CN101331900B - Four kinds of lactobacillus for generating bilesalt hydrolase and extracellular polysaccharide and functionality yoghourt production technique thereof - Google Patents
Four kinds of lactobacillus for generating bilesalt hydrolase and extracellular polysaccharide and functionality yoghourt production technique thereof Download PDFInfo
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Abstract
The invention belongs to a production technology of four lactobacillus producing bile salt hydrolase and extracellular polysaccharide as well as functional yoghurt production technology. The technology is adaptable to the production of functional yoghurt and microbial ecological agents in fermented dairy products for lowering cholesterol. The technology adopts high throughput screening technique and an ortho-phthalaldehyde method to obtain bacterial strains which can efficiently reduce the cholesterol, to get the bacterial strains which can produce the bile salt hydrolase with high yield by an Oxford cup method and to gain the bacterial strains which can produce the extracellular polysaccharide with high yield by a phenol-sulfuric acid method. All the four kinds of screened lactobacillus have excellent fermentation performance. The yogurt with fine quality is prepared by lactobacillus casei KL1 (CGMCC No1809) and J1 (CGMCC No1808), which have the bile salt hydrolase with high efficiency in lowering the cholesterol as well as high yield and the extracellular polysaccharide, lactococcus lactis subsp.lactis KS4 (CGMCC No1807) and streptococcus thermophilus Tx(CGMCC No1810), therefore, the yogurt not only has the function of lowering serum cholesterol, but also utilizes the extracellular polysaccharide produced by thalli to replace stabilizer, thus solving the separating problem of yogurt whey. The technology has simple fermentation process in producing the functional yoghurt, low fermentation cycle and lower cost, so the technology is suitable for industrialized production inlarge scale.
Description
Technical field
The present invention relates to four kinds and produce bile salt hydrolase (Bile Salt Hydrolase, BSH) and exocellular polysaccharide (exopolysaccharide, EPS) milk-acid bacteria and functional yoghourt production technology thereof are applicable to the production of the functional yoghourt and the probiotics of decreasing cholesterol in the cultured milk prod.
Background technology
Probiotic bacterium (Probiotics) is meant that a class grows improved effect host privileged site microecological balance surely by enteron aisle and have the microorganism of some other useful physiological functions concurrently.They are by improving the enteric microorganism eubiosis, and the control intestinal tract infections reduces serum cholesterol level, improve the utilization of lactose intolerance people to lactose, and can stimulate specificity or nonspecific immune reaction, have the effect of immunostimulant.The viable bacteria of probiotic composition should metabolic stability, and activity is stronger, by large number of viable behind the digestive tube, brings into play probiotic action after entering enteron aisle.The excellent species that is used for producing probiotic agent at present has Lactococcus lactis subsp.lactis of genus bifidobacterium, lactobacillus, lactococcus etc.
Just confirm that absorbing the sour milk that contains certain Bacterium lacticum or bifidobacterium fermentation has the content that reduces serum cholesterol as far back as people in 1963.The seventies in 20th century, scientist is successively by to drinking the research of the African Massai human serum cholesterol of cultured milk prods such as yogurt in a large number, to neonatal research, American investigation to normal drink yogurt, and, find that all milk-acid bacteria has the effect that reduces the human serum cholesterol directly to the research of yogurt etc.So far, the investigator remains in different viewpoints for the mechanism of action of milk-acid bacteria reducing cholesterol both at home and abroad, mainly concentrates on following 3 points: the direct assimilation cholesterol of (1) milk-acid bacteria somatic cells; (2) the bile salt hydrolase activity of milk-acid bacteria makes the combined cholate be degraded to the free state cholate, and latter's solubleness descends and cholesterol generation coprecipitation effect; (3) other theories are as assimilation and co-precipitation combined action.Bile salt hydrolase (BSH) is the meta-bolites that milk-acid bacteria produces.This enzyme can produce amino acid and the lower free state cholate of solubleness with the degraded of combined cholate in the circulation of human body liver sausage.The latter can combine the formation sediment composite with cholesterol and excrete, thereby reduces the content of serum total cholesterol.
There is the bibliographical information milk-acid bacteria to optimize under the growth conditions,, can secretes exocellular polysaccharide (EPS) for provide protection to self in the Asia.Whitfield etc. studies show that being formed with of exocellular polysaccharide is beneficial to microorganism opposing poor environment factor, as the invasion and attack of drying, osmotic pressure condition, microbiotic or poisonous substance effect, scavenger cell or phage etc.Since people such as nineteen eighty-two Japan scholar Shiomi report that milk-acid bacteria EPS has antitumor action, caused many scholars' attention.From the result of study of report, milk-acid bacteria EPS mainly contains the function of the following aspects: improve the cultured milk prod structural state, the synersis of control sour milk prevents that whey from separating out; Improve the non-specific adhesion of bacterial strain, strengthen decide the grow effect of bacterial strain enteron aisle to intestinal mucosa; Antitumor action; Regulate the immunity system effect; The pair cell body has provide protection; Suppress the breeding of enteron aisle spoilage organism, prevent and treat the effect of colorectal carcinoma.Think at present that the mechanism of milk-acid bacteria EPS antitumor action has following side and: (1) influences blood supply, and in view of bacteriocin can cause tumor cell tissue's ischemic necrosis, the antitumor action of once imagining polysaccharide may influence the blood supply of tumour; (2) stimulate certain organ or tissue, secrete a kind of material and attack tumour cell; (3) cytolemma contact inhibition.Tumor cell surface has very strong negative charge, and some polysaccharide can be in conjunction with these electric charges, make cell surface by " neutralization " thus helping the cell received signal stops dividing.
Domestic research about lactobacterium casei mainly is on the health-care effecies such as its reducing blood-fat, hypertension, treatment anaphylactic disease, as " the lactobacterium casei Bd-II bacterial strain and in the application aspect the blood fat reducing " of Shanghai Bright Dairy ﹠ Food Co., Ltd application, number of patent application is: CN03128995.9; Lactobacterium casei LC2W bacterial strain and the application number of patent application aspect hypertension thereof are: two patents of CN03129450.2; Jingyue Biological Science and Technology Co., Ltd. application: new microbial strain lactobacillus paracasei GM-080 and treat the purposes of irritated relative disease, number of patent application is: CN200410038566.X.
Though abroad the research about lactobacterium casei reduction serum cholesterol effect also has a patent report: Lactobacillus casei bd-II stain and used to reduce blood cholesterol (20060127380), but, abroad almost have nothing to do at present and produce the patent report of bile salt hydrolase in bacterial strains such as Lactococcus lactis subsp.lactis, thermophilus streptococcuses.Though the domestic patent report that " containing functional yoghourt of lactobacillus reuteri and preparation method thereof (200310112715.8) " arranged, domestic nothing utilize the milk-acid bacteria of decreasing cholesterol and high yield bile salt hydrolase and exocellular polysaccharide to be applied to related article report and patent report that functional yoghourt is produced.
Summary of the invention
First purpose of the present invention provides four kinds of milk-acid bacterias with efficient decreasing cholesterol and high yield bile salt hydrolase and exocellular polysaccharide.
Milk-acid bacteria provided by the present invention is: lactobacterium casei (Lactobacillus casei) KL1 bacterial strain, lactobacterium casei (Lactobacillus casei) J1 bacterial strain, Lactococcus lactis subsp.lactis (Lactococcus lactissubsp.lactis) KS4, thermophilus streptococcus (Streptococcus thermophilus) Tx has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on September 11st, 2006.Preserving number is respectively: CGMCC № 1809 (bacterial strain KL1), CGMCC № 1808 (bacterial strain J1), CGMCC № 1807 (bacterial strain KS4), CGMCC № 1810 (bacterial strain Tx).
Lactobacterium casei (Lactobacillus casei) KL1, lactobacterium casei (Lactobacillus casei) J1, Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) KS4, thermophilus streptococcus (Streptococcus thermophilus) Tx, separation screening from Jilin, the Tibetan of Huhehaote City's average family spirit mushroom (claiming the Kai Feier grain again).
Containing on the MRS selective medium of lime carbonate and tennecetin the bacterium colony size 2～3mm of lactobacterium casei (Lactobacillus casei) KL1 and J1 bacterial strain, surface irregularity such as flakes, the edge is irregular, and tarnish is flat, canescence, translucent, periphery of bacterial colonies has dissolving CaCO
3Transparent circle, bacterium colony has than high viscosity; It is shaft-like that individual morphology is, varying length, and the short chain shape is arranged, and thalline is spherical in shape sometimes, G
+Sporeless bacterium; The bacterium colony size of Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) KS4 and thermophilus streptococcus (Streptococcus thermophilus) Tx bacterial strain is 1～2mm, smooth surface, neat in edge, rounded, tarnish, projection, canescence, translucent, periphery of bacterial colonies has dissolving CaCO
3Transparent circle, bacterium colony has than high viscosity.Spherical in shape or the oval of individual morphology, general paired or catenation, G
+Facultative anaerobe.
Aforesaid method obtains having the Lactococcus lactis subsp.lactis KS4 of efficient decreasing cholesterol and high yield bile salt hydrolase and exocellular polysaccharide, thermophilus streptococcus Tx, and lactobacterium casei KL1 and J1 belong to protection domain of the present invention.
Second purpose of the present invention provides four kinds of milk-acid bacterias and prepares the functional yoghourt production technology.
The method for preparing functional yoghourt provided by the present invention is by the lactobacterium casei with efficient decreasing cholesterol and high yield bile salt hydrolase and exocellular polysaccharide (Lactobacillus casei) KL1 and J1, Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) KS4, four kinds of milk-acid bacterias of thermophilus streptococcus (Streptococcus thermophilus) Tx make.
In the described method, the technological condition for fermentation that is used to prepare functional yoghourt is: leavening temperature is 37～43 ℃, inoculum size is 2%～6%, Tx/KS4/KL1 (or J1) inoculative proportion is 1: 1: 2,2: 1: 1,2: 1: 3, fermentation time is 3.5～5.5h, and the sucrose addition is 4%～8% (w/v).
The best technological condition for fermentation of preparation functional yoghourt is in the described method: leavening temperature is 40 ℃, and the starter inoculum size is 4%, and Tx/KS4/KL1 (or J1) inoculative proportion is 2: 1: 3, and fermentation time is 4.0h, sucrose addition 4～5% (w/v).Under this optimization of fermentation conditions, the finished product sour milk curdled milk time is short, the fixed reality of curdling, and no whey is separated out, and sticky fine and smooth children is sliding for mouthfeel, and sweet acidity is moderate, and strong ester fragrant breeze flavor is arranged.
Utilize the probiotic bacterium that produces exocellular polysaccharide to substitute yoghourt stabilizer or thickening material, can obviously improve sour milk stability and rheological characteristics, can utilize the probiotic strain scale operation functional yoghourt that produces bile salt hydrolase and exocellular polysaccharide again.
Aforesaid method obtains best technological condition for fermentation of functional yoghourt and products thereof and also belongs to protection domain of the present invention with special preparing strain.
Description of drawings
Using Lactobacillus casei KL1 and J1, Lactococcus lactis subsp. lactis KS4 and streptococcus thermophilus Tx production functional yoghourt process chart is described as follows:
(1) raw milk: the raw milk of making sour milk requires the bacterium number generally to be lower than 104Individual/mL, do not contain antibiotic and disinfectant, should not select the trouble Bovine Mastitis Milk. (2) purify: remove leucocyte and other macroscopic impurity in the cow's milk with centrifuge. (3) standardization: the main component index of raw milk should meet National standards of food sanitation GB 5408-85. Its total solids should be not less than 11.5%, fat content is adjusted into 4 kinds substantially according to the product difference: 3.2%, 2.5%, 1.0% and＜0.1%, can by remove cream add 1%～3% skimmed milk power or rare cream to adjust total solids or fat content. (4) preheating, batching and filtration: raw milk is heated to about 60 ℃, adds the sucrose of 4%～5% (w/v), filtering and impurity removing after the dissolving. (5) homogeneous: in homogenizer, under 8～10MPa pressure, the raw milk of preheating is carried out homogeneous and process. Purpose is to make curdling admittedly even, and quality is finer and smoother, level and smooth, prevents fat floating and fat globule is diminished. (6) sterilization: raw milk is heated to 90 ℃, insulation 5～10min. (7) cooling: the raw milk after sterilization is cooled to rapidly 40 ℃, waits to inoculate. (8) inoculation: the total inoculum concentration that adds single working stock culture is 4%, and the inoculative proportion of the single fermentation agent of t bacteria x, KS4 and KL1 (or J1) is 2: 1: 3. (9) packing: invade for avoiding miscellaneous bacteria, the operation that is sub-packed in plastic small container should be carried out in desinfection chamber fast. (10) heat-preservation fermentation: place fermenting cellar to keep 40 ℃ of fermentation temperatures plastic small container. When acidified milk acidity reaches 65 ° of T, when curdling solidity shape is good, i.e. fermenting-ripening. Fermentation time is generally 4h. (11) cooling: the container that will contain sour milk takes out from fermenting cellar, is cooled to rapidly below 10 ℃ with cold wind. (12) refrigeration and after-ripening: through the sour milk of cooling processing, preserve in 0～5 ℃ of refrigerating chamber and preserve, until drink. Purpose is to prevent that sour milk from continuing fermentation and acid and causing pH to cross low and affect mouthfeel, and prevents that living contaminants from breeding. After-ripening under the refrigerated condition is conducive to the formation of yoghourt-flavored material, finally obtains soft, the strong functional finished product sour milk of local flavor.
Lactobacillus casei (Lactobacillus casei) KL1 and J1, Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) KS4, streptococcus thermophilus (Streptococcus thermophilus) Tx separation screening is in the Tibetan of northeast average family spirit mushroom (claiming again the Kai Feier grain), has reliable security, the functional yoghourt curdled milk of producing with these four kinds of lactic bacteria strains is solid, no whey is separated out, sticky, fine and smooth children is sliding for mouthfeel, sweet acidity is moderate, and strong ester Flavor is arranged.
The present invention makes the raw material sources convenience of functional yoghourt, and zymotechnique is simple, and fermentation period is short, and simple to operate, low for equipment requirements, cost is lower, is suitable for suitability for industrialized production.
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.
Percentage composition among the following embodiment if no special instructions, is volumn concentration.
Lactobacterium casei (Lactobacillus casei) KL1 and the J1 of embodiment 1, efficient decreasing cholesterol and high yield bile salt hydrolase and exocellular polysaccharide, Lactococcus lactis subsp.lactis (Lactococcus lactissubsp.lactis) KS4, screening and the evaluation of thermophilus streptococcus (Streptococcus thermophilus) Tx
1, the screening of efficient decreasing cholesterol and high yield bile salt hydrolase bacterial strain
Get and hide the 45mL stroke-physiological saline solution that clever mushroom filtrate 5mL injects the band granulated glass sphere, the 30min that fully vibrates makes bacteria suspension, and then 10 times of gradient dilutions become 10
-4～10
-6Dilution bacterium liquid.Dull and stereotyped with the inoculation of dilution tilt-pour process, pour the MRS selective medium that contains lime carbonate and tennecetin into, cultivate 24h for 37 ℃, picking has single bacterium colony streak inoculation of dissolving circle in the MRS slant medium, cultivates 24h in 37 ℃.Gramstaining is looked its individual morphology and purity.Change and be inoculated in the MRS liquid nutrient medium, 37 ℃ increase bacterium and cultivated for 2～3 generations, transfer into the I substratum that contains cholesterol with 1%～2% inoculum size again, behind 37 ℃ of cultivation 20h, with content of cholesterol before and after the fermentation in the o-phthalaldehyde(OPA) colorimetric method for determining I substratum, and calculate cholesterol reduced rate (%).Determine the bacterial strain of efficient decreasing cholesterol according to the cholesterol reduced rate.Concrete measuring method: get 0.4mL and cultivate the fermented liquid of front and back in colorimetric cylinder, add 0.2mL o-phthalaldehyde(OPA) (1mg/mL) and 4mL mixing acid (Glacial acetic acid, vitriol oil balanced mix), room temperature leaves standstill 10min behind the thorough mixing, with the I substratum that does not contain cholesterol is blank accent 0, measure light absorption value (A) in 550nm, on cholesterol-light absorption value typical curve, check in fermentation front and back content of cholesterol (the results are shown in Table 1).
The primary dcreening operation test-results of the different lactobacillus strain reducing cholesterol of table 1
Studies show that the milk-acid bacteria of adopting high flux screening technology and o-phthalaldehyde method to obtain efficient reducing cholesterol is KS4, J1, KL1 and Tx bacterial strain.Wherein, KS
4Bacterial strain reducing cholesterol ability is the strongest, secondly is J1, KL1 and Tx bacterial strain.They belong to the strong bacterial strain of reducing cholesterol ability in relevant bibliographical information.
With the bacterial strain that obtains behind the primary dcreening operation with efficient decreasing cholesterol; on the MRS culture medium flat plate that contains 0.1% cholesterol and 0.3% Bile Salts (respectively in contrast) with the MRS substratum of the MRS substratum that contains 0.1% cholesterol, 0.3% Bile Salts; symmetry is placed the Oxford cup; add 0.1mL MRS and cultivate bacterium liquid; after 37 ℃ of anaerobism are cultivated 3～4d; around observing the Oxford cup whether the silver color deposited phenomenon is arranged, it the results are shown in Table 2.Positive person detects the hydrolysate whether cholate is arranged in the silver color throw out with ninhydrin method---the amino acid existence, determine according to the bluish voilet reaction is preliminary whether bacterial strain produces bile salt hydrolase.The streak inoculation of ninhydrin reaction positive strain in the MRS slant tube, is cultivated the back and preserved standby.
Table 2 produces the bile salt hydrolase lactobacillus strain and sieves test-results again
Annotate: silver color deposited phenomenon is in various degree arranged around the cup of "+" expression Oxford; The no deposited phenomenon of "-" expression.
Show that by table 1 and table 2 result the size of precipitation circle is directly proportional with the vigor of bile salt hydrolase and the percentage of reducing cholesterol.Multiple sieve test is relatively overall, and bacterial strain KS4, J1, KL1 and Tx can the high yield bile salt hydrolases, and wherein bacterial strain KS4 enzymatic productivity is the strongest, secondly is J1, KL1 and Tx bacterial strain.The lactobacillus strain of efficient reducing cholesterol comes from its bile salt hydrolase that produces volume or high vigor.Can utilize the further functional yoghourt of developing efficient decreasing cholesterol of lactobacillus strain of these high yield bile salt hydrolases.
2, the screening of high-yield extracellular polysaccharide strains
Get and hide the 45mL stroke-physiological saline solution that clever mushroom filtrate 5mL injects the band granulated glass sphere, the 30min that fully vibrates makes bacteria suspension, and then 10 times of gradient dilutions become 10
-4～10
-6Dull and stereotyped with dilution tilt-pour process inoculation, pour the MRS selective medium that contains lime carbonate and tennecetin into, cultivate 24h for 37 ℃, picking has the higher single bacterium colony streak inoculation of dissolving circle, stickiness in the MRS slant medium, cultivates 24h in 37 ℃.Gramstaining is looked its individual morphology and purity.Change to be inoculated in the MRS liquid nutrient medium, 37 ℃ increase bacterium and cultivated for 2～3 generations, transfer in the MRS liquid nutrient medium with 1%～2% inoculum size again, 37 ℃ cultivate 12h after, with the content of EPS in the phenolsulfuric acid colorimetric method for determining nutrient solution.To record sample absorbancy y value substitution typical curve formula y=0.8649x (R
2=0.9991) promptly obtain EPS concentration x value (100mg/L), it the results are shown in Table 3.According to the EPS content in the MRS substratum, determine to produce the bacterial strain of exocellular polysaccharide.The bacterial strain streak inoculation of producing exocellular polysaccharide in the MRS slant tube, is cultivated the back and preserved standby.
Table 3 isolated strains produces the exocellular polysaccharide test result
Studies show that the milk-acid bacteria of adopting high flux screening technology and phenolsulfuric acid method to obtain higher synthetic EPS is KS4, KL1, J1 and Tx bacterial strain.Wherein the output of KS4 bacterial strain EPS is the highest, is KL1, J1 and Tx bacterial strain secondly.They all belong in relevant bibliographical information produces the stronger bacterial strain of EPS ability.
3, screening strain fermentation Performance Testing
KS4, KL1, J1 and the Tx strain culture of screening are transferred in the sterilization skimming milk 2～3 generations of continuous passage activation, and 37 ℃ of overnight incubation to cow's milk solidify.Transfer with 1% inoculum size again and contain in the triangular flask of skimming milk, after 37 ℃ of overnight incubation to cow's milk solidify, refrigerate after-ripening again and spend the night, measure the vigor (comprising curdled milk time, acidity, viscosity, tough reddish black time of reduction, viable lactic acid bacteria quantity) of starter.After solidifying respectively at 37 ℃, 40 ℃, 43 ℃ overnight incubation to cow's milk, carry out the sense organ Comprehensive Assessment, it the results are shown in Table 4.According to the vigor and the sense organ Comprehensive Assessment mark height of starter, determine the bacterial strain that leavening property is good.Bile salt hydrolase, exocellular polysaccharide and the good MRS inclined-plane bacterial strain of leavening property are produced in screening, be inoculated in various physics and chemistry and identify that 37 ℃ of cultivations are identified in the substratum.
The leavening property test result of table 4 screening lactobacillus strain
Annotate: data are the mean value of n=3 mensuration in the table.
Studies show that bacterial strain KS4, KL1, J1 and Tx all have good leavening property.It is the highest wherein to prepare the viscosity of starter by the Tx bacterial strain, and the curdled milk time is the shortest, and the sense organ comprehensive grading is the highest, secondly is J1, KL1 and KS4 bacterial strain.Can utilize these lactobacillus strains that produce EPS further to develop functional yoghourt with good stability.
4, the evaluation of efficient decreasing cholesterol and high yield bile salt hydrolase and extracellular polysaccharide strains
Conventional physiological and biochemical test is identified: glycitols fermentation test, salt tolerant test (7.5%NaCl), differing temps test (10 ℃, 40 ℃).Biolog full automatic microorganism assessing instrument is identified: the single bacterium colony streak inoculation of the purpose on the picking MRS flat board (contains 5% aseptic defiber sheep blood) on the BUA+B flat board, after 37 ℃ of cultivations obtain single bacterium colony, adjust turbidity to the 65 ± 2%T of bacteria suspension with the special-purpose inoculation liquid of Biolog, be inoculated in the AN trace identification plate, after 35～37 ℃ of anaerobism are cultivated 24～48h, on assessing instrument, read qualification result.Qualification result is as shown in table 5.Identify that bacterial strain KL1 and J1 are lactobacterium casei (Lactobacillus casei), bacterial strain KS4 is Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis), and bacterial strain Tx is thermophilus streptococcus (Streptococcus thermophilus).
Certified variety and the result of table 5 bacterial strain KL1, J1, KS4, Tx
Annotate: "+" bacterial strain more than 95% is positive; "-" bacterial strain more than 95% is negative.
Embodiment 2, utilize efficient decreasing cholesterol and high yield bile salt hydrolase and extracellular polysaccharide strains to produce functional yoghourt
The used yoghurt production special bacteria of the present invention is lactobacterium casei (Lactobacillus casei) KL1 and J1, Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) KS4, thermophilus streptococcus (Streptococcus thermophilus) Tx.They are the functional bacterial classification of high yield bile salt hydrolase and exocellular polysaccharide.Wherein lactobacterium casei KL1 and J1 bacterial strain stomach juice-resistant, anti-biliary salts ability are stronger, have the contrary environmental characteristics of anti-alimentary tract, can arrive the intestinal secretion bile salt hydrolase smoothly, and the degraded of combined cholate is produced amino acid and the lower free state cholate of solubleness.The latter can combine the formation sediment composite with cholesterol and excrete, thereby reduces the content of serum total cholesterol.Utilize small white mouse to do the functional yoghourt per os and irritate the stomach test, result after 1 month shows, the ldl concn of model trial group mice serum is than the obvious reduction of the blank group of model, and from stool in mice, separate a large amount of viable bacterias of bacterial strain KL1, illustrate that lactobacterium casei (Lactobacillus casei) KL1 can arrive the efficient effect that reduces serum cholesterol of small intestine performance by the contrary environment of digestive tube smoothly.Produce the functional yoghourt of serum cholesterol-lowering so can utilize these bacterial strains.
1, the preparation of single fermentation agent
With the 10mL skimming milk test tube of packing into, through 0.07Mpa sterilization 20min, be cooled to 37 ℃, the lactobacterium casei KL1 or the J1 of bile salt hydrolase and exocellular polysaccharide, Lactococcus lactis subsp.lactis KS4, thermophilus streptococcus Tx strain excellent are produced in inoculation respectively, 37 ℃ are cultured to curdling admittedly, make starter with 2% inoculum size again, it is solid to be cultured to curdling under 37 ℃, is the single culture starter.
2, functional yoghourt production Optimizing Conditions of Fermentation
Carry out three factors, three level [L according to influencing the bigger leavening temperature of functional yoghourt quality, inoculative proportion and inoculum size
9(3
4)] orthogonal test (seeing Table 6), and to solid time of the curdling of finished product sour milk, viscosity, sense organ comprehensive grading (total points 80 minutes, wherein color and luster 5 minutes, flavour and gas 30 minutes, structural state 15 minutes, several 10 minutes of viable lactic acid bacteria, stickiness 20 minutes), determine its best technological condition for fermentation by range analysis and variance analysis to test-results.
Table 6 functional yoghourt optimization of fermentation conditions orthogonal experimental design level of factor table
Table 7 fermentation condition optimization result and initial fermentation condition result contrast
In order to obtain the functional yoghourt of bigger vigor and fine quality, optimize at its fermentation condition.Pass through orthogonal test, with curdled milk time, viscosity and sense organ comprehensive grading is evaluation index, determine that the functional yoghourt optimal conditions of fermentation is: leavening temperature is 40 ℃, the starter inoculum size is 4%, the inoculative proportion of Tx/KS4/KL1 (J1) is 2: 1: 3, fermentation time is 4.0h, and the sucrose addition is 4%～5% (w/v).(see Table 7) under this optimization of fermentation conditions, finished product sour milk viscosity is higher, and the curdled milk time is shorter, and curdled milk is solid, and no whey is separated out, and mouthfeel is sticky, fine and smooth and the children is sliding, and sweet acidity is moderate, and strong ester fragrant breeze flavor is arranged.Refrigeration 1 its viable count of all backs is higher to be 9.6 * 10
6Cfu/mL, viscosity are still higher, illustrate that the exocellular polysaccharide that milk-acid bacteria produces has certain protective role to lactic bacteria activity, have improved the resistance of producing bacterial classification.Its whey amounts of separating out of refrigeration two week backs keep lower level, reduce approximately 50% than the common sour milk whey amount of separating out, and have solved the problem that soured milk whey is separated out.So utilize the probiotic bacterium that produces exocellular polysaccharide to substitute yoghourt stabilizer or thickening material, can obviously improve sour milk stability and rheological characteristics, can utilize the probiotic strain scale operation functional yoghourt that produces bile salt hydrolase and exocellular polysaccharide again.
Project under this patent: the open problem " bacterial screening of Kai Feier grain and the applied research in functional fermentation cow's milk thereof " of agricultural application new technology Beijing key lab of Beijing Education Commission
Item number: KF2003-06
The project beginning and ending time: 2004.01-2006.12
Project leader: Liu Hui
Claims (3)
1. lactobacterium casei (Lactobacillus casei) KL1 CGMCC No.1809.
2. one kind is utilized lactobacterium casei (Lactobacillus casei) KL1, Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) KS4, the functional yoghourt of three kinds of milk-acid bacteria preparations of thermophilus streptococcus (Streptococcus thermophilus) Tx, it is characterized in that: the preserving number of lactobacterium casei (Lactobacillus casei) KL1 is CGMCC No.1809, the preserving number of Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) KS4 is CGMCC No.1807, and the preserving number of thermophilus streptococcus (Streptococcus thermophilus) Tx is CGMCC No.1810; Technological condition for fermentation is: leavening temperature is 40 ℃, and the starter inoculum size is 4%, and the inoculative proportion of Tx/KS4/KL1 is 2: 1: 3, and fermentation time is 4.0h, and the sucrose addition is: 4%～5% (w/v).
3. one kind is utilized lactobacterium casei (Lactobacillus casei) J1, Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) KS4, the functional yoghourt of three kinds of milk-acid bacteria preparations of thermophilus streptococcus (Streptococcus thermophilus) Tx, it is characterized in that: the preserving number of lactobacterium casei (Lactobacillus casei) J1 is CGMCC No.1808, the preserving number of Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) KS4 is CGMCC No.1807, and the preserving number of thermophilus streptococcus (Streptococcus thermophilus) Tx is CGMCC No.1810; Technological condition for fermentation is: leavening temperature is 40 ℃, and the starter inoculum size is 4%, and the inoculative proportion of Tx/KS4/J1 is 2: 1: 3, and fermentation time is 4.0h, and the sucrose addition is: 4%～5% (w/v).
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| CN101642274B (en) * | 2009-08-21 | 2012-08-01 | 北京农学院 | Method for preparing cholesterol reducing egg milk fermenting drink by utilizing lactobacillus and microzyme which produce bile salt hydrolase |
| CN102321550B (en) * | 2011-08-03 | 2013-04-10 | 甘肃农业大学 | Lactococcus lactis fermented at low temperature and method for preparing yoghurt by using Lactococcus lactis |
| CN102399730B (en) * | 2011-12-07 | 2012-12-26 | 天津科技大学 | High-extracellular-polysaccharide-yield Lactobacillus, application and extracellular polysaccharide thereof |
| CN102586137B (en) * | 2012-01-10 | 2013-10-30 | 北京农学院 | Method for preparing bile salt hydrolase lactobacillus powder starter |
| CN103131654B (en) * | 2013-03-05 | 2015-07-15 | 北京农学院 | Application of Lactobacillus casei KL1 for producing bile salt hydrolase in functional sour milk |
| CN103146607B (en) * | 2013-03-05 | 2014-12-17 | 北京农学院 | Preparation method of active microbial preparation of lactobacillus casei KL1 of produced bile salt hydrolase |
| CN103355406B (en) * | 2013-06-20 | 2015-08-19 | 宁波大学 | A kind of production method being rich in active polysaccharide Fermented Soybean Milk |
| US10959444B2 (en) | 2015-12-22 | 2021-03-30 | Chr. Hansen A/S | EPS gene cluster of texturizing lactic acid bacteria |
| CN107557303A (en) * | 2017-09-08 | 2018-01-09 | 天津大学 | A kind of ocean thraustochytriale Isolation and screening of bacterial strain method of high-yield extracellular polysaccharide |
| CN108004167B (en) * | 2017-12-17 | 2020-11-03 | 石家庄君乐宝乳业有限公司 | Streptococcus thermophilus JMCC0019 capable of producing exopolysaccharides, and separation and purification method and application thereof |
| CN109182186B (en) * | 2018-09-19 | 2022-02-01 | 内蒙古大学 | Lactobacillus extracellular polysaccharide and immunologic adjuvant |
| CN110699273B (en) * | 2019-07-29 | 2021-06-22 | 贵州亮欢寨生物科技有限公司 | Lactobacillus casei and application thereof |
| CN110973244B (en) * | 2019-12-17 | 2023-08-29 | 江苏省农业科学院 | Method for reducing precipitation of fermented cow whey and application thereof |
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