physiological saline solution, continue to add stroke-physiological saline solution and be diluted to 10
-3-10
-7, draw 0.2ml and coat on improvement M17 plate culture medium, 37 DEG C of aerobic cultivation 24-48h.Select the further separation and purification of bacterium colony of Gram-positive, negative catalase, until obtain pure bacterial strain.Select on 37 degree of cultivations flat board of 24 hours, colony edge is smooth, smooth, oyster white, and colony diameter is 1-2mm, the slightly tiny projection of middle portion, and 37 degree of cultivations are after 48 hours, and bacterium colony is flats, and surface is slightly yellow, and colony diameter expands as 2-3mm.The bacterial strain obtained by primary dcreening operation carries out bread application experiment (step see embodiment 2), through multiple sieve (specific standards is: measure application experiment obtain bread phenyllactic acid comparision contents high (improving more than 10%), that flavor taste evaluates fermented flavour is softer, sour does not stimulate, bread specific volume increases by more than 10% and evaluate and quality guaranteed period experiment) obtain bacterial strain.
Improvement M17 slat chain conveyor based formulas: soy peptone 10g, yeast leaching powder 2.5g, extractum carnis 5g, glucose 5g, sodium ascorbate 0.5g, MgSO40.25g, agar 17g, distilled water 1000ml, regulate pH6.5 ± 0.2.
2, identify
1) 16srDNA qualification
The band of 1 treaty 1.5kb is obtained with 16srDNA universal primer pcr amplification from the genomic dna of HM6055 of bacterium, amplified production is checked order, obtain sequence and carry out homologous sequence retrieval in GenBank, result shows in the 16SrDNA sequence of the higher bacterial strain of 100 similaritys retrieving, and 90% is
lactobacilluspalntarum; Belong to kind of a 16SrDNA sequence construct phylogenetic tree to relevant, through sequence analysis find bacterial strain HM6055 with
lactobacilluspalntarum16SrDNA sequence homology more than 99%, therefore determine that it is plant lactobacillus.
2) Microbiological Characteristics
l.plantarumHM6055bacterial strain is Gram-positive, amphimicrobian, and glucose fermentation type is facultative heterofermentation, and catalase is negative, and poly-peptide sugar composition B group is negative, and form is regular shaft-like, judges that it is lactobacillus; Its pectinose, melizitose, wood-sugar fermentation are positive (utilization ratio is 11-89%), judge that it is plant lactobacillus.
Microscopic examination form: in regular shaft-like, become short chain in liquid medium within, spore of not sprouting;
Judge that it belongs to plant lactobacillus and belongs to according to " common bacteria system identification handbook " (the wonderful English version of 2001 eastern elegant pearl Cai).
embodiment 2:plant lactobacillus
l.plantarumHM6055application in food
It is below the amount of 1000 weight parts.
1, contain
l.plantarumHM6055the preparation of milk-acid bacteria dough:
The milk-acid bacteria dough (in scattered paste shape) be stirred is inserted 37 DEG C of constant temperature water baths and cultivate 3h15min, obtain lactobacillus-fermented dough.Now the pH of dough should be 6.05 ± 0.05.Milk-acid bacteria dough formula (flour 171g, whole-fat milk powder 19g, glucose 2g, sucrose 8g, AN lipase 0.03g, PiccantaseR enzyme 0.03g,
l.plantarumHM6055freeze-dried vaccine powder 0.1g(is containing 6 × 10
10cFU's
l.plantarumHM6055), dechlorination tap water 179g).
2, yeast containing dough powder mix and the preparation containing saccharomycetic mixing solutions:
First flour and whole-fat milk powder are added agitator mixing, obtain yeast containing dough powder mix (flour 686g, whole-fat milk powder 76g);
Being dissolved in 345g dechlorination tap water by other auxiliary material (sucrose, Kaoliang spirit, AN lipase, PiccantaseR enzyme, active dry yeast) makes containing saccharomycetic mixing solutions, containing saccharomycetic mixing solutions formula (sucrose 38g, Kaoliang spirit (38 degree) 1.5g, AN lipase 0.11g, PiccantaseR enzyme 0.11g, Angel dry yeast 9.5g, dechlorination tap water (4 DEG C) 345g).
3, contain
l.plantarumHM6055milk-acid bacteria dough and the mixing of yeast dough proof:
Step 1 gained lactobacillus-fermented dough and step 2 gained yeast containing dough powder mix (flour and whole-fat milk powder) are added agitator, the more saccharomycetic mixing solutions that contains step 2 prepared slowly adds agitator, 180rpm stirs 20min.Now dough should be smooth surface exquisiteness, can be stretched as transparent film, neither too hard, nor too soft.
Dough is divided into 50 ± 0.5g weight part, shaping sabot under room temperature (about 30 DEG C), process is about 10min.
Then proof, proofing temperature: 35 DEG C ± 2 DEG C relative humidity: 70-80%, proofing period is 1h, and proofing terminal pH is 5.90 ± 0.05.
Dough leavening completes rear surface brush liquid whole egg baking, face fire: 170 DEG C, the fire in a stove before fuel is added: 190 DEG C; Baking time 35min.
Preparation-obtained bread products bread specific volume is 3.40 ± 0.16, pH is 5.82 ± 0.04, and titration acidity is 2.40 ± 0.08.Bread sour is moderate not to stimulate, and ferment strongly fragrant pleasant, wheat fragrance is dense, and overall fragrance is pure and fresh.
With the bread of blank group (except
l.plantarumHM6055outside bacterium powder does not add, other compositions are all identical with step, condition) compare, the results are shown in
fig. 1with
fig. 2.Wherein
fig. 1it is the outward appearance of blank group bread.
fig. 2be by
l.plantarumHM6055bacterium powder fermentation is for the outward appearance of the bread of gained.Can find out, the present embodiment by
l.plantarumHM6055bacterium powder fermentation for gained bread (
fig. 2shown in) surface is in golden yellow, uniform color is consistent, glossy, and bread be circular, and outwardly rouse convex, tangent plane pore is evenly fine and closely woven, and without macroscopic void, the pure white and high resilience of endoplasm, organizes fluffy spongy, the nothing life heart.
Will be by under room temperature
l.plantarumHM6055under bacterium powder fermentation is put in normal temperature (temperature 28 degree, humidity 55%) for the bread of gained with the blank bread organized, result is, by
l.plantarumHM6055bacterium powder fermentation obviously extends shelf-lives for the bread of gained, and blank starts to find obviously visible bacterial plaque under these conditions on the 3rd day, and adds the present invention
l.plantarumHM6055the bread of bacterium powder starts to occur obviously visible bacterial plaque the 7th talent, and by the 12nd day, blank group bacterial plaque spread all over whole bread; The bacterial plaque of adding bacterium powder group can also be inhibited.
This shows, by
l.plantarumHM6055bacterium powder fermentation does not stimulate for the bread sour of gained is moderate, and ferment strongly fragrant pleasant, wheat fragrance is dense, and overall fragrance is pure and fresh; Surface is in golden yellow, and uniform color is consistent, glossy, and bread be circular, and outwardly rouse convex, tangent plane pore is evenly fine and closely woven, and without macroscopic void, the pure white and high resilience of endoplasm, organizes fluffy spongy, the nothing life heart; Can effective Shelf-life.
embodiment 3: the comparison test of phenyllactic acid content
The addition of testing sequence and each composition is with embodiment 2, and difference is: contrast to add
l.plantarumHM6055replace to and add moral formula Bacterium lacticum De Shi subspecies; Product group is added
l.plantarumHM6055.addition is
l.plantarumHM6055 oradd the freeze-dried vaccine powder 0.1g(of moral formula Bacterium lacticum De Shi subspecies containing 6 × 10
10cFU's
l.plantarumHM6055or moral formula Bacterium lacticum De Shi subspecies).Blank is then that this step does not add any bacterium.Preparation-obtained bread carries out phenyllactic acid assay.
Phenyllactic acid measuring method:
1. laboratory apparatus and reagent:
High performance liquid chromatograph (Agilent1200, USA), the desk-top high-speed refrigerated centrifuge of Sigma3-18K4 degree (German Sigma company), phenyllactic acid (purity >=98%, Sigma-Alidrich company of the U.S.), acetonitrile (chromatographically pure, Fisher company of the U.S.), methyl alcohol (chromatographically pure, Fisher company of the U.S.), phosphoric acid (concentration more than 85% analytical pure, Tianjin Fengchuan Chemical Reagent Science & Technology Co., Ltd.), Milli-Q ultra-pure water;
2. phenyllactic acid standard working solution preparation:
Accurately take 100mg phenyllactic acid standard substance, be settled to 100ml with dissolve with methanol, namely concentration is the standard reserving solution of 1g/L; Drawing phenyllactic acid standard reserving solution, take methyl alcohol as solvent constant volume, compound concentration is respectively 0,5,10,20,40,80, the phenyllactic acid standard working solution of 200mg/L.
3. detect the preparation of sample:
Get 5g Bread Samples, use 10ml(water after pulverizing: acetonitrile=4:1) solvent soaking, 6000r/min, 4 degree of centrifugal 10min after immersion 10h, after using 0.45um aluminium membrane filtration supernatant liquor, provide HPLC to analyze.
4. efficient liquid phase chromatographic analysis:
Chromatographic column: AgilentZorbaxSB-C18(4.6mm × 150mm, 5um) chromatographic column; Moving phase: A0.5% phosphate aqueous solution (V/V), B:0.5% phosphoric acid acetonitrile solution (V/V); Flow velocity 1ml/min; Column temperature: 30 degree; Sample size: 5uL; Determined wavelength 210nm; Elution program is shown in
table 1.
table 1elution program
table
| Time (min) | 0.5% phosphate aqueous solution | 0.5% phosphoric acid acetonitrile solution | Flow velocity (ml/min) |
| 0 | 80 | 20 | 1 |
| 12 | 80 | 20 | 1 |
| 13 | 0 | 100 | 1 |
| 15 | 0 | 100 | 1 |
Going out the sample time is about 8.8min.
Its phenyllactic acid assay the results are shown in
table 2.
table 2phenyllactic acid assay result
table
| Sample ID | Phenyllactic acid content (mg/kg) |
| Contrast | 0 |
| Strain control (adding moral formula Bacterium lacticum De Shi subspecies) | 12 |
| Product (adds L.plantarum HM6055） | 32 |
Note: 1, moral formula Bacterium lacticum De Shi subspecies are the lactobacillus representative strain introduced in " common bacteria system identification handbook " (the wonderful English version of 2001 eastern elegant pearl Cai), and the present embodiment uses Chinese industrial microbial strains
preservationthe shared bacterial strain of administrative center, its bacterial strain
preservationbe numbered CICC20258.
2, moral formula Bacterium lacticum De Shi subspecies and the present invention
l.plantarumHM6055addition is consistent.
Phenyllactic acid is a kind of
novelantibacterial substance, spoilage organism, pathogenic bacterium can be suppressed, particularly to the infection of fungi.From
table 2can find out, use of the present invention
l.plantarumHM6055the bread prepared and blank ratio, with moral formula Bacterium lacticum De Shi subspecies ratio, can produce higher phenyllactic acid, illustrates that use is of the present invention
l.plantarumHM6055the bread prepared more can suppress spoilage organism, pathogenic bacterium, particularly to the infection of fungi, thus Shelf-life.
Although illustrate and describe embodiments of the invention above, be understandable that, above-described embodiment is exemplary, can not be interpreted as limitation of the present invention, those of ordinary skill in the art can change above-described embodiment within the scope of the invention when not departing from principle of the present invention and aim, revising, replacing and modification.
Claims (8)
1. a plant lactobacillus
l.plantarumHM6055, it is characterized in that, deposit number is CGMCCNo.11213.
2. plant lactobacillus described in claim 1
l.plantarumHM6055purposes in probiotics, food.
3. purposes described in claim 2 is plant lactobacillus
l.plantarumHM6055for generation of the purposes of the phenyllactic acid of high level.
4. a probiotics, is characterized in that: described probiotics comprises plant lactobacillus described in claim 1
l.plantarumHM6055.
5. a food, is characterized in that: described food contains plant lactobacillus described in claim 1
l.plantarumHM6055or adopt plant lactobacillus described in claim 1
l.plantarumHM6055prepare.
6. food described in claim 5, is characterized in that, described food is foodstuff additive.
7. food described in claim 5, is characterized in that, described food is dough.
8. food described in claim 5, is characterized in that, described food is bread.
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Cited By (5)
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|---|---|---|---|---|
| CN106399208A (en) * | 2016-12-05 | 2017-02-15 | 厦门和美科盛生物技术有限公司 | Lactobacillus fermentation broth suitable for food processing application, preparing method and application of lactobacillus fermentation broth |
| CN106520625A (en) * | 2016-12-05 | 2017-03-22 | 厦门和美科盛生物技术有限公司 | Symbiosis lactobacillus fermentation broth, preparation method and application thereof |
| CN108624533A (en) * | 2018-05-10 | 2018-10-09 | 浙江工商大学 | A kind of method that phenyl-lactic acid is isolated and purified in lactobacillus plantarum |
| CN111996146A (en) * | 2020-08-31 | 2020-11-27 | 山东宝来利来生物工程股份有限公司 | Lactobacillus plantarum for high yield of phenyllactic acid and application thereof |
| CN112168846A (en) * | 2018-07-03 | 2021-01-05 | 大江生医股份有限公司 | Lactobacillus plantarum TCI378 strain and application of metabolite thereof in fat reduction |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399208A (en) * | 2016-12-05 | 2017-02-15 | 厦门和美科盛生物技术有限公司 | Lactobacillus fermentation broth suitable for food processing application, preparing method and application of lactobacillus fermentation broth |
| CN106520625A (en) * | 2016-12-05 | 2017-03-22 | 厦门和美科盛生物技术有限公司 | Symbiosis lactobacillus fermentation broth, preparation method and application thereof |
| CN106520625B (en) * | 2016-12-05 | 2019-09-10 | 厦门和美科盛生物技术有限公司 | A kind of symbiosis streptococcus acidi lactici fermented solution, preparation method and its usage |
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| CN108624533B (en) * | 2018-05-10 | 2021-03-09 | 浙江工商大学 | Method for separating and purifying phenyl lactic acid from lactobacillus plantarum |
| CN112168846A (en) * | 2018-07-03 | 2021-01-05 | 大江生医股份有限公司 | Lactobacillus plantarum TCI378 strain and application of metabolite thereof in fat reduction |
| CN111996146A (en) * | 2020-08-31 | 2020-11-27 | 山东宝来利来生物工程股份有限公司 | Lactobacillus plantarum for high yield of phenyllactic acid and application thereof |
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