CN102604851B - Lactococcus lactis capable of lowering cholesterol and producing extracellular polysaccharide - Google Patents
Lactococcus lactis capable of lowering cholesterol and producing extracellular polysaccharide Download PDFInfo
- Publication number
- CN102604851B CN102604851B CN201110025200.9A CN201110025200A CN102604851B CN 102604851 B CN102604851 B CN 102604851B CN 201110025200 A CN201110025200 A CN 201110025200A CN 102604851 B CN102604851 B CN 102604851B
- Authority
- CN
- China
- Prior art keywords
- lactococcus lactis
- acid bacteria
- milk
- lactic acid
- cholesterol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Dairy Products (AREA)
Abstract
The invention discloses a lactococcus lactis separation strain which is capable of lowering cholesterol and producing extracellular polysaccharide and an application of the lactococcus lactis separation strain, belonging to the field of biotechnology, in particular to the field of functional lactic acid bacteria development. A method for preparing the lactococcus lactis separation strain comprises the following steps: (1) obtaining a lactic acid bacteria from the traditional Tibetan fermented milk product Kefir by using a culturing medium; and (2) screening out the lactococcus lactis which is capable of lowering cholesterol and producing extracellular polysaccharide by identifying stains by adopting the o-phthalaldehyde colorimetry, the phenol-sulfuric acid method and the thin-layer chromatography. The fermented milk product prepared from the lactococcus lactis has good flavor characteristics and organoleptic properties, and in addition, the lactic acid bacteria powder prepared from the lactic acid bacteria is safe and nontoxic. The lactococcus lactis can be produced by adopting a simple process and has a good development prospect.
Description
Technical field:
The present invention relates to biological technical field, particularly functional lactobacillus production development field.
Technical background:
Hypercholesterolemia is one of important factor causing blood fat to raise, when cholesterol metabolic generation obstacle, and long-term Excess free enthalpy, and body inner cholesterol normal regulating is out of hand, cause blood cholesterol level too high, just likely deposit on arterial blood tube wall, draw its a series of cardiovascular and cerebrovascular diseases.A survey according to the U.S. finds: the cardiovascular disorder caused by hyperlipidaemia is that current harm humans is healthy, causes dead primary factor.
Probiotic bacterium extensively distributes at nature, because it is useful to host health, is thus widely used.All contain profitable probliotics in many functional foodstuffs, they can improve intestinal microflora, reduce the quantity of harmful bacteria, prevent diarrhoea; Improve immunity of organisms; Reduce serum cholesterol; Improve the resistance of oxidation of body, delay senility and preventing cancer function etc.Therefore, probiotic bacterium, as a frontier, is applied further extensive in food, except being applied to cultured milk prod, also be just progressively applied to the various functional foodstuff of exploitation, as fruit juice, candy, ice-creams, frozen yogurt etc., the nutrition of the mankind and health had great importance.
The exocellular polysaccharide of bacterium is that bacterium is secreted into glue polysaccharide outside cell walls or capsular polysaccharide in growth metabolism process, it both can promote the growth of other probiotic bacteriums in enteron aisle as probiotics, again with antitumor, anti-fistula, immunomodulatory, the decreasing cholesterol of probiotic bacterium or regulate blood pressure effect relevant.The product exocellular polysaccharide milk-acid bacteria of food grade can be used as physiological function regulator, directly enters human body with the form of active bacteria formulation, saves the techniques such as fermentation, extraction, and produce exocellular polysaccharide milk-acid bacteria also has certain influence to fermented-milk quality and cheese quality simultaneously.Therefore, the milk-acid bacteria that polysaccharide characteristic is produced in exploitation has obvious economic implications, particularly with practical value in whisk yoghout, the slipperiness improving Yoghourt and minimizing synersis.
Kefir is suggested very early as a kind of functional beverage of health, and it not only has higher trophism, and containing a large amount of probiotic bacterium in Kefir grain, comprises milk-acid bacteria, yeast, and a small amount of acetic bacteria etc.Therefore, hospital or the sanatorium in Russia and some Europe extensively utilize Kefir, be directed to the patients such as gastrointestinal disorder, metabolic disturbance disease, diabetes, hypertension, hypercholesterolemia, local anemia, allergy, heart trouble and obesity especially, apply in sitotherapy, also extensively rise the R and D to Kefir source function probiotic bacterium both at home and abroad.
Therefore, from traditional zymotic milk-product Kefir, screening has the milk-acid bacteria of good characteristic, not only has broad application prospects, and also has important social effect simultaneously.
Summary of the invention:
The object of the invention is that invention one strain has decreasing cholesterol and produces the Kefir source Lactococcus lactis of exocellular polysaccharide ability, to further exploitation.
Lactococcus lactis of the present invention is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica on December 24th, 2010.Preserving number is: CGMCC No:4496.
Through experimental verification, Lactococcus lactis 4496 of the present invention is a kind of functional lactobacillus, in reduction cholesterol and product exocellular polysaccharide, there is good characteristic especially, as Lactococcus lactis 4496 of the present invention is developed to protective foods or medicine, long-term taking can improve intestinal microflora, reduce serum cholesterol, improve immunity of organisms; Or the present invention is put in cultured milk prod or cheese product, also can improve quality and the local flavor of product.
Another object of the present invention is to the preparation method inventing above-mentioned Lactococcus lactis 4496, comprise the following steps:
(1) from the traditional zymotic milk-product Kefir of Tibet, separation screening obtains milk-acid bacteria;
(2) by gramstaining, catalase experiment, thalli morphology and API identification system, screening obtains a strain Lactococcus lactis 4496;
(3) by o-phthalaldehyde(OPA) colorimetry, phend-sulphuric acid and tlc, determine that bacterial strain-Lactococcus lactis 4496 of the present invention has good decreasing cholesterol and produces exocellular polysaccharide ability.
The third object of the present invention is the application of inventing described Lactococcus lactis 4496:
Can be used for preparing the cultured milk prod with decreasing cholesterol function, product has good flavor characteristic and organoleptics property;
Also can be used for preparing the milk-acid bacteria bacterium powder with decreasing cholesterol function, in finished product, viable count can reach 1 ~ 9 × 10
11cfu/g.
Also be a kind ofly well to select for the preparation of other protective foods or medicine, long-term taking can reduce serum cholesterol, improves immunity of organisms, is particularly suitable for gastrointestinal disorder, hypercholesterolemia and metabolic disturbance patient, and without any side effects.
Accompanying drawing illustrates:
Fig. 1 is the colonial morphology figure of Lactococcus lactis 4496;
Fig. 2 is the thalline microscopic morphology figure of Lactococcus lactis 4496;
Fig. 3 is that the API 50CHL of Lactococcus lactis 4496 identifies photo;
Fig. 4 is that Lactococcus lactis 4496 grows the relation with degrading rate of cholesterol;
Fig. 5 is the thin-layer chromatography result (1. the glucose 2. semi-lactosi 3. exocellular polysaccharide aqueous solution 4. exocellular polysaccharide acid hydrolysis liquid) of exocellular polysaccharide in Lactococcus lactis 4496 fermented liquid;
Fig. 6 is Lactococcus lactis 4496 preservation stability analysis;
Fig. 7 is the total ion current journey figure that Lactococcus lactis 4496 fermented-milk GC-MS analyzes.
Embodiment:
One, sample source
This routine bacterium source is in China Tibet traditional zymotic milk-product-Kefir.Kefir is a kind of time-honored containing ethanol fermentation breast, prosper in the mountain area, Caucasia of USSR (Union of Soviet Socialist Republics), is a kind of cultured milk prod of preciousness.
Two, from Kefir, separation screening obtains milk-acid bacteria
Get a certain amount of Kefir grain broken in sterilizing mortar in the ratio of 1: 10, get broken rear gained homogenate 1mL salts solution and (comprise the K of 4.5g/L
2hPO
4, the L-cysteine hydrochloride of 0.5g/L, the Na of 6g/L
2hPO
4, the Tween80 of 0.5g/L) and dilution, diluent cultivates 2-3 days in 30--37 DEG C, picking feature bacterium colony after being inoculated in M17 flat board, and is further purified, and then saves backup.
Three, bacterial strain is identified
(1) gramstaining, catalase experiment
Gramstaining: the bacterium colony on picking flat board carries out smear, fixing, Viola crystallina just contaminates, mordant dyeing, decolouring, washing, sarranine are redyed, dry, microscopy.
Catalase is tested: bacterium to be measured, prior to exposing 30min in air, draws a small amount of 3%H with kapillary
2o
2drip the bacterium colony grown in planar surface.If aerogenesis bubble is not then negative catalase.
This bacterial strain gramstaining is positive, and catalase experiment, for negative, tentatively can be considered as milk-acid bacteria.
(2) colonial morphology, thalline microscopic morphology are observed
The colony morphology characteristic of visual inspection bacterial strain, observations is as Fig. 1, and this bacterial strain colony colour is oyster white, and colony edge is neat, rounded convex, and colony diameter≤2.0mm, meets milk-acid bacteria colony characteristics; After gramstaining, in the cell morphological characteristic of opticmicroscope oil Microscopic observation bacterial strain, observations is as Fig. 2, and its cellular form is spherical in shape, and paired clustering phenomena is obvious, therefore tentatively judges that bacterial strain 4496 is as Lactococcus lactis.
(3) the API 50CHL identification systems of French Biomerieux company are used to carry out sugar-fermenting experiment
After bacterial strain 4496 was activated for 2 generations, be inoculated on MRS plate culture medium, 30 DEG C of Anaerobic culturel 24--48h, the bacteria suspension being equivalent to 2McFarland turbidity is made in picking list bacterium colony access suspension substrate, then bacterium liquid is inoculated in API 50CHL reagent strip, and with whiteruss (mineral oil) sealing, is placed in 30 DEG C and cultivates 24h, detect bacterial strain to 49 kinds of sugared fermentation capacities according to reagent strip colour-change, the results are shown in Figure 3.
API is used to identify the software analysis sugar-fermenting ability of this bacterial strain.Result shows, and the sugar-fermenting ability of this bacterial strain is similar to Lactococcus lactis (see table 1), and similarity is 92.4%.
The API 50CHL identification systems reaction result of table 1 Lactococcus lactis 4496
Note: "+" represents positive reaction; "-" represents negative reaction.
Four, the decreasing cholesterol characteristic of o-phthalaldehyde(OPA) colorimetric method for determining Lactococcus lactis 4496 is adopted
Being inoculated in by bacterial strain 4496 containing cholesterol final concentration is in the M17 liquid nutrient medium of 30mg/100mL, 30 DEG C of Anaerobic culturel, every 4h sampling.With the degradation rate of the OD of o-phthalaldehyde(OPA) colorimetric method for determining fermented liquid (600nm) value and cholesterol.
As shown in Figure 4, the degrading rate of cholesterol of Lactococcus lactis 4496 and the growth of thalline are proportionate result, and when 24h, the degradation rate of cholesterol can reach 30. ~ 40%.
Five, the product exocellular polysaccharide characteristic of phend-sulphuric acid and tlc determination galactococcus 4496 is adopted
Be inoculated in whey medium by Lactococcus lactis 4496, leave standstill the centrifugal 15min of Anaerobic culturel 24h, 10000g at 30 DEG C and remove thalline, Aspirate supernatant loads dialysis tubing, dialyses in 4 DEG C of distilled water, measures exocellular polysaccharide content in dialysis tubing with phend-sulphuric acid.Result shows, Lactococcus lactis 4496 transposon mutagenesis amount reaches 160 ~ 280mg/L.
Further use tlc verifies that this bacterial strain produces exocellular polysaccharide ability.After bacterial strain activation in access whey liqs substratum, cultivate 24h, 10000g centrifugal segregation thalline for 30 DEG C, collect supernatant liquor, alcohol settling, carries out lyophilize after distill water dialysis, then namely obtains EPS crude product with trichloroacetic acid method removing protein.As shown in Figure 5, the exocellular polysaccharide aqueous solution before acid hydrolysis is not containing glucose and semi-lactosi any one wherein for result, but the but spottiness appearance of the aqueous solution after acid hydrolysis, and this may be glucose, also may be semi-lactosi or both mixtures.Due to the restriction of method itself, can not conclude and form accurately.But there is any to conclude: this sugar itself containing monose, does not create monose after acid hydrolysis.Therefore, the thick exocellular polysaccharide extracted is polymerization polysaccharide, is namely separated and possesses the ability of producing exocellular polysaccharide from the Lactococcus lactis 4496 of Kefir.
Six, the preservation Stability Determination of Lactococcus lactis 4496
By activated 4496 with in 1% inoculum size access M17 substratum, be placed in 30 DEG C of Anaerobic culturel to logarithmic growth latter stage, put into the preservation of 4 DEG C, refrigerator, carried out viable plate counting at the the the 0th, 5,10,15 day respectively, represent with cfu/mL.As shown in Figure 6,4496 viable counts have decline to a certain degree to result when preservation temperature is 4 DEG C, and its reason may be the dual function of fermented liquid acidic conditions and somatic cells natural apoptosis.But the survival rate recorded after 15 days is still higher, only than reducing a number magnitude during fermentation termination, show good stability.
Seven, bacterial strain preservation
Bacterial strain of the present invention is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica on December 24th, 2010.Preserving number is: CGMCC No:4496.
Application example
Embodiment 1: the fermented-milk containing Lactococcus lactis 4496
(1) preparation method
Dose volume mark is the whole-fat milk powder aqueous solution of 12%, after sterilizing in 115 DEG C, 15 minutes, be cooled to about 30 DEG C, by the volume fraction inoculating lactic acid galactococcus 4496 of 2%, in 30 DEG C of anaerobically fermentings to curdled milk, namely makes solidified-type fermented milk first product.Through stirring after fermentation ends, adding white sugar, various jam fruit juice etc., namely making the yogurt containing active lactic acid galactococcus 4496.
(2) detection of fermented-milk flavor substances
Adopt solid-phase microextraction-gas chromatography-mass spectrum (Vavian 4000GC/MS) coupling technique, measure the flavour substances in fermented-milk.Chromatographic condition is: chromatographic column is VF-5ms 30m × 0.25mm × 0.25 μm; Temperature programming, initial temperature 40 DEG C, keeps 3min, is warmed to 150 DEG C with 4 DEG C/min, keeps 1min, is warmed to 250 DEG C with 8 DEG C/min, keeps 6min.Detector: injector temperature 250 DEG C, splitting ratio is 20: 1, and carrier gas is helium, and column flow rate is 1mL/min.Mass Spectrometry Conditions: interface temperature 280 DEG C, ion source temperature 220 DEG C, scan mode is full scan, and sweep limit is 43-500m/z.Mass spectral database is NIST 05.
The total ion current journey figure result that Lactococcus lactis 4496 fermented-milk GC-MS analyzes as shown in Figure 7, in total ion current journey figure, the qualification result at each peak is as shown in table 2, Lactococcus lactis 4496 fermented-milk sample identifies 25 kinds of flavour substancess altogether after gas-chromatography is separated, wherein: Ester 3 kinds, and alcohols material 6 kinds, aldehyde material 5 kinds, heterogeneous ring compound 4 kinds, aromatic compound 2 kinds, furans lopps material a kind, short chain volatile lipid acid and ketone alkene class material totally 3 kinds, di-acetyl class material a kind.Result shows, this bacterial strain is in flavor characteristics, and the production advantage is obvious, can independently or with other lactic acid bacteria culturers fit applications in the production of producing fragrant cultured milk prod.
Table 2 Lactococcus lactis 4496 fermented-milk GC-MS flavour substances qualification result
Embodiment 2: the making method of bacterial classification powdery product of the present invention
Lactococcus lactis 4496 yogurt is refrigerated to-40 DEG C ~-60 DEG C, drying, be 3%-5% to water content, be crushed to Powdered, powdery product can be added oligose, maltodextrin, milk powder, flavour agent etc. and make Powdered or sheet bulk product, for daily edible or as medicine, healthcare products.
The product of strain fermentation provided by the invention can play decreasing cholesterol effect above, the milk of this strain fermentation, the various fermented milk prods etc. through lactobacillus-fermented of acquisition, all obtains the decreasing cholesterol effect had in various degree.This product long-term taking is without any side effects, is particularly suitable for gastrointestinal disorder, hypercholesterolemia and metabolic disturbance patient and takes.
Claims (3)
1. a strain has decreasing cholesterol and produces Lactococcus lactis (Lactococcus lactis) WH-C1 of exocellular polysaccharide ability, this bacterial strain is in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, and preserving number is CGMCC No:4496.
2. the purposes that the Lactococcus lactis WH-C1 having decreasing cholesterol as claimed in claim 1 and produce exocellular polysaccharide ability has in the fermented-milk of excellent flavor feature in preparation.
3. the Lactococcus lactis WH-C1 as claimed in claim 1 with decreasing cholesterol and product exocellular polysaccharide ability is preparing the purposes in the food or medicine with decreasing cholesterol function.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110025200.9A CN102604851B (en) | 2011-01-24 | 2011-01-24 | Lactococcus lactis capable of lowering cholesterol and producing extracellular polysaccharide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110025200.9A CN102604851B (en) | 2011-01-24 | 2011-01-24 | Lactococcus lactis capable of lowering cholesterol and producing extracellular polysaccharide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102604851A CN102604851A (en) | 2012-07-25 |
| CN102604851B true CN102604851B (en) | 2015-01-07 |
Family
ID=46522628
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110025200.9A Active CN102604851B (en) | 2011-01-24 | 2011-01-24 | Lactococcus lactis capable of lowering cholesterol and producing extracellular polysaccharide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102604851B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104560761A (en) * | 2013-10-11 | 2015-04-29 | 中国计量学院 | Marine leuconostoc lactis for producing exopoly saccharides |
| CN109105491B (en) * | 2018-09-12 | 2021-07-23 | 内蒙古农业大学 | Lactococcus lactis IMAU11823 capable of producing sticky cheese fragrance and application thereof |
| CN113234645B (en) * | 2021-06-30 | 2022-07-01 | 四川农业大学 | Lactococcus lactis, screening method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101235360A (en) * | 2008-01-31 | 2008-08-06 | 南开大学 | Lactic acid galactococcus with urea conversion capability and its domestication method and application |
| CN101586111A (en) * | 2008-05-22 | 2009-11-25 | 北京三元基因工程有限公司 | Method for preparing product of active lactic acid galactococcus |
-
2011
- 2011-01-24 CN CN201110025200.9A patent/CN102604851B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101235360A (en) * | 2008-01-31 | 2008-08-06 | 南开大学 | Lactic acid galactococcus with urea conversion capability and its domestication method and application |
| CN101586111A (en) * | 2008-05-22 | 2009-11-25 | 北京三元基因工程有限公司 | Method for preparing product of active lactic acid galactococcus |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102604851A (en) | 2012-07-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110272842B (en) | Lactobacillus plantarum LP104 with weight-losing and lipid-lowering functions | |
| CN104073455B (en) | One strain has the plant lactobacillus reducing cholesterol ability | |
| CN102533588B (en) | Lactobacillus brevis for producing extracellular exopolysaccharide and application thereof | |
| CN104430851B (en) | A kind of norcholesterol acidified milk and preparation method thereof | |
| CN102899276B (en) | Streptococcus thermophilus capable of lowering cholesterol levels and application thereof | |
| WO2023098678A1 (en) | High-protein saccharomyces cerevisiae and application thereof | |
| CN111471604A (en) | Application of saccharomyces cerevisiae Z L G-6 and lactobacillus plantarum Picp-2 | |
| CN107227278A (en) | A kind of Lactobacillus plantarum A11 and its application | |
| CN106754542A (en) | One plant has the lactobacillus plantarum SR9 3 and purposes for reducing cholesterol and antioxidation activity | |
| Gayathry | Production of nata de coco-a natural dietary fibre product from mature coconut water using Gluconacetobacter xylinum (sju-1) | |
| CN116286468A (en) | Lactobacillus mucilaginosus LF-ONLLY with antioxidant function and application thereof in fermented food | |
| CN108018248B (en) | Lactobacillus casei capable of regulating flora structural disorder caused by antibiotics | |
| CN102604851B (en) | Lactococcus lactis capable of lowering cholesterol and producing extracellular polysaccharide | |
| CN108902601B (en) | Litchi chinensis endogenous lactic acid bacteria and fermented fruit juice beverage thereof | |
| Liamngee et al. | Microbial analysis of soyabean milk sold in Makurdi metropolis | |
| Praia et al. | Coconut water-based probiotic drink proposal: evaluation of microbio-logical stability and lactic acid estimation | |
| CN101144064B (en) | Lactobacillus brevis with anti-mutagenesis active and capable of producing exopolysaccharide and use thereof | |
| CN112813006A (en) | Lactobacillus plantarum and application thereof in fruit and vegetable juice fermentation | |
| CN105820968B (en) | One plant of lactobacillus paracasei and its application | |
| CN112708577B (en) | Lactobacillus fermentum DALI02 with high intestinal adhesion and immunoregulation function and application thereof | |
| CN106011023B (en) | One plant of acetic acid bacteria for being isolated from traditional fermented food acid congee and its application | |
| CN114085791A (en) | Pediococcus pentosaceus He10-a-1 and application thereof | |
| CN113604385A (en) | Lactobacillus delbrueckii with butter degrading capability and application thereof | |
| CN110179031A (en) | The production method of Wine brewing yeast strain and foaming type apple enzyme beverage | |
| CN104726380B (en) | One plant of Lactobacillus casei and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230522 Address after: Building 15, Haitian Nanyuan, northeast of the intersection of Outer Ring Auxiliary Road and Xianfeng River, Shuanggang Town, Jinnan District, Tianjin, 300350-1,2-320 Patentee after: Haitai Hejia (Tianjin) Information Technology Co.,Ltd. Address before: 300457 No. thirteenth, 29 Avenue, Tianjin economic and Technological Development Zone Patentee before: TIANJIN University OF SCIENCE AND TECHNOLOGY |