CN101331900A - 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 in large 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) lactic acid bacteria and functional yoghourt production technology thereof are applicable to the production of the functional yoghourt and the probiotics of norcholesterol in the fermented dairy product.
Background technology
Probio (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 ecological balance, and the control enteric infection reduces serum cholesterol level, improve the utilization of lactose intolerance people to lactose, and can stimulate specificity or nonspecific immune reaction, have the effect that immunity strengthens.The viable bacteria of probiotic composition should metabolic stability, and activity is stronger, by large number of viable behind the alimentary canal, brings into play probiotic action after entering enteron aisle.The excellent species that is used for producing probiotics at present has Lactococcus lactis lactic acid subspecies of Bifidobacterium, lactobacillus, lactococcus etc.
Just confirm that absorbing the sour milk that contains certain lactobacillus 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 the research to the African Massai human serum cholesterol of drinking fermented dairy product such as yogurt in a large number, to neonatal research, American investigation to normal drink yogurt, and, find that all lactic acid bacteria has the effect that reduces the human serum cholesterol directly to the research of yogurt etc.So far, the researcher remains in different viewpoints for the mechanism of action that lactic acid bacteria reduces cholesterol both at home and abroad, mainly concentrates on following 3 points: the direct absorption and assimilation cholesterol of (1) lactic acid bacteria somatic cells; (2) the bile salt hydrolase activity of lactic acid bacteria makes in conjunction with the attitude cholate and is degraded to the free state cholate, and latter's solubility descends and cholesterol generation co-precipitation effect; (3) other theories are as absorption and assimilation and co-precipitation synergy.Bile salt hydrolase (BSH) is the metabolite that lactic acid bacteria produces.This enzyme can will produce amino acid and the lower free state cholate of solubility in conjunction with the degraded of attitude 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 lactic acid bacteria to optimize under the growth conditions,, can secretes exocellular polysaccharide (EPS) for protective effect 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, antibiotic or poisonous substance effect, macrophage or bacteriophage etc.Since people such as nineteen eighty-two Japan scholar Shiomi report that lactic acid bacteria EPS has antitumor action, caused many scholars' attention.From the result of study of report, lactic acid bacteria EPS mainly contains the function of the following aspects: improve the fermented dairy product structural state, the syneresis 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 immune system effect; The pair cell body has protective effect; Suppress the breeding of enteron aisle spoilage organisms, prevent and treat the effect of colon cancer.Think at present that the mechanism of lactic 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) cell membrane contact inhibition.Tumor cell surface has very strong negative electrical charge, and some polysaccharide can be in conjunction with these electric charges, make cell surface by " neutralization " thus helping the cell acknowledge(ment) signal stops dividing.
Domestic research about Lactobacillus casei mainly is on the health-care efficacies such as its reducing blood lipid, anti-hypertension, treatment anaphylactia, as " the Lactobacillus casei Bd-II bacterial strain and in the application that reduces lipid aspects " of Shanghai Bright Dairy ﹠ Food Co., Ltd application, number of patent application is: CN03128995.9; Lactobacillus casei LC2W bacterial strain and the application number of patent application aspect anti-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 relevant disease, number of patent application is: CN200410038566.X.
Though abroad the research about Lactobacillus 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 lactic acid subspecies, streptococcus thermophiluses.Though the domestic patent report that " containing functional yoghourt of lactobacillus reuteri and preparation method thereof (200310112715.8) " arranged, domestic nothing utilize the lactic acid bacteria of norcholesterol 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 lactic acid bacterias with efficient norcholesterol and high yield bile salt hydrolase and exocellular polysaccharide.
Lactic acid bacteria provided by the present invention is: Lactobacillus casei (Lactobacillus casei) KL 1 bacterial strain, Lactobacillus casei (Lactobacillus casei) J1 bacterial strain, Lactococcus lactis lactic acid subspecies (Lactococcus lactissubsp.lactis) KS4, streptococcus thermophilus (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).
Lactobacillus casei (Lactobacillus casei) KL 1, Lactobacillus casei (Lactobacillus casei) J1, Lactococcus lactis lactic acid subspecies (Lactococcus lactis subsp.lactis) KS4, streptococcus thermophilus (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 calcium carbonate and natamycin the bacterium colony size 2～3mm of Lactobacillus casei (Lactobacillus casei) KL1 and J1 bacterial strain, rough surface 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 lactic acid subspecies (Lactococcus lactis subsp.lactis) KS4 and streptococcus thermophilus (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.
Said method obtains having the Lactococcus lactis lactic acid subspecies KS4 of efficient norcholesterol and high yield bile salt hydrolase and exocellular polysaccharide, streptococcus thermophilus Tx, and Lactobacillus casei KL1 and J1 belong to protection domain of the present invention.
Second purpose of the present invention provides four kinds of lactic acid bacterias and prepares the functional yoghourt production technology.
The method for preparing functional yoghourt provided by the present invention is by the Lactobacillus casei with efficient norcholesterol and high yield bile salt hydrolase and exocellular polysaccharide (Lactobacillus casei) KL1 and J1, Lactococcus lactis lactic acid subspecies (Lactococcus lactis subsp.lactis) KS4, four kinds of lactic acid bacterias of streptococcus thermophilus (Streptococcus thermophilus) Tx make.
In the described method, the technological condition for fermentation that is used to prepare functional yoghourt is: fermentation temperature is 37～43 ℃, inoculum concentration 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: fermentation temperature is 40 ℃, and the leavening inoculum concentration 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 probio that produces exocellular polysaccharide to substitute yoghourt stabilizer or thickener, can obviously improve sour milk stability and rheological behavior, can utilize the probiotic strain large-scale production functional yoghourt that produces bile salt hydrolase and exocellular polysaccharide again.
Said 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: with centrifuge remove in the cow's milk leucocyte and Other macroscopic impurity. (3) standardization: the main component index of raw milk should meet food hygiene state The standard GB 5408-85 of family. Its total solids should be not less than 11.5%, and fat content is substantially different according to product Be adjusted into 4 kinds: 3.2%, 2.5%, 1.0% and＜0.1%, can be by removing cream or adding 1%～3% Skimmed milk power or rare cream to adjust total solids or fat content. (4) preheating, batching and filtration: will Raw milk is heated to about 60 ℃, adds the sucrose of 4%～5% (w/v), filtering and impurity removing after the dissolving. (5) equal Matter: in homogenizer, under 8～10MPa pressure, the raw milk of preheating is carried out homogeneous and process. Purpose is to make breast Solidify evenly, quality is finer and smoother, level and smooth, fat globule is diminished and prevents fat floating. (6) kill Bacterium: raw milk is heated to 90 ℃, insulation 5～10min. (7) cooling: the raw milk after sterilization is rapid Be cooled to 40 ℃, wait to inoculate. (8) inoculation: the total inoculum concentration that adds single working stock culture is 4%, bacterial classification The inoculative proportion of the single fermentation agent of Tx, KS4 and KL1 (or J1) is 2: 1: 3. (9) packing: for avoiding assorted Bacterium is invaded, and the operation that is sub-packed in plastic small container should be carried out in desinfection chamber fast. (10) heat-preservation fermentation: will Plastic small container places fermenting cellar to keep 40 ℃ of fermentation temperatures. When acidified milk acidity reaches 65 ° of T, curdling is solid When proterties is good, i.e. fermenting-ripening. Fermentation time is generally 4h. (11) cooling: the container that will contain sour milk from Fermenting cellar takes out, and is cooled to rapidly below 10 ℃ with cold wind. (12) refrigeration and after-ripening: through the acid of cooling processing Milk is preserved in 0～5 ℃ of refrigerating chamber and is preserved, until drinks. Purpose is to prevent that sour milk from continuing fermentation and acid and making One-tenth pH crosses low and affects mouthfeel, and prevents the living contaminants breeding. After-ripening under the refrigerated condition is conducive to sour milk wind The formation of flavor 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 has reliable security in the Tibetan of northeast average family spirit mushroom (claiming again the Kai Feier grain), uses this The functional yoghourt curdled milk of four kinds of lactic bacteria strain productions is solid, and no whey is separated out, and 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, behaviour Do simply, low for equipment requirements, cost is lower, is suitable for suitability for industrialized production.
The specific embodiment
Experimental technique among the following embodiment if no special instructions, is conventional method.
Percentage composition among the following embodiment if no special instructions, is volumn concentration.Lactobacillus casei (Lactobacillus casei) KL1 and the J1 of embodiment 1, efficient norcholesterol and high yield bile salt hydrolase and exocellular polysaccharide, Lactococcus lactis lactic acid subspecies (Lactococcus lactissubsp.lactis) KS4, screening and the evaluation of streptococcus thermophilus (Streptococcus thermophilus) Tx
1, the screening of efficient norcholesterol and high yield bile salt hydrolase bacterial strain
Get and hide the 45mL SPSS that clever mushroom filtrate 5mL injects the band bead, 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 calcium carbonate and natamycin into, cultivate 24h for 37 ℃, picking has single bacterium colony streak inoculation of dissolving circle in the MRS slant medium, cultivates 24h in 37 ℃.Gram is looked its individual morphology and purity.Change and be inoculated in the MRS fluid nutrient medium, 37 ℃ increase bacterium and cultivated for 2～3 generations, transfer into the I culture medium that contains cholesterol with 1%～2% inoculum concentration again, behind 37 ℃ of cultivation 20h, with content of cholesterol before and after the fermentation in the OPA colorimetric method for determining I culture medium, and calculate cholesterol reduced rate (%).Determine the bacterial strain of efficient norcholesterol according to the cholesterol reduced rate.Concrete assay method: get 0.4mL and cultivate the zymotic fluid of front and back in colorimetric cylinder, add 0.2mL OPA (1mg/mL) and 4mL mixed acid (glacial acetic acid, concentrated sulfuric acid mixed in equal amounts), fully mix the back room temperature and leave standstill 10min, with the I culture medium that does not contain cholesterol is blank accent 0, measure light absorption value (A) in 550nm, on cholesterol-light absorption value calibration curve, check in fermentation front and back content of cholesterol (the results are shown in Table 1).
The different lactic bacteria strains of table 1 reduce the primary dcreening operation result of the test of cholesterol
Studies show that adopting high flux screening technology and o-phthalaldehyde method to obtain the efficient lactic acid bacteria that reduces cholesterol is KS4, J1, KL1 and Tx bacterial strain.Wherein, KS
4It is the strongest that bacterial strain reduces cholesterol ability, secondly is J1, KL1 and Tx bacterial strain.They belong to the strong bacterial strain of reduction cholesterol ability in relevant bibliographical information.
With the bacterial strain that obtains behind the primary dcreening operation with efficient norcholesterol; on the MRS culture medium flat plate that contains 0.1% cholesterol and 0.3% Bile Salts (respectively in contrast) with the MRS culture medium of the MRS culture medium 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 sediment with ninhydrin method---the amino acid existence, determine according to the bluish violet 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 lactic bacteria strain and sieves result of the test 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 percentage of the vigor of bile salt hydrolase and reduction 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 efficient lactic bacteria strain that reduces cholesterol comes from the bile salt hydrolase that it produces volume or high vigor.Can utilize the further functional yoghourt of developing efficient norcholesterol of lactic bacteria strain of these high yield bile salt hydrolases.
2, the screening of high-yield extracellular polysaccharide strains
Get and hide the 45mL SPSS that clever mushroom filtrate 5mL injects the band bead, 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 calcium carbonate and natamycin 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 ℃.Gram is looked its individual morphology and purity.Change to be inoculated in the MRS fluid nutrient medium, 37 ℃ increase bacterium and cultivated for 2～3 generations, transfer in the MRS fluid nutrient medium with 1%～2% inoculum concentration again, 37 ℃ cultivate 12h after, with the content of EPS in the phenolsulfuric acid colorimetric method for determining nutrient solution.To record sample absorbance y value substitution calibration 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 culture medium, 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 lactic 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 skimmed milk 2～3 generations of continuous passage activation, and 37 ℃ of overnight incubation to cow's milk solidify.Transfer with 1% inoculum concentration again and contain in the triangular flask of skimmed 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 leavening.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 leavening, determine the bacterial strain that fermenting property is good.Bile salt hydrolase, exocellular polysaccharide and the good MRS inclined-plane bacterial strain of fermenting property are produced in screening, be inoculated in various physics and chemistry and identify that 37 ℃ of cultivations are identified in the culture medium.
The fermenting property test result of table 4 screening lactic bacteria 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 fermenting property.It is the highest wherein to prepare the viscosity of leavening 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 lactic bacteria strains that produce EPS further to develop functional yoghourt with good stability.
4, the evaluation of efficient norcholesterol 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), different temperatures 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 Lactobacillus casei (Lactobacillus casei), bacterial strain KS4 is Lactococcus lactis lactic acid subspecies (Lactococcus lactis subsp.lactis), and bacterial strain Tx is streptococcus thermophilus (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 norcholesterol and high yield bile salt hydrolase and extracellular polysaccharide strains to produce functional yoghourt
The used Yoghourt Production special bacteria of the present invention is Lactobacillus casei (Lactobacillus casei) KL1 and J1, Lactococcus lactis lactic acid subspecies (Lactococcus lactis subsp.lactis) KS4, streptococcus thermophilus (Streptococcus thermophilus) Tx.They are the functional bacterial classification of high yield bile salt hydrolase and exocellular polysaccharide.Wherein Lactobacillus casei KL1 and J1 bacterial strain stomach juice-resistant, anti-bile salt ability are stronger, have the contrary environmental characteristics of anti-alimentary tract, can arrive the intestinal secretion bile salt hydrolase smoothly, will produce amino acid and the lower free state cholate of solubility in conjunction with the degraded of attitude cholate.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 test 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 Lactobacillus casei (Lactobacillus casei) KL1 can arrive the efficient effect that reduces serum cholesterol of small intestine performance by the contrary environment of alimentary canal 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 skimmed milk test tube of packing into, through 0.07Mpa sterilization 20min, be cooled to 37 ℃, the Lactobacillus casei KL1 or the J1 of bile salt hydrolase and exocellular polysaccharide, Lactococcus lactis lactic acid subspecies KS4, streptococcus thermophilus Tx strain excellent are produced in inoculation respectively, 37 ℃ are cultured to curdling admittedly, make leavening with 2% inoculum concentration again, it is solid to be cultured to curdling under 37 ℃, is the single culture leavening.
2, functional yoghourt production Optimizing Conditions of Fermentation
Carry out three factors, three level [L according to influencing the bigger fermentation temperature of functional yoghourt quality, inoculative proportion and inoculum concentration
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 result of the test.
Table 6 functional yoghourt optimization of fermentation conditions Orthogonal Experiment and Design factor level 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: fermentation temperature is 40 ℃, the leavening inoculum concentration 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 lactic 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 reduced levels, 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 probio that produces exocellular polysaccharide to substitute yoghourt stabilizer or thickener, can obviously improve sour milk stability and rheological behavior, can utilize the probiotic strain large-scale production functional yoghourt that produces bile salt hydrolase and exocellular polysaccharide again.
Project under this patent: Beijing Education Commission agricultural open problem " bacterial screening of Kai Feier grain and the application study in functional fermentation cow's milk thereof " of Beijing key lab that uses of the new technology
Bullets: KF2003-06
The project beginning and ending time: 2004.01-2006.12
Project leader: Liu Hui
Claims (3)
1, four kinds of lactic acid bacteria and functional yoghourt production technologies thereof of producing bile salt hydrolase and exocellular polysaccharide, its technical characterictic is: screening obtains the lactic acid bacteria of four kinds of high yield bile salt hydrolases (BSH) and exocellular polysaccharide (EPS) from hide clever mushroom, and they all have efficient reduction cholesterol and excellent fermentation performance.Four kinds of lactic acid bacterias that utilization has efficient reduction cholesterol and high yield bile salt hydrolase and exocellular polysaccharide make colory sour milk, both had the function that reduces serum cholesterol, the exocellular polysaccharide that can utilize thalline to produce again substitutes stabilizing agent, can obviously improve the stability of functional yoghourt.
2, according to claim 1ly be used to produce four kinds of genus lactubacillus of functional yoghourt in the scope of protection of the invention with efficient reduction cholesterol and high yield bile salt hydrolase and exocellular polysaccharide.The classification name of these four kinds of lactic acid bacterias is: bacterial strain KL1 is a Lactobacillus casei (Lactobacillus casei) (CGMCC № 1809), bacterial strain J 1 is a Lactobacillus casei (Lactobacillus casei) (CGMCC № 1808), bacterial strain KS4 is Lactococcus lactis lactic acid subspecies (Lactococcus lactis subsp.lactis) (CGMCC № 1807), and bacterial strain Tx is a streptococcus thermophilus (Streptococcus thermophilus) (CGMCC № 1810).
3, the best technological condition for fermentation that four kinds of lactic acid bacterias with efficient reduction cholesterol and high yield bile salt hydrolase and exocellular polysaccharide prepare colory functional yoghourt and products thereof that utilizes according to claim 1 also belongs to the scope of protection of the invention with special preparing strain.Promptly utilize Lactobacillus casei (Lactobacilluscasei) KL1 and J1, Lactococcus lactis lactic acid subspecies (Lactococcus lactis subsp.lactis) KS4, the best technological condition for fermentation that four kinds of lactic acid bacterias of streptococcus thermophilus (Streptococcus thermophilus) Tx prepare functional yoghourt is: fermentation temperature is 40 ℃, the leavening inoculum concentration is 4%, the inoculative proportion of Tx/S4/KL1 (J1) is 2: 1: 3, fermentation time is 4.0h, and the sucrose addition is 4%～5% (w/v).
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