CN104489085A - Fermented milk rich in beta-glucan and preparation process thereof - Google Patents
Fermented milk rich in beta-glucan and preparation process thereof Download PDFInfo
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Abstract
The invention discloses fermented milk rich in beta-glucan and a preparation process thereof and belongs to the technical field of bio-fermentation. The fermented milk is prepared by process steps of fermenting, cooling, after-ripening and the like by taking a mutation-bred beta-glucan high-producing strain streptococcus thermophilus and lactobacillus bulgaricus as a fermenting strain. The yield of the fermented milk rich in beta-glucan disclosed by the invention reaches 268 microgram/ml, and the content of beta-glucan in the fermented milk prepared by the original strain is 238 microgram/ml which is improved by 12.2% compared with that of the original strain-yielded beta-glucan. Moreover, the flavor is not affected.
Description
Technical field
The present invention relates to technical field of biological fermentation, particularly relate to a kind of biofermentation with lactic acid bacteria for Dairy fermentation.
Background technology
Along with lactic acid bacteria is revealed to the mechanism of action that health is useful, latic acid-fermented food is subject to people's attention day by day.And the lactic acid bacteria for making fermented food can form exocellular polysaccharide in growth metabolism process, just containing beta glucan in these exocellular polysaccharides, beta glucan is raw-food material low in calories, not easily digested and assimilated by people after edible, the increase of Glucose in Blood by Cyclic can be reduced, diabetes and obesity controlling disease, diabetes can be prevented, reduce body weight, prevent the generation of angiocardiopathy.Because beta glucan has the character of imbibition, under one's belt can imbibition after people eats, make people produce satiety, delay the speed entering small intestine from stomach.The beta glucan entering enteron aisle only has 25% digested absorption, and all the other 60% continuation are moved to intestines bottom, promote intestines peristalsis, in addition in colon osmosis, prevent constipation, shorten the time of discarded object by enteron aisle, decrease carcinogenic substance in intestines and, to the pollution of intestinal tube, reach the effect of anti-cancer.The beta glucan content utilizing lactobacillus-fermented to produce in most of fermented dairy products in current China market is lower, the high yield beta glucan that screening energy orientation fermentation beta glucan content is higher produces bacterial strain to raising people quality of life, and the exploitation carrying out functional lactate goods is significant.
Summary of the invention
The object of the invention is the deficiency overcoming existing bacterial classification, a kind of acidified milk prepared with lactobacillus bulgaricus (Lb) by the beta glucan superior strain streptococcus thermophilus (St) of mutagenic and breeding is provided, in gained acidified milk, is rich in a large amount of beta glucan.
A kind of acidified milk being rich in beta glucan of the present invention
,obtain through the beta glucan superior strain streptococcus thermophilus of mutagenic and breeding and bulgaria lactobacillus fermentation, specifically obtained by following preparation technology:
(1) the beta glucan superior strain streptococcus thermophilus of mutagenic and breeding is mixed with both lactobacillus bulgaricus, through overactivation, spread cultivation, be prepared into leavening, for subsequent use;
(2) whole-fat milk powder is modulated into 12% reconstituted milk, then adds sucrose, after dissolving at 90 DEG C-95 DEG C sterilization 5min, then be cooled to 45 DEG C;
(3) leavening prepared by step (1) is inoculated in the reconstituted milk of cooling in step (2), carries out fermented and cultured;
(4) eventually pass cooling, after-ripening, obtained finished product, refrigerates, to obtain final product at 4 DEG C;
In described step (1), the beta glucan superior strain streptococcus thermophilus of lactobacillus bulgaricus and mutagenic and breeding mixes with the bacterial classification ratio of 1:3,1:2,1:1,2:1 or 3:1;
In described step (2), sucrose addition mass percent is 1%, 3%, 5%, 6%, 7% or 9%;
In described step (3), strain inoculation amount is 2%, 4%, 5%, 6%, 8% or 10%;
Fermentation temperature in described step (3) is 37 DEG C, 39 DEG C, 41 DEG C, 43 DEG C or 45 DEG C.
In described step (1), the bacterial classification mixed proportion of the beta glucan superior strain streptococcus thermophilus of lactobacillus bulgaricus and mutagenic and breeding is 2:1.
In described step (2), sucrose addition is 6%.
In described step (3), strain inoculation amount is 5%.
Fermentation temperature in described step (3) is 41 DEG C.
The preparation method of the beta glucan superior strain streptococcus thermophilus of described mutagenic and breeding is as follows:
The streptococcus thermophilus selected is cultivated as starting strain using utilizing lactic acid bacteria, cultivated by the selection of different culture media and purifying is carried out to it, performance test is carried out to the bacterial strain that purifying obtains simultaneously, the good bacterial strain of selectivity makes unicellular thallus suspension, carries out suspension colony counting by slat chain conveyor; Utilize ultraviolet mutagenesis and nitrosoguanidine mutagenesis, mutagens process is carried out to thallus suspension liquid, utilize lactic acid bacteria to cultivate and colony counting is carried out to the thallus suspension liquid after mutagens process, calculate fatal rate; Utilize middle cultivation and dull and stereotyped coating to carry out mutant strain to be separated, isolated bacterial strain is screened further, first carries out primary dcreening operation, multiple sieve is carried out to the bacterial strain filtered out, genetic stability is carried out after measured to the bacterial strain sifted out again, to obtain final product.
The wavelength that described ultraviolet mutagenesis uses the ultraviolet mutagenesis lamp of manual manufacture to send is the ultraviolet of 253.7 nm, and mutation time is 120 seconds.
Described NTG mutant treatment dosage is 0.3mg/ml, and mutation time is 50 minutes.
The preparation method of the acidified milk of beta glucan is rich in the present invention, specifically comprises following processing step:
(1) the beta glucan superior strain streptococcus thermophilus of mutagenic and breeding is mixed with both lactobacillus bulgaricus, through overactivation, spread cultivation, be prepared into leavening, for subsequent use;
(2) whole-fat milk powder is modulated into 12% reconstituted milk, then adds sucrose, after dissolving at 90 DEG C-95 DEG C sterilization 5min, then be cooled to 45 DEG C;
(3) leavening prepared by step (1) is inoculated in the reconstituted milk of cooling in step (2), carries out fermented and cultured;
(4) eventually pass cooling, after-ripening, obtained finished product, refrigerates, to obtain final product at 4 DEG C;
In described step (1), the beta glucan superior strain streptococcus thermophilus of lactobacillus bulgaricus and mutagenic and breeding mixes with the bacterial classification ratio of 1:3,1:2,1:1,2:1 or 3:1;
In described step (2), sucrose addition mass percent is 1%, 3%, 5%, 6%, 7% or 9%;
In described step (3), strain inoculation amount is 2%, 4%, 5%, 6%, 8% or 10%;
Fermentation temperature in described step (3) is 37 DEG C, 39 DEG C, 41 DEG C, 43 DEG C or 45 DEG C.
The preparation method of beta glucan superior strain streptococcus thermophilus (St) and check specific as follows.
(1) determination of starting strain
1, because Bromocresol purple is in acid condition in yellow, in the basic conditions in purple, this experiment is 6.5 with the pH of MRS culture medium, therefore in purple after adding Bromocresol purple, lactic acid bacteria (thermophilus bacterial strain) is cultivated in this culture medium, because decomposition glucose produces lactic acid, make bacterium colony be yellow, the culture medium of periphery of bacterial colonies also becomes yellow.Illustrate if flat board becomes yellow this bacterial strain of explanation from original purple can produce acid.
2, in culture medium described in 1, CaCO is added
3, the lactic acid that lactic acid bacteria produces can dissolve the CaCO in culture medium
3, form obvious transparent circle in periphery of bacterial colonies, both easily identified, again by the size of transparent circle diameter, semi-quantitatively can judge the height of this bacterial strain acid producing ability, the bacterial strain transparent circle producing acid amount high is large, and the transparent circle yielded poorly is little.
3, by described in 1,2, bromocresol purple-CaCO is selected in test
3plating medium carries out the Isolation and screening of bacterial strain.
4, degreasing milk medium: formula: skimmed milk powder 12g, distilled water 100mL, 121 DEG C, autoclaving 15min.Containing a large amount of compound nitrogen source, lactose (4.8%), mineral matter (0.5%) and enzyme in skimmed milk, be the good substrates lactic acid bacteria activation of lactobacter growth, fermentation medium.
5, lactic acid bacteria (streptococcus thermophilus) is accessed in MRS fluid nutrient medium activate three generations, then carry out gradient dilution.Bromocresol purple described in 3-calcium carbonate plating medium carries out coating be separated, cultivate after 2 days for 37 DEG C, select transparent circle and the large single bacterium colony of variable color circle, be inoculated in degreasing milk medium described in 4, cultivate 2 days for 37 DEG C.
6, select the good bacterial strain of curdled milk described in 5 to carry out Congo red detection, determine that beta glucan output is high and produce the starting strain of the stable bacterial strain of acid as mutagenesis.Method is: by the fermentation broth agitation of heat sterilization, then leaves standstill and makes protein denaturation precipitation.Centrifugal 10min under 5000r/min condition, add Powdered Activated Carbon 1.5%, abundant stirring, 30-40min is left standstill with adsorbing contaminant at 50 DEG C, active carbon is removed afterwards by vacuum filtration method, then adopt freeze crystallization to remove most of calcium lactate, according to the calcium content that EDTA-Ca salt method measures, add quantitative Na
2cO
3generate CaCO
3pelleting centrifugation removing calcium ion, obtains lactic fermentation extract.Get 4 brace plug test tubes, at room temperature add the phosphate buffer of 3ml0.2mol/LPH8 successively, the Congo red solution 0.1ml of 25mg/L, wherein 3 test tubes add the zymotic fluid that 0.4mlPH is adjusted to 8 successively, and remaining volume deionized water polishing is to 4ml, directly use deionized water polishing to 4ml for other 1, mixing, reacts 15min under 20 DEG C of conditions, surveys absorbance at 550nm wavelength place, average, look into the content that calibration curve obtains actual beta glucan in zymotic fluid.
(2) ultraviolet (UV) mutagenesis
1, the determination of UV mutagenesis starting strain exponential phase
By bromocresol purple-CaCO
3plate screening starting strain is out inoculated in MRS test tube fluid nutrient medium, and constant temperature 37 DEG C of Secondary Culture activate, and adjustment viable count is 10
8cfu/mL, the inoculum concentration by 3% is inoculated in the triangular flask that MRS fluid nutrient medium is housed, 37 DEG C of constant temperature culture.Getting zymotic fluid at interval of 2h, measure its OD value under 600nm wavelength, is abscissa with time, with OD
600be worth for ordinate mapping, the exponential growing curve (each measured value does three repetitions, gets its mean value) of starting strain can be obtained.The bacterial strain choosing the logarithmic growth middle and later periods prepares single-cell suspension liquid.
2, the preparation of single-cell suspension liquid
The starting strain zymocyte liquid 4000rpm of exponential phase will be cultured to, 5min is centrifugal obtains thalline, abandoning supernatant washes twice, suitably dilute with physiological saline again, shake 20min in the triangular flask of bead and be namely prepared into cell suspending liquid, stepwise dilution also carries out colony counting on MRS flat board.General mutagenic treatment bacterium, adjusting its vegetative cell concentration is 10
7-10
8cfu/mL.
3, UV mutagenic treatment
Mutagenic processes should be carried out in darkroom, and illumination adopts red light bubble.Ultraviolet wavelength is 136-390nm, and its medium wavelength is that the ultraviolet sterilization ability of 260 nm is the strongest.The ultraviolet wavelength that the ultraviolet mutagenesis lamp of manual manufacture sends is 253.7 nm, and not only sterilizing ability is strong for it, and stable, and Mutagenic Effect is good.
Get the sterilized petri dishes that 5-10mL thallus suspension liquid is placed in Φ 90, sterilizing pin one piece is put in plate central authorities, opens uviol lamp (20W, irradiation distance 30cm) preheating 20-30min, and light wave is stablized.Open magnetic stirring apparatus, then open ware lid, irradiate 30s respectively, 60s, 90s, 120s, 150s, 180s and 210s.Comparing with the bacterium liquid without ultraviolet irradiation, getting ultraviolet irradiation and undosed bacterium liquid 1mL respectively, through suitably diluting separation.Bacterium liquid after mutagenic treatment, to be immersed in ice-water bath 37 DEG C of constant temperature culture 2h after 2-3h.Getting 0.1mL bacterium liquid is coated with dull and stereotyped, often organizes three plates, and flat board black cloth or brown paper parcel are placed in insulating box and cultivate.
4, the calculating of fatal rate
Count after the bacterium liquid of process to be mutagenic grows bacterium colony, get the mean value of three plates, compare with the bacterium liquid without ultraviolet irradiation, according to following formulae discovery fatal rate.
According to the fatal rate curve of starting strain after Ultraviolet radiation measured, determine that best mutation time is 120 seconds, ultraviolet fatal rate, between 80%-90%, can ensure that farthest sudden change occurs thalline.
5, the screening of UV mutagenic mutant
Be 10 by vegetative cell concentration
7-10
8cfu/mL carries out ultraviolet mutagenesis to starting strain, the screening of beta glucan enhanced variant is carried out after mutagenesis, by inoculation in above-mentioned degreasing milk medium, carry out 41-43 DEG C, 4-6h cultivates, select the good bacterial strain of curdled milk to do congo red method and detect beta glucan output, select the highest bacterial strain of beta glucan output as the bacterial strain continuing mutagenic treatment.
(3) nitrosoguanidine (NTG) mutagenesis
1, the determination of NTG mutagenesis starting strain exponential phase
By Uv-induced screening inoculation out in MRS fluid nutrient medium, draw the growth curve of NTG mutagenesis starting strain, the bacterial strain choosing the logarithmic growth middle and later periods prepares single-cell suspension liquid.
2, the preparation of single-cell suspension liquid
The NTG mutagenesis starting strain zymocyte liquid 4000rpm of exponential phase will be cultured to, 5min is centrifugal obtains thalline, abandoning supernatant washes twice, phosphate buffer (pH=8) is used to dilute again, shake 20min in the triangular flask of bead and be namely prepared into cell suspending liquid, stepwise dilution also carries out colony counting on MRS flat board, and adjusting its vegetative cell concentration is 10
7-10
8cfu/mL.
3, the determination of NTG mutagenic treatment time
Test selects the processing time to be respectively 30 min, 40 min, 50 min and 60 min, with 0.3mg/mLNTG process thallus suspension liquid.Bacterium liquid after mutagenic treatment, is immersed in 2-3h in ice-water bath, then at 37 DEG C of constant temperature culture 2h.Getting 0.1mL is coated with dull and stereotyped, and often organize three plates, flat board is placed in insulating box 37 DEG C of constant temperature culture 2h.Count after growing bacterium colony, get the mean value of three plates, calculate fatal rate, determine that the Best Times of NTG mutagenic treatment is 50 minutes according to fatal rate.
4, the determination of NTG mutagenic treatment dosage
Accurately take NTG 2 mg respectively, 4 mg, 6 mg, 8 mg, 10mgNTG and acetone dissolve in the ratio of 10mg:1mL.NTG test solution is moved in 20mL thallus suspension liquid respectively, concentration is respectively: 0.1,0.2,0.3,0.4 and 0.5mg/mL.With the NTG best mutagenic treatment time, at 37 DEG C, concussion process on the shaking table of 100 rpm, the bacterium liquid after mutagenic treatment, is immersed in 2 ~ 3h in ice-water bath, then 37 DEG C of constant temperature culture 2h.Get 0.1mL spread plate, often organize three plates, flat board is placed in insulating box and cultivates.Count after growing bacterium colony, get the mean value of three plates, calculate fatal rate, determine that NTG mutagenic treatment optimal dose is 0.3mg/ml according to fatal rate.
5, the screening of NTG mutagenic mutant
According to best NTG mutant treatment time and dosage, nitrosoguanidine mutagenesis is carried out to starting strain, the screening of beta glucan high productive mutant is carried out after mutagenesis, by inoculation in above-mentioned degreasing milk medium, carry out 41-43 DEG C, 4-6h cultivates, select the good bacterial strain of curdled milk to do congo red method and detect beta glucan output, select the highest bacterial strain of beta glucan output as the bacterial strain continuing mutagenic treatment.
(4) ultraviolet and nitrosoguanidine complex mutation
1, the determination of complex mutation starting strain exponential phase
The inoculation screened by nitrosoguanidine mutagenesis is in MRS fluid nutrient medium, and draw the growth curve of complex mutation starting strain, the bacterial strain choosing the logarithmic growth middle and later periods prepares single-cell suspension liquid.
2, multiple mutated
With the best Induced dosage of both ultraviolet and nitrosoguanidine, mutagenesis is carried out to thallus suspension liquid.Thallus suspension liquid through the best Induced dosage process of NTG is proceeded to plate and keeps sample path length to be less than 2mm, aseptically, irradiates at 30cm place, below with 20W uviol lamp.Bacterium liquid after mutagenic treatment suitably dilutes, and is immersed in ice-water bath after 2-3h, 37 DEG C of constant temperature culture 2h.Then MRS solid medium is coated with dull and stereotyped, flat board black cloth or brown paper parcel are placed in 37 DEG C of insulating boxs to be cultivated, after growing bacterium colony, by colony inoculation in above-mentioned degreasing milk medium, carry out 41-43 DEG C, 4-6h cultivates, and select the good bacterial strain of curdled milk to do congo red method and detect beta glucan output, the higher bacterial strain of beta glucan level is produced in screening.
3, mutagenesis repeatedly
After first round multiple mutated, the higher bacterial strain of beta glucan level takes turns mutagenesis starting strain as second is produced in screening, with the best Induced dosage of both ultraviolet and nitrosoguanidine, second is carried out to thallus suspension liquid and take turns complex mutation, repeat the operation of 2, the production performance of mutagenic strain between more different round, filters out Optimal Production bacterial strain.
(5) high yield beta glucan produces the screening of bacterial strain
Test adopts primary dcreening operation and sieves the screening in two stages again, and primary dcreening operation, based on amount, namely selects the bacterium colony that mutagenic treatment rear plate is separated in a large number, expands screening scope as far as possible; Multiple sieve, based on matter and essence, namely a small amount of strain excellent obtained through primary dcreening operation, filters out optimum bacterial strain further.
1, primary dcreening operation
Bacterium liquid through mutagenesis is coated on the MRS flat board that sucrose is carbon source successively, filters out the bacterial strain of growth; Then at maltose be carbon source MRS flat board on be coated with, screen the bacterial strain do not grown on maltose plates, namely filter out the bacterial strain of lactic acid bacteria; To the bacterial strain screened, at sucrose be repeatedly carbon source MRS flat board on go down to posterity, purifying bacterial strain also strengthens Mutagenic Effect.
2, multiple sieve
The bacterial strain that primary dcreening operation obtains is numbered in order, then adjusts viable count and be about 108cfu/mL, proceed to degreasing milk medium with 3% inoculum concentration, 41-43 DEG C of constant temperature culture, observe the curdled milk effect of skimmed milk, directly eliminate the bacterial strain of not curdled milk.The good bacterial strain of vigor is gone down to posterity 3 times (carrying out skimmed milk powder medium culture), carries out beta glucan determination of yield one by one.
(6) mensuration of genetic stability
The genetic stability of the beta glucan superior strain screened after investigating complex mutation by the method for thalline continuous passage.By this bacterial strain low-temperature preservation in 4 DEG C of refrigerators, go down to posterity continuously 6 times in degreasing milk medium, per in generation, does 5 Duplicate Samples, measures beta glucan output.
(7) the comparing of superior strain and starting strain
Adjust the viable count of superior strain and starting strain respectively to about 10
8cfu/mL, is then seeded to degreasing milk medium with the inoculum concentration of 3%, 41 DEG C of ferment at constant temperature, measures beta glucan output respectively every 1h sampling, produces beta glucan situation before and after contrast strain mutagenesis.
(8) comparison of fermenting property
Under the same conditions, respectively with 3% inoculum concentration, the 41-43 DEG C of test carrying out superior strain and starting strain fermented skim milk, compares acidity, viscosity, whey amount of precipitation, viable count and the structural state when curdled milk time of bacterial strain, curdled milk.
(9) Preliminary Identification of superior strain
According to " uncle Jie Shi Bacteria Identification handbook " the 8th edition about the authentication method of bacterial classification, by cultivating the bacterial strain of mutagenic and breeding, observing its bacterium colony and morphological features, investigating its fermentation character and physiological and biochemical property, determine its kind.
1, morphological observation
10 are carried out after being activated by superior strain
1-10
580% normal saline dilution is separated, and is coated on MRS solid plate, cultivates 48h for 37 DEG C, observes colony characteristics.The typical bacterium colony of picking carries out smear, carries out Gram's staining, examines under a microscope thalli morphology.Lactic acid bacteria to be identified is adopted semi-solid percutaneous puncture-inoculation, observes its motion conditions.
2, physio-biochemical characteristics qualification
1. Gram's staining
Get that to cultivate 1d bacterium liquid a small amount of, through smear on slide with after fixing, Gram's staining, first just contaminates by oxalic acid ammonia violet staining, 1min after washing, then carries out mordant dyeing with dye liquor, 1min after washing, blots.Use 95% ethanol decolorization again, until the alcohol dripped is not in purple, the general time is 30s, washes, blots.Finally with 0.5% sarranine dyeing liquor dyeing 10-30s, washing, dry, microscopy.Observe coloration result, it is gram-positive bacteria that thalline is dyed to purple.
2. catalase test
The H of 3%-15% is dripped on the bacterium colony being grown on agar medium
2o
2, whether observe and have bubble to produce, if bubble-free, be negative catalase.
3. reduction test of Meilan
In 10mL milk sample, add 1mL methylene blue, 35-40 DEG C of water-bath, observe the change of color, curdled milk.
4. litmus milk test
Add the reindeer moss solution that 4mL concentration is 25g/L in 100mL skimmed milk, after mixing, color is with lilac purple for appropriateness, and packing test tube, the fresh bacterial classification of 1% inoculation, thermophilic is cultivated 1 ~ 3d observation litmus milk and produced acid and Hirschfeld-Klinger reaction.Continue to cultivate 7-14d observation milk and peptonize result.
5. gelatin liquefaction test
Peptone water medium packing test tube 10mL, often pipe adds gelatin 0.6g, 115 DEG C of autoclaving 15-20min, and after cooling, puncture method is inoculated, and thermophilic is cultivated, and two nonvaccinated test-tube culture mediums need be had during cultivation to compare.Observed result in ice bath, as control tube solidify time, inoculated tube liquefaction is for positive reaction, and solidifying simultaneously or liquefy is negative findings.
6. voges-Proskauer test (V-P test)
Bacterium is activated is inoculated in peptone water medium, and after 37 DEG C of cultivation 1-2d, get the KOH that 1mL culture adds equivalent 40%, a small amount of creatine, be incubated accelerated reaction in vibration 30min or boiling water bath, aobvious redness is positive.
7. hydrogen sulfide production test
Bacterium can be decomposed sulfur-containing amino acid (cystine, cysteine) and be produced hydrogen sulfide, if add heavy metallic salt in protein culture medium, inoculated bacteria is observed after cultivating, if produce hydrogen sulfide, then occurs vulcanized lead or the iron sulfide of black.
Get H
2s test culture medium, with puncture method inoculation, is placed in 37 DEG C and cultivates 24h, observe and produce with or without black precipitate.
8. nitrate reduction test
Activated being inoculated in nitrate fluid nutrient medium of bacterial classification (does the pipe do not inoculated to compare), cultivates 1-2d for 37 DEG C.Get nutrient solution a little in colorimetric disc, get the contrast nutrient solution do not inoculated simultaneously, wherein respectively each reagent A liquid and B liquid, when nutrient solution become pink, rose, orange or brown time be that nitrate reduction is positive.
9. indole test is produced
I.e. indole test.Bacterial strain is activated to be inoculated in containing in protein culture medium, and thermophilic slowly adds several indole reagent along test tube wall, forms red person be the positive at liquid layer interface after cultivating 1-2d, is then feminine gender as being still yellow.
10. casein hydrolysis experiment
Added by 5g skimmed milk powder in 50mL distilled water, claim 1.2g agar to be dissolved in 50mL distilled water, two liquid separate sterilizing, in time being chilled to 45-50 DEG C, two liquid mixings are down flat plate milk dull and stereotyped.The activated dibbling of bacterial classification, on flat board, cultivates 1-5d for 37 DEG C, observe periphery of bacterial colonies and below casein oneself is decomposed and transparent.What periphery of bacterial colonies had transparent circle is the casein hydrolysis positive.
3, carbohydrate fermentation test
Carbohydrate fermentation test is the most frequently used biochemical reaction, most microorganism can utilize carbohydrate, but the utilization of different microorganisms to different carbohydrate is selectable, and capacity of decomposition is also different, some microorganism can make sugar decomposition produce sour aerogenesis, what have can only produce acid and not aerogenesis, produces acid and whether can be changed by color and judges.
Be connected to by bacterial strain in MRS fluid nutrient medium, after 37 DEG C of activation culture, adopt biochemical identification pipe to carry out fermentation test, study the utilization power of bacterial strain 16 kinds of carbohydrate, bacterial strain only produces acid not aerogenesis.
Above bio-chemical characteristics and carbohydrate fermentation test are all with blank cultures in contrast.
The acidified milk beta glucan output that the present invention selects the beta glucan superior strain streptococcus thermophilus (St) of mutagenic and breeding and lactobacillus bulgaricus (Lb) to prepare reaches 268 μ g/ml, the acidified milk beta glucan content 238 μ g/ml that former bacterial strain makes, produce beta glucan ability than former bacterial strain and improve 12.2%, and local flavor does not change.
Accompanying drawing explanation
Fig. 1 is preparation technology's flow chart of acidified milk;
Fig. 2 is the mutagenesis screening flow chart of beta glucan superior strain streptococcus thermophilus;
Fig. 3 is the mutagenesis pedigree chart of test lactic acid bacteria strains;
Fig. 4 is the primary dcreening operation flow process that high yield beta glucan produces bacterial strain;
Fig. 5 is the multiple sieve flow process that high yield beta glucan produces bacterial strain;
Fig. 6 is the calibration curve of glucose.
Detailed description of the invention
The present invention is further illustrated below by embodiment.Embodiments of the invention are only used for the present invention is described, instead of limitation of the present invention, under concept thereof of the present invention, all belong to the scope of protection of present invention to the simple modifications of preparation method of the present invention.
embodiment 1 is rich in the preparation of the acidified milk of beta glucan
(1) by the beta glucan superior strain streptococcus thermophilus (St) of mutagenic and breeding and lactobacillus bulgaricus (Lb), the two mixes with the ratio of 2:1, through overactivation, spreads cultivation, is prepared into leavening, for subsequent use;
(2) whole-fat milk powder is modulated into 12% reconstituted milk, then adds sucrose 6%, after dissolving at 90 DEG C-95 DEG C sterilization 5min, then be cooled to 45 DEG C;
(3) be inoculated into by leavening prepared by step () in the reconstituted milk of cooling in step (two), inoculum concentration is 5%, at 41 DEG C, carry out fermented and cultured;
(4) eventually pass cooling, after-ripening, obtained finished product, refrigerates at 4 DEG C.
embodiment 2 finished product detection
1. acidified milk organoleptic indicator detects
With reference to GB19302-2010, the subjective appreciation method of employing is as follows: evaluate marking by 10 people, and getting its mean value is sensory evaluation scores.Standards of grading are in Table 2-3.
Table 1 sensory evaluation scores standard
Standards of grading are as follows:
Color: milky or micro-yellow, 20 points; Yellow, 10 points; Yellowish-brown, 5 points.
Structural state: grumeleuse uniformity, high resilience, the phenomenons such as surface is flawless, bubble-free, have thickness sense after provoking with glass bar, no whey is separated out, and tilt container curdled milk does not flow gently, 20 points; Grumeleuse uniform and smooth, high resilience, have a small amount of whey to separate out, tilt container curdled milk slightly flows, 15 points; Grumeleuse is even, and flexible, slightly chap in surface, has whey to separate out, and a large amount of curdled milk of tilt container flows out, 10 points; Grumeleuse slightly vibrates and all scatters, 5 points.
Smell: have the smell that Yoghourt is intrinsic, without other peculiar smell, gives off a strong fragrance, 20 points; There is the intrinsic fragrance of Yoghourt, but fragrance is lighter, 15 points; Slightly Yoghourt fragrance, without other peculiar smell, 10 points; Slightly other smells, but can accept, 5 points.
Mouthfeel: there is fine and smooth smooth mouthfeel, long times of aftertaste, 20 points; Delicate mouthfeel, little have granular sensation, long times of aftertaste, 15 points; Delicate mouthfeel is smooth, and aftertaste is not enough, 10 points; Delicate mouthfeel, fragrance is written in water, slightly puckery mouth after drink, 5 points.
Flavour: there is the intrinsic fermented flavour of Yoghourt, without peculiar smell such as stale flavor, bitter taste, mould ferment tastes, sour-sweet moderate, 20 points; There is the flavour that Yoghourt is intrinsic, slightly meta-acid acid or partially sweet, free from extraneous odour, 15 points; There is the intrinsic flavour of lighter Yoghourt, free from extraneous odour, 10 points; There is the intrinsic flavour of Yoghourt, simultaneously with other lighter flavours, but can accept, 5 points.
2. Yoghourt physical and chemical index detects
I, acidity: detect with dairy products acidity assaying according to GB5413.34-2010 breast.
Take 10g(and be accurate to 0.001g) style that mixed, being placed in 150ml conical flask, adding 20ml and newly boil the water being cooled to room temperature, mixing, is terminal with the constant-current titration of 0.1000mol/l standard solution of sodium hydroxide to PH8.3; Or in dissolving the instructions phenolphthalein solution adding 0.5% of 2.0ml in the sample after mixing, after mixing, be titrated to blush with standard solution of sodium hydroxide, and colour-fast in 30s, the Standard Volumetric Solutions for Sodium Hydroxide milliliter number that record consumes.
Acidity (° T)=(molar concentration × 100 of the NaOH solution milliliter number × standard solution of sodium hydroxide consumed during titration)/(quality grams × 0.1 of sample)
(note: 0.1 is the molar concentration of acidity theoretical definition NaOH)
II, fat: according to the mensuration of fat in GB5413.3-2010 infant food dairy products.
The ρ of 10ml is first added in lid Bo Shi butyrometer
20about 1.84g/L sulfuric acid, carefully 10.75ml sample is accurately added again along tube wall, sample is not mixed with concentrated acid, then 1ml isoamyl alcohol is added, rubber stopper beyond the Great Wall, make bottleneck downward, wrap up in case go out with cloth simultaneously, make in even brown liquid with forced oscillation, leave standstill several minutes (bottleneck is downward), put 5min in 65 DEG C of-70 DEG C of water-baths, take out and be placed on the centrifugal 5min of the rotating speed of 1100 revs/min in butterfat centrifuge, then be placed in 65 DEG C-70 DEG C water-bath 5min(and notice that the water-bath water surface should higher than butyrometer fat deposit).Take out, reading immediately, be the percentage of fat.
III, protein: measure according to GB/T 5009.5-2010.
Protein in food is decomposed under catalysis heating condition, and the ammonia of generation is combined with sulfuric acid and generates ammonium sulfate.Alkalization distillation makes ammonia dissociate, and with sulfuric acid or Hydrochloric Standard Titration titration after absorbing with boric acid, the consumption according to acid is multiplied by conversion coefficient, is the content of protein.
IV, non-fat solid: measure according to GB5413.39-2010.
In flat ware box, add 20g quartz sand or extra large sand, dry 2h in the drying box of 100 DEG C ± 2 DEG C, in drier cooling 0.5h, weigh, and be repeatedly dried to constant weight.Taking 5.0g(and be accurate to 0.0001g) sample is in the ware of constant weight, put evaporate to dryness in water-bath, that wipes outside ware is water stain, dry 3h in 100 DEG C ± 2 DEG C drying boxes, taking-up is put into drier and is cooled 0.5h, weigh, then in 100 DEG C ± 2 DEG C drying boxes dry 1h, weigh after taking out cooling, to front and back, twice mass difference is no more than 1.0mg.
The content of total solid={ quality of (ware box, extra large sand add the quality of quality-ware box after samples dried, extra large sand) × 100}/sample
The content of the content-sucrose of the content-fat of the content=total solid of non-fat solid
3. acidified milk microbiological indicator
I, lactic acid bacteria inspection: measure according to GB4789.35-2010.
II, Escherichia coli: measure according to GB4789.3-2010 colony counting method;
III, staphylococcus aureus: according to GB4789.10-2010 qualitative reaction;
IV, salmonella: check according to GB4789.4-2010;
V, yeast, mould: check according to GB4789.15-2010.
4. the mensuration of beta glucan
By the fermentation broth agitation of heat sterilization, then leave standstill and make protein denaturation precipitation.Centrifugal 10min under 5000r/min condition, add Powdered Activated Carbon 1.5%, abundant stirring, 30-40min is left standstill with impurity such as adsorpting pigments at 50 DEG C, active carbon is removed afterwards by vacuum filtration method, then adopt freeze crystallization to remove most of calcium lactate, according to the calcium content that EDTA-Ca salt method measures, add quantitative Na
2cO
3generate CaCO
3pelleting centrifugation removing calcium ion, obtains lactic fermentation extract, wherein containing the residual sugar such as reduced sugar, polysaccharide.
The mensuration of beta glucan in zymotic fluid, get 4 brace plug test tubes, at room temperature add the phosphate buffer of 3ml0.2mol/LPH8 successively, the Congo red solution 0.1ml of 25mg/L, wherein 3 test tubes add the zymotic fluid that 0.4mlPH is adjusted to 8 successively, remaining volume with deionized water polishing to 4ml, directly use deionized water polishing to 4ml for other 1, mixing, 15min is reacted under 20 DEG C of conditions, survey absorbance at 550nm wavelength place, average, look into the content that calibration curve obtains actual beta glucan in zymotic fluid.
5. the technical indicator of beta glucan cultured milk is rich in
(1) organoleptic indicator
Be rich in beta glucan acidified milk smooth surface, be creamy white and uniformity; There is the distinctive flavour of acidified milk and smell; Sour and sweet palatability, fragrance is dense; Tissue is fine and smooth, evenly, without sense organ visible particle, no whey is separated out.
(2) physical and chemical index
Table 2 physical and chemical index
(3) microbiological indicator
Table 3 microbiological indicator
before and after embodiment 3 mutagenesis cultured milk index contrast situation
Accurate absorption 0. 1 mgml
-1standard glucose solution 0 .2,0 .4,0 .6,0 .8,1 .0,1 .2 ml, put in 6 brace plug scale test tubes, adding distil water to 2 .0 ml; Separately get distilled water 2 .0 mi to put in another test tube and do blank.Precision adds 4 respectively. and the Congo red solution of 0 ml, shakes up, and puts 20C water-bath 10 min, take blank solution as contrast, measures absorbance (Y) at 550nm.Obtain the calibration curve (as accompanying drawing 6) of glucose.As can be seen from Figure 6 when the concentration of glucose is at 0-300 μ g/ml, present good linear relationship.
The mensuration of beta glucan in zymotic fluid: get 4 brace plug test tubes, at room temperature add the phosphate buffer of 3ml 0.2mol/LPH8 successively, the Congo red solution 0.1ml of 25mg/L, wherein 3 test tubes add the zymotic fluid that 0.4mlPH is adjusted to 8 successively, remaining volume with deionized water polishing to 4ml, directly use deionized water polishing to 4ml for other 1, mixing, 15min is reacted under 20 DEG C of conditions, absorbance is surveyed at 550nm wavelength place, average, obtain the content of actual beta glucan in zymotic fluid according to regression equation.(table 4)
Table 4 congo red method is surveyed beta glucan and is obtained content (%)
Draw from table 4, the beta glucan content of first three fermented sample of mutagenesis is respectively 3.53%, 3.79%, 3.23%, and its mean value is: 3.52%; After mutagenesis, the beta glucan content of three fermented samples is respectively 4.54%, 3.53%, 3.79%, and its mean value is: 3.95%.Relatively the beta glucan content of mutagenesis after fermentation liquid comparatively improves 12.2% before mutagenesis.After mutagenesis, beta glucan content is 268 μ g/ml, and former bacterial strain produces beta glucan 238 μ g/ml.
Claims (9)
1. be rich in an acidified milk for beta glucan, it is characterized in that: described acidified milk obtains through the beta glucan superior strain streptococcus thermophilus of mutagenic and breeding and bulgaria lactobacillus fermentation, is specifically obtained by following preparation technology:
(1) the beta glucan superior strain streptococcus thermophilus of mutagenic and breeding is mixed with both lactobacillus bulgaricus, through overactivation, spread cultivation, be prepared into leavening, for subsequent use;
(2) whole-fat milk powder is modulated into 12% reconstituted milk, then adds sucrose, after dissolving at 90 DEG C-95 DEG C sterilization 5min, then be cooled to 45 DEG C;
(3) leavening prepared by step (1) is inoculated in the reconstituted milk of cooling in step (2), carries out fermented and cultured;
(4) eventually pass cooling, after-ripening, obtained finished product, refrigerates, to obtain final product at 4 DEG C;
In described step (1), the beta glucan superior strain streptococcus thermophilus of lactobacillus bulgaricus and mutagenic and breeding mixes with the bacterial classification ratio of 1:3,1:2,1:1,2:1 or 3:1;
In described step (2), sucrose addition mass percent is 1%, 3%, 5%, 6%, 7% or 9%;
In described step (3), strain inoculation amount is 2%, 4%, 5%, 6%, 8% or 10%;
Fermentation temperature in described step (3) is 37 DEG C, 39 DEG C, 41 DEG C, 43 DEG C or 45 DEG C.
2. be rich in the acidified milk of beta glucan as claimed in claim 1, it is characterized in that: in described step (1), the bacterial classification mixed proportion of the beta glucan superior strain streptococcus thermophilus of lactobacillus bulgaricus and mutagenic and breeding is 2:1.
3. be rich in the acidified milk of beta glucan as claimed in claim 1, it is characterized in that: in described step (2), sucrose addition is 6%.
4. be rich in the acidified milk of beta glucan as claimed in claim 1, it is characterized in that: in described step (3), strain inoculation amount is 5%.
5. be rich in the acidified milk of beta glucan as claimed in claim 1, it is characterized in that: the fermentation temperature in described step (3) is 41 DEG C.
6. be rich in the acidified milk of beta glucan as claimed in claim 1, it is characterized in that: the preparation method of the beta glucan superior strain streptococcus thermophilus of described mutagenic and breeding is as follows:
The streptococcus thermophilus selected is cultivated as starting strain using utilizing lactic acid bacteria, cultivated by the selection of different culture media and purifying is carried out to it, performance test is carried out to the bacterial strain that purifying obtains simultaneously, the good bacterial strain of selectivity makes unicellular thallus suspension, carries out suspension colony counting by slat chain conveyor; Utilize ultraviolet mutagenesis and nitrosoguanidine mutagenesis, mutagens process is carried out to thallus suspension liquid, utilize lactic acid bacteria to cultivate and colony counting is carried out to the thallus suspension liquid after mutagens process, calculate fatal rate; Utilize middle cultivation and dull and stereotyped coating to carry out mutant strain to be separated, isolated bacterial strain is screened further, first carries out primary dcreening operation, multiple sieve is carried out to the bacterial strain filtered out, genetic stability is carried out after measured to the bacterial strain sifted out again, to obtain final product.
7. be rich in the acidified milk of beta glucan as claimed in claim 6, it is characterized in that: the wavelength that described ultraviolet mutagenesis uses the ultraviolet mutagenesis lamp of manual manufacture to send is the ultraviolet of 253.7 nm, and mutation time is 120 seconds.
8. be rich in the acidified milk of beta glucan as claimed in claim 6, it is characterized in that: described NTG mutant treatment dosage is 0.3mg/ml, mutation time is 50 minutes.
9. one kind is rich in the preparation method of the acidified milk of beta glucan as claimed in claim 1, it is characterized in that: described acidified milk obtains through the beta glucan superior strain streptococcus thermophilus of mutagenic and breeding and bulgaria lactobacillus fermentation, specifically comprises following processing step:
(1) the beta glucan superior strain streptococcus thermophilus of mutagenic and breeding is mixed with both lactobacillus bulgaricus, through overactivation, spread cultivation, be prepared into leavening, for subsequent use;
(2) whole-fat milk powder is modulated into 12% reconstituted milk, then adds sucrose, after dissolving at 90 DEG C-95 DEG C sterilization 5min, then be cooled to 45 DEG C;
(3) leavening prepared by step (1) is inoculated in the reconstituted milk of cooling in step (2), carries out fermented and cultured;
(4) eventually pass cooling, after-ripening, obtained finished product, refrigerates, to obtain final product at 4 DEG C;
In described step (1), the beta glucan superior strain streptococcus thermophilus of lactobacillus bulgaricus and mutagenic and breeding mixes with the bacterial classification ratio of 1:3,1:2,1:1,2:1 or 3:1;
In described step (2), sucrose addition mass percent is 1%, 3%, 5%, 6%, 7% or 9%;
In described step (3), strain inoculation amount is 2%, 4%, 5%, 6%, 8% or 10%;
Fermentation temperature in described step (3) is 37 DEG C, 39 DEG C, 41 DEG C, 43 DEG C or 45 DEG C.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108774627A (en) * | 2018-06-29 | 2018-11-09 | 昆山佰生优生物科技有限公司 | Lactobacillus bulgaricus and streptococcus thermophilus mixed fungus fermentation preparation process and its application in ferment agent for sour milk freeze-dried powder |
| CN110178897A (en) * | 2019-05-27 | 2019-08-30 | 安徽工程大学 | A kind of fermented grain cream of hypoglycemia patient and preparation method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101333519A (en) * | 2007-06-28 | 2008-12-31 | 北京农学院 | Process for extracting bilesalt hydrolase and exopolysaccharide from lactococcus lactis and streptococcus thermophilus |
| CN102210345A (en) * | 2010-09-07 | 2011-10-12 | 北京博益众科技有限公司 | Congealing type yogurt and preparation method thereof |
| CN102399730A (en) * | 2011-12-07 | 2012-04-04 | 天津科技大学 | High-extracellular-polysaccharide-yield Lactobacillus, application and extracellular polysaccharide thereof |
| CN102453689A (en) * | 2011-12-06 | 2012-05-16 | 光明乳业股份有限公司 | Lactobacillus plantarum strain producing extracellular polysaccharide, and application thereof |
| CN102965318A (en) * | 2012-11-26 | 2013-03-13 | 吉林省农业科学院 | Streptococcus thermophilus generating exopolysaccharides and applications of streptococcus thermophilus |
-
2014
- 2014-12-31 CN CN201410848145.7A patent/CN104489085A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101333519A (en) * | 2007-06-28 | 2008-12-31 | 北京农学院 | Process for extracting bilesalt hydrolase and exopolysaccharide from lactococcus lactis and streptococcus thermophilus |
| CN102210345A (en) * | 2010-09-07 | 2011-10-12 | 北京博益众科技有限公司 | Congealing type yogurt and preparation method thereof |
| CN102453689A (en) * | 2011-12-06 | 2012-05-16 | 光明乳业股份有限公司 | Lactobacillus plantarum strain producing extracellular polysaccharide, and application thereof |
| CN102399730A (en) * | 2011-12-07 | 2012-04-04 | 天津科技大学 | High-extracellular-polysaccharide-yield Lactobacillus, application and extracellular polysaccharide thereof |
| CN102965318A (en) * | 2012-11-26 | 2013-03-13 | 吉林省农业科学院 | Streptococcus thermophilus generating exopolysaccharides and applications of streptococcus thermophilus |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108774627A (en) * | 2018-06-29 | 2018-11-09 | 昆山佰生优生物科技有限公司 | Lactobacillus bulgaricus and streptococcus thermophilus mixed fungus fermentation preparation process and its application in ferment agent for sour milk freeze-dried powder |
| CN108774627B (en) * | 2018-06-29 | 2022-07-01 | 善恩康生物科技（苏州）有限公司 | Preparation method of yogurt starter culture freeze-dried powder |
| CN110178897A (en) * | 2019-05-27 | 2019-08-30 | 安徽工程大学 | A kind of fermented grain cream of hypoglycemia patient and preparation method thereof |
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