CN105820968A - Lactobacillus paracasei and application thereof - Google Patents
Lactobacillus paracasei and application thereof Download PDFInfo
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- CN105820968A CN105820968A CN201510005280.XA CN201510005280A CN105820968A CN 105820968 A CN105820968 A CN 105820968A CN 201510005280 A CN201510005280 A CN 201510005280A CN 105820968 A CN105820968 A CN 105820968A
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Abstract
The invention relates to Lactobacillus paracasei, which is named as Lactobacillus paracasei AR-93, and a preservation number is CGMCC 9443. The Lactobacillus paracasei AR-93 is taken as a low-fat yoghurt starter and as a raw material for extracting exopolysaccharide. The acid production speed of the Lactobacillus paracasei AR-93 during a low-fat yoghurt fermentation process is slow, so that the yoghurt products have the advantages of soft acidity and rich flavor, the fermented low-fat yoghurt is viscous. The exopolysaccharide produced by Lactobacillus paracasei can obviously improve the texture properties such as structural state of the yoghurt, precipitation of milk serum is minimized, a stabilizing agent is not required for the product, and the requirement of the low-fat yoghurt without addictives by the consumers is satisfied.
Description
Technical field
The present invention relates to microorganism, particularly to strain Lactobacillus paracasei and an application thereof.
Technical background
Yoghourt is a kind of curdled milk goods utilizing the fermented cultivation of specific lactic acid bacteria to produce, and nutritive value is higher, excellent flavor, and Yoghourt can improve Tiny ecosystem structure of community in intestinal, the immunity of enhancing body, has various active function.At present, in addition to pasteurization milk, increase production one of the fastest milk product during Yoghourt Production becomes China's liquid milk.Along with increasing of the diseases such as obesity and diabetes, the consciousness of health diet is also gradually stepped up by consumer, and content fatty in conventional dairy product has caused the concern that people are more, and people are more willing to tend to low fat milk goods.
Butter oil plays an important role for forming milk product distinctive matter structure, local flavor and mouthfeel.Low fat sour milk is relatively low due to fat content, the bad phenomenon such as viscosity is low, structural state is coarse, milk surum separates out, mouthfeel difference usually occurs.Therefore, the simple dairy fat content reduced in dispensing can reduce the acceptability of product.Exopolysaccharides Produced by Lactic Acid Bacteria (EPS) refers to that lactic acid bacteria is in its growth, the capsular polysaccharide being secreted into outside born of the same parents in metabolic process or mucus, its composition and structure are complicated, can be applied to the numerous areas such as food, pharmacy as thickening agent, stabilizer, water binding agent etc..In milk product, extracellular polysaccharide produces viscosity, the stability that the extracellular polysaccharide of bacterium and generation thereof has, and can effectively improve rheological properties and the texture characteristic of fermentation milk, it is to avoid milk surum separates out；Meanwhile, lubrication sense and local flavor can be improved.High-yield extracellular polysaccharide lactic acid bacteria can be effectively combined moisture unnecessary in Yoghourt, improves the viscosity of Yoghourt, and reduces the mechanical processing process matter structure damage to Yoghourt products, reduces the syneresis defect of product.
Additionally, the extracellular polysaccharide that high-yield extracellular polysaccharide strains is produced also has good physiological function, breast strengthens intestinal mucosa absorbability, regulation immunity, reduction cholesterol and blood pressure etc..Therefore, carry out the screening operation of high-yield extracellular polysaccharide lactic acid bacteria, for improving the production and processing of milk product, the Yoghourt product of exploitation characteristic, there is very important economic benefit and social benefit.
Summary of the invention
The purpose of the present invention, it is aiming in prior art lacking the deficiency of the lactic acid bacteria of high-yield extracellular polysaccharide, there is provided the strain can the Lactobacillus paracasei of high extracellular polysaccharide and application thereof, the bacterial strain of the present invention ferments lowfat milk under optimum conditions, the Yoghourt acidity obtained after fermentation is soft, local flavor is full, and matter structure is good.
In order to realize the purpose of the present invention, present invention employs techniques below scheme: a strain Lactobacillus paracasei (Lactobacillusparacasei), named Lactobacillus paracasei AR-93, this bacterial strain on July 11st, 2014 in China Committee for Culture Collection of Microorganisms's common micro-organisms center (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3) preservation, its deposit number is CGMCC9443, and its gene order is as shown in SEQIDNO.1.
Above-mentioned Lactobacillus paracasei AR-93 screens from Kefir grains granule.Kefir grains granule is carried out separation screening, utilize gradient dilution method, sample liquid after dilution is coated MRS flat board, 37 DEG C of constant temperature culture, select and produce the bacterium colony that viscosity can be good, after line separates, the high yield Acarasiales strain to separating acquisition carries out morphologic observation, and combining with fermentation experiment screening determines high-yield extracellular polysaccharide strains.Bacterial strain high-yield extracellular polysaccharide of the present invention, its yield of extracellular polysaccharide is 206.4mg/L.
Lactobacillus paracasei AR-93 is as the application of low fat sour milk leaven.
Above-mentioned Lactobacillus paracasei AR-93 is as the application of low fat sour milk leaven, and method is as follows:
(1) Lactobacillus paracasei AR-93 is activated；
(2) will activation after strain be inoculated in the container containing low fat milk by the inoculum concentration of 5-10%, 37 DEG C of standing for fermentation to curdled milk, 4 DEG C of after fermentation 16h, obtain low fat sour milk.
Activation described in step (1) refers to: being accessed in sterilized non-fat breast culture medium by 5% inoculum concentration by the Lactobacillus paracasei AR-93 being preserved in skimmed milk culture medium, 37 DEG C of quiescent culture 48h activate.
Utilizing the Lactobacillus paracasei AR-93 fermentation low fat sour milk that the present invention filters out, acidity is soft, and few milk surum separates out, and low fat sour milk viscosity is 3060mPa s, and Yoghourt retention ability is 20.9%, and acidity is 80 ° of T.The bacterial strain utilizing the present invention carries out low fat sour milk fermentation, can effectively keep the matter structure of Yoghourt, improves the stability of Yoghourt.
Lactobacillus paracasei AR-93 is as the application of the raw material extracting extracellular polysaccharide.
Above-mentioned Lactobacillus paracasei AR-93 is as the application of the raw material extracting extracellular polysaccharide, be the fermentation liquid of Lactobacillus paracasei AR-93 is boiled after centrifugal to remove thalline and coagulated protein, supernatant trichloroacetic acid method precipitation removes protein precipitation, then precipitate by the alcohol sedimentation method, take and dialyse with water again after being precipitated and dissolved in water, obtain extracellular polysaccharide.
The described centrifugal centrifugal condition to remove thalline and coagulated protein is 4 DEG C, 9000g, centrifugal 20min.
The fermentation liquid of described Lactobacillus paracasei AR-93 is with the inoculum concentration of 5% (v/v), Lactobacillus paracasei AR-93 to be inoculated in skimmed milk powder cultivate 40h under the conditions of 37 DEG C and the fermentation liquid that obtains.
Described trichloroacetic acid method is that interpolation trichloroacetic acid, to final concentration 4% (w/v), stands overnight in supernatant；4 DEG C, 9000g, centrifugal 20min remove protein precipitation.
The described alcohol sedimentation method are to add ethanol to final concentration 75% (v/v), and 4 DEG C stand 24h；4 DEG C, 9000g, centrifugal 20min take precipitation.
It is the slowest that the Lactobacillus paracasei AR-93 that the present invention filters out produces acid during fermentation low fat sour milk so that Yoghourt products acidity is soft, and local flavor is full.The low fat sour milk thickness of fermentation gained, the extracellular polysaccharide that this bacterial strain produces can significantly improve the texture characteristics such as the structural state of Yoghourt, reduces milk surum and separates out, and makes product without adding stabilizer, meets consumer to the demand without adding low fat sour milk.High-yield extracellular polysaccharide lactic bacteria strain and low fat sour milk sweat thereof that the present invention provides are simple, it is possible to achieve heavy industrialization is applied.
Accompanying drawing explanation
Fig. 1 is the bacterium colony figure of Lactobacillus paracasei AR-93；
Fig. 2 is the growth curve of Lactobacillus paracasei AR93.
Specific implementation method
The present invention is expanded on further below in conjunction with specific embodiment.For concrete grammar used in embodiment or material, those skilled in the art on the basis of the technology of the present invention thinking, can carry out the replacement of routine and select according to existing technology, are not limited solely to the concrete record of the embodiment of the present invention.
Experimental technique used in following embodiment if no special instructions, is conventional method；The material that used, reagent etc., if no special instructions, the most commercially obtain.
MRS solid medium: peptone 10g/L, Carnis Bovis seu Bubali cream 10g/L, yeast extract 5g/L, K2HPO42g/L, dibasic ammonium citrate 2g/L, sodium acetate 5g/L, glucose 20g/L, Tween 80 1mL, MgSO4·7H2O0.58g/L, MnSO4·4H2O0.25g/L, agar 20g/L, regulate pH to 6.4.
Skimmed milk culture medium: skimmed milk powder 100g/L, 115 DEG C of sterilizing 15min.
The screening of embodiment 1 Lactobacillus paracasei AR93 and qualification
1.1 sample diluting liquid preparation and strains separation
Gather the Kefir grains granule from pasturing area in Tibet, pulverize, add 15mL sterilized water, acutely shake 20min.Using Concentraton gradient dilution method (10-1-10-4), take diluent 100uL and coat on MRS agar culture medium flat board, 37 DEG C of Anaerobic culturel 48h, the bacterial strain that choosing colony is big, growth is fast, Pick Wire viscosity is big, line is isolated and purified further.
1.2 bacterial strain primary dcreening operations
By in inoculation big for Pick Wire viscosity in step 1.1 to MRS fluid medium, 37 DEG C of quiescent culture 24h, it is prepared as seed liquor, inoculum concentration according to 5% accesses in the MRS fluid medium of bacterium of having gone out, 37 DEG C of quiescent culture 48h, measure extracellular polysaccharide content in fermentation liquid, and experimental result is as shown in table 1.
Table 1 primary dcreening operation bacterial strain extracellular polysaccharide content
1.3 bacterial strains sieve again
1.3.1 curdled milk experiment
The lactic acid bacteria in skimmed milk culture medium will be preserved in, access in sterilized non-fat Lac Bovis seu Bubali culture medium by 5% inoculum concentration, cultivate 48h for 37 DEG C to activate, inoculation after activation is in containing in 5mL low fat milk culture medium test tube, 37 DEG C of quiescent culture are to Coagulation of milk, be inoculated in containing in 150mL low fat milk triangular flask by the inoculum concentration of 8%, 37 DEG C of quiescent culture to Coagulation of milk, observe and record curdled milk state in time between.
1.3.2 the mensuration of viscosity
The mensuration of viscosity: use flow graph (Bo Lifei company DV-III+ type).Tested viscosity at 4 DEG C, rotor model is RV5, measures three times respectively under the speed of 16r/min, and once, each sample does two parallel samples to every 15s value, and result calculates arithmetic mean of instantaneous value.
1.3.3 subjective appreciation
As shown in table 2, by subjective appreciation, obtain that a strain curdled milk is fast, quality thickness and the bacterial strain of excellent in flavor, numbered AR-93.
Table 2 sieves strain characteristic evaluation result again
1.4 identification of strains
By a strain bacterium name AR93 of isolated, bacterium colony is as it is shown in figure 1, ovalize or similar round, neat in edge, and bacterium colony size is generally 2～3mm, milky.By 16SrDNA sequence comparative analysis, result shows that this sequence reaches 99% with the homology of Lactobacillus paracasei in ncbi database, can identify that this bacterium is Lactobacillus paracasei (Lactobacillusparacasei), named Lactobacillus paracasei (Lactobacillusparacasei) AR-93, this Lactobacillus paracasei AR-93 bacterial strain on July 11st, 2014 at China Committee for Culture Collection of Microorganisms's common micro-organisms center (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3) preservation, its numbered CGMCC9443.
1.5 strain growth characteristics
Accessing in MRS fluid medium by the Lactobacillus paracasei AR-93 activated by 1% (v/v) inoculum concentration, 37 DEG C of quiescent culture 24h, the bacterium measuring culture fluid at 600nm every 2h is dense.Being plotted against time, so that bacterium is dense, the growth curve obtaining strains A R-93 in MRS, its result is as shown in Figure 2.Test result indicate that, Lactobacillus paracasei AR-93 grows rapidly in MRS culture medium, enters logarithmic (log) phase at about 2h, and about 10h enters stable phase.Along with the prolongation of incubation time, strain growth produces acid, and pH constantly reduces, and enters stable phase.
Embodiment 2 Lactobacillus paracasei AR-93 prepares low fat sour milk
The lactic acid bacteria AR-93 that will be preserved in skimmed milk culture medium, is accessed in sterilized non-fat Lac Bovis seu Bubali culture medium by 5% inoculum concentration, and 37 DEG C of quiescent culture 48h activate.Bacterial strain after activation is inoculated in 150mL low fat milk triangular flask by the inoculum concentration of 5%, and 37 DEG C of quiescent culture are to Coagulation of milk, and 4 DEG C keep 16h, obtain the Yoghourt being not required to add thickening agent.The acidity of gained Yoghourt is soft, and few milk surum separates out, and low fat sour milk viscosity is 1347mPa s, and Yoghourt retention ability is 15.7%, and acidity is 77oT.
Embodiment 3 Lactobacillus paracasei AR-93 prepares low fat sour milk
The lactic acid bacteria AR-93 that will be preserved in skimmed milk culture medium, is accessed in sterilized non-fat Lac Bovis seu Bubali culture medium by 5% inoculum concentration, and 37 DEG C of quiescent culture 48h activate.Bacterial strain after activation is inoculated in 150mL low fat milk triangular flask by the inoculum concentration of 8%, and 37 DEG C of quiescent culture are to Coagulation of milk, and 4 DEG C keep 16h, obtain the Yoghourt being not required to add thickening agent.The acidity of gained Yoghourt is soft, and few milk surum separates out, and low fat sour milk viscosity is 3060mPa s, and Yoghourt retention ability is 20.9%, and acidity is 80oT.
Embodiment 4 Lactobacillus paracasei AR-93 prepares low fat sour milk
The lactic acid bacteria AR-93 that will be preserved in skimmed milk culture medium, is accessed in sterilized non-fat Lac Bovis seu Bubali culture medium by 5% inoculum concentration, and 37 DEG C of quiescent culture 48h activate.Bacterial strain after activation is inoculated in 150mL low fat milk triangular flask by the inoculum concentration of 10%, and 37 DEG C of quiescent culture are to Coagulation of milk, and 4 DEG C keep 16h, obtain the Yoghourt being not required to add thickening agent.The acidity of gained Yoghourt is soft, and few milk surum separates out, and low fat sour milk viscosity is 2824mPa s, and Yoghourt retention ability is 19.5%, and acidity is 84oT.
The extraction of embodiment 5 Lactobacillus paracasei AR-93 extracellular polysaccharide
(1) actication of culture: be inoculated in MRS fluid medium by Lactobacillus paracasei AR-93 strain, cultivates 12-16h under the conditions of 37 DEG C and activates, two generations of activation continuously.
(2) seed culture: after Lactobacillus paracasei AR-93 is activated, is inoculated in skimmed milk, and after 115 DEG C of sterilizing 15min, under the conditions of 37 DEG C, cultivation 14-16h is to curdled milk, cultivates two generations of activation continuously, is used as mother's fermentation liquid.
(3) fermentation culture: be inoculated in skimmed milk with the inoculum concentration of 5% (v/v) by Lactobacillus paracasei AR-93, cultivates 40h under the conditions of 37 DEG C.
null(4) extraction purification of EPS: the fermentation liquid of above-mentioned preparation is first passed around boiling water bath 10min，To inactivate the enzyme of degradable polysaccharide，It is then centrifuged for (20min，9000g，4 DEG C) remove thalline and coagulated protein，Supernatant concentration is to the 1/3 of original volume，Add 80% (w/v) trichloroacetic acid to final concentration 4% (w/v)，Stand overnight，Centrifugal (20min，9000g，4 DEG C) remove protein precipitation，(v/v) ethanol that concentrated solution adds 95% is to final concentration 75% (v/v)，4 DEG C stand 24h，Centrifugal (20min，9000g，4℃)，Take precipitation deionized water dissolving，Centrifugal (20min，9000g，4 DEG C) go precipitation，Supernatant deionized water dialysis 72h，Every 8h changes water once，Lyophilization obtains crude polysaccharides sample.
Claims (10)
1. a strain Lactobacillus paracasei (Lactobacillusparacasei), named Lactobacillus paracasei AR-93, its deposit number is CGMCC9443, and its gene order is as shown in SEQIDNO.1.
2. Lactobacillus paracasei AR-93 is as the application of low fat sour milk leaven.
Lactobacillus paracasei AR-93 the most according to claim 2 is as the application of low fat sour milk leaven, it is characterised in that:
Application process is as follows:
(1) Lactobacillus paracasei AR-93 is activated；
(2) will activation after strain be inoculated in the container containing low fat milk by the inoculum concentration of 5-10%, 37 DEG C of standing for fermentation to curdled milk, 4 DEG C of after fermentation 16h, obtain low fat sour milk.
Lactobacillus paracasei AR-93 the most according to claim 3 is as the application of low fat sour milk leaven, it is characterized in that: the activation described in step (1) refers to: being accessed in sterilized non-fat breast culture medium by 5% inoculum concentration by the Lactobacillus paracasei AR-93 being preserved in skimmed milk culture medium, 37 DEG C of quiescent culture 48h activate.
5. Lactobacillus paracasei AR-93 is as the application of the raw material extracting extracellular polysaccharide.
The most according to claim 5, Lactobacillus paracasei AR-93 is as the application of the raw material extracting extracellular polysaccharide, it is characterized in that: centrifugal to remove thalline and coagulated protein after the fermentation liquid of Lactobacillus paracasei AR-93 is boiled, supernatant trichloroacetic acid method precipitation removes protein precipitation, then precipitate by the alcohol sedimentation method, take and dialyse with water again after being precipitated and dissolved in water, obtain extracellular polysaccharide.
The most according to claim 6, Lactobacillus paracasei AR-93 is as the application of the raw material extracting extracellular polysaccharide, it is characterised in that: the described centrifugal centrifugal condition to remove thalline and coagulated protein is 4 DEG C, 9000g, centrifugal 20min.
The most according to claim 6, Lactobacillus paracasei AR-93 is as the application of raw material extracting extracellular polysaccharide, it is characterised in that: the fermentation liquid of described Lactobacillus paracasei AR-93 is with the inoculum concentration of 5% (v/v), Lactobacillus paracasei AR-93 to be inoculated in skimmed milk powder cultivate 40h under the conditions of 37 DEG C and the fermentation liquid that obtains.
The most according to claim 6, Lactobacillus paracasei AR-93 is as the application of the raw material extracting extracellular polysaccharide, it is characterised in that: described trichloroacetic acid method is that interpolation trichloroacetic acid, to final concentration 4% (w/v), stands overnight in supernatant；4 DEG C, 9000g, centrifugal 20min remove protein precipitation.
The most according to claim 6, Lactobacillus paracasei AR-93 is as the application of raw material extracting extracellular polysaccharide, it is characterised in that: the described alcohol sedimentation method are to add ethanol to final concentration 75% (v/v), and 4 DEG C stand 24h；4 DEG C, 9000g, centrifugal 20min take precipitation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109706098A (en) * | 2019-01-21 | 2019-05-03 | 北京农学院 | Lactobacillus paracasei and its application |
| CN114736835A (en) * | 2022-04-24 | 2022-07-12 | 北京工商大学 | Lactobacillus paracasei strain SS-01, exopolysaccharide prepared by strain, preparation method and application |
| CN116769673A (en) * | 2023-08-04 | 2023-09-19 | 云南农业大学 | Lactic acid bacteria with high extracellular polysaccharide yield and application thereof in preparation of wiredrawing yoghourt |
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| WO2012069546A1 (en) * | 2010-11-23 | 2012-05-31 | Chr. Hansen A/S | Use of glycosidase in preparation of a milk product |
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| WO2012069546A1 (en) * | 2010-11-23 | 2012-05-31 | Chr. Hansen A/S | Use of glycosidase in preparation of a milk product |
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| RIHUA XU ET AL.: "Screening, identification and statistic optimization of a novel exopolysaccharide producing Lactobacillus paracasei HCT", 《 AFRICAN JOURNAL OF MICROBIOLOGY RESEARCH》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109706098A (en) * | 2019-01-21 | 2019-05-03 | 北京农学院 | Lactobacillus paracasei and its application |
| CN114736835A (en) * | 2022-04-24 | 2022-07-12 | 北京工商大学 | Lactobacillus paracasei strain SS-01, exopolysaccharide prepared by strain, preparation method and application |
| CN116769673A (en) * | 2023-08-04 | 2023-09-19 | 云南农业大学 | Lactic acid bacteria with high extracellular polysaccharide yield and application thereof in preparation of wiredrawing yoghourt |
| CN116769673B (en) * | 2023-08-04 | 2023-12-29 | 云南农业大学 | Lactic acid bacteria with high extracellular polysaccharide yield and application thereof in preparation of wiredrawing yoghourt |
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