CN104357359A - Production technology of bile salt hydrolase-yielding lactobacillus paracasei dry powder living bacterium preparation - Google Patents
Production technology of bile salt hydrolase-yielding lactobacillus paracasei dry powder living bacterium preparation Download PDFInfo
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- C12N1/04—Preserving or maintaining viable microorganisms
Abstract
The invention relates to a preparation method of a bile salt hydrolase-yielding lactobacillus paracasei dry powder living bacterium preparation, wherein the preparation method is suitable for the production of functional probiotic lactic acid bacterium leavening agents and microecologics for eating or feeding. The method comprises the following steps: carrying out activation, cultivation enlargement, high-density fermentation and centrifugal concentration on bile salt hydrolase-yielding lactobacillus paracasei KL1-Liu CGMCC No.7029 screened from Tibetan kefir, adding a freeze-drying protective additive, pre-freezing, freeze-drying and the like. According to the method, a nitrogen source in an improved MRS culture medium is completely replaced with rapeseed peptone, so that the production cost is lowered; and production and fermentation conditions and a formula of the freeze-drying protective additive are optimized, so that the number of living bacteria of the KL1-Liu strain can reach above 10<10>CFU/g, and the survival rate can reach above 85%. The prepared living bacterium preparation is low in cost and high in living bacterium content, and is a high-quality probiotic living bacterium preparation/leavening agent with a serum cholesterol lowering function.
Description
Technical field
The present invention relates to a kind of production technology of producing bile salt hydrolase lactobacillus paraceasi KL1-Liu dry powder active bacteria formulation, be applicable to the production of functional probiotic lactic acid bacterium leavening agent, edible or feeding micro-ecological preparation, functional foodstuff, functional leavened food.
Background technology
Lactobacillus paraceasi (Lactobacillus paracasei) KL1-Liu that the present invention relates to be from Kefir grain separation screening obtain a kind ofly produce bile salt hydrolase and there is the milk-acid bacteria reducing serum cholesterol effect.
" Kefir grain " (Tibetan Kefir, TK) is also known as Kefir granule, and 19th century were found by the mankind.Kefir granule originates from area, Russian Ciscaucasia, cow's milk or goat dairy are injected sheepskin pocket and produce kephir through spontaneous fermentation by local herdsman, its residue adds cow's milk again or goat dairy continues fermentation, after long-term fermentation, in skin pocket, form irregular particle shape object, be Kefir granule.After this people are just direct adds in cow's milk by Kefir granule, makes it to be fermented into the nourishing drink with frank tart flavour and whipability, is Kefir grains.Kefir grains has various health care functions.The growth of viable bacteria in Kefir grains to the pathogenic bacteria such as Shigellae, Salmonellas has strongly inhibited effect; Yeast wherein has stronger anti-microbial activity to tubercule bacillus, intestinal bacteria.Therefore Kefir grains has good curative effect to intestinal tract disease, pulmonary tuberculosis.In addition, the meta-bolites that the milk-acid bacteria in Kefir grains produces, as bile salt hydrolase, exocellular polysaccharide etc. effectively can prevent the generation of hyperlipidemia and tumour.
The little ecosystem of microflora mainly based on milk-acid bacteria, yeast and a small amount of acetic bacteria in the mucopolysaccharide matrix of Kefir grain.The present invention's lactobacillus paraceasi used KL1-Liu is a kind of probiotic lactobacillus that separation screening obtains from Kefir grain, the bile salt hydrolase of a large amount of high vigor can be produced in growth metabolism process, prove that there is the effect reducing body serum total cholesterol level through external Oxford cup test and experimentation on animals.It reduces cholesterol mechanism is the bile salt hydrolase (Bile Salt Hydrolase, BSH) produced because of lactobacillus paraceasi KL1-Liu metabolism.The meta-bolites that BSH is genus bifidobacterium in enteron aisle, multiple lactic-acid-bacterium in lactobacillus, lactococcus produces in process of growth.Experimental results demonstrate, bile salt hydrolase will can decompose in conjunction with cholate in hepato-enteric circulation, produces amino acid and the lower free cholate of solubleness.The latter can be combined with cholesterol and forms sediment composite and excrete, thus reduces the content of serum total cholesterol.
Just confirm that the Yoghourt of picked-up containing certain Bacterium lacticum or bifidobacterium fermentation has the effect reducing serum cholesterol content as far back as people in 1963.Up to now, the research of Chinese scholars to the milk-acid bacteria decreasing cholesterol mechanism of action remains in different viewpoints, 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 combined cholate be degraded to free state cholate, and the latter's solubleness declines and cholesterol generation coprecipitation effect; (3) other are theoretical, as assimilation and co-precipitation combined action.
At present, the patent of domestic application various active bacteria formulation preparation method is more, and " active clostridium butyrium agent and preparation method thereof " (CN101032527) applied for as Jiangsu Institute of Microbiology Co., Ltd Tang Baoying and Zhu Xiaohui is the dual-purpose active bacteria formulation of people beast prepared by Butylic acid bacteria; " Bifid Triple Viable and preparation method " (CN98110623.4) of Shanghai Xinyi Pharmaceutical Co., Ltd Chen Bin China and Yao Yiqiang application is by bifidumbacterium bifidum, Lactobacillus acidophilus and streptococcus faecium lyophilize viable bacteria powder; " a kind of composite microorganism viable bacteria preparation and its preparation method and application " (ZL200810034746) of Shanghai Eco-well Bioscience Co., Ltd. Zhang Qin and old Lu of building application is coccus, yeast and genus bacillus fluid enlargement culture respectively; then in required ratio mixing, through a kind of composite microorganism viable bacteria preparation that solid state fermentation, drying, pulverizing obtain.But prepare active bacteria formulation about the lactobacillus paraceasi KL1-Liu of the product bile salt hydrolase utilizing separation screening in Kefir grain, utilize Fermentation high density fermentation, and the method utilizing rapeseed protein peptone to substitute nitrogenous source in modified MRS culture medium completely there is not yet domestic and international pertinent literature report and patent report.
Summary of the invention
The object of this invention is to provide a kind of production technology of producing bile salt hydrolase lactobacillus paraceasi KL1-Liu dry powder active bacteria formulation.
The present invention is achieved by the following technical solutions:
In described method, lactobacillus paraceasi (Lactobacillus paracasei) KL1-Liu separation screening from Jilin, the Kefir grain (also known as Kefir granule) of Huhehaote City's average family, and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 25th, 2012, its preserving number is: CGMCC No.7029 (bacterial strain KL1-Liu), preservation address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.On the MRS selective medium containing calcium carbonate and tennecetin, the bacterium colony size 2 ~ 3mm of lactobacillus paraceasi (Lactobacillus paracasei) KL1-Liu bacterial strain, bacterium colony has comparatively high viscosity, surface irregularity is as flakes, edge is irregular, matt, flat, canescence, translucent; Individual morphology is shaft-like, varying length, short catenation, G
+sporeless bacterium.
In described method, the lactobacillus paraceasi KL1-Liu bacterial strain of skimming milk test tube conservation is cultivated 16h with modified MRS culture medium in 37 DEG C, after so activating for 2 ~ 3 generations, refrigerates for subsequent use.
In described method, modified MRS cultivates formula composition: substituted with 5g milk casein hydrolyzate and 5g Tryptones by the 10g peptone in MRS substratum, other components unchanged, regulates pH to be 6.5.
In described method, single factor experiment is adopted to optimize modified MRS culture medium, the 501 rapeseed protein peptone modified MRS culture medium formula compositions optimized: glucose 20g, 501 rapeseed protein peptone 25g, dibasic ammonium citrate 2g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, bitter salt 0.58g, four anhydrous manganese 0.25g, tween-80 1mL, distilled water 1 000mL, regulate pH to be 7.0 (after sterilizing, pH is down to 6.5).
In described method, on the basis of above-mentioned test, by the bacterium liquid that activated with in inoculum size culture transferring to the 501 rapeseed protein peptone modified MRS culture medium of 3% (V/V, volume fraction), cultivate 16h in 37 DEG C, obtain scale-up medium.
In described method, on above-mentioned test-results basis, adopt four factor three horizontal quadrature assay optimization lab scale fermentation conditions.2L 501 rapeseed protein peptone modified MRS culture medium is joined in 5L automatic fermenter, utilizes fermentor tank sterilising system in 115 DEG C of sterilizing 20min, utilize cooling-water machine that the substratum in fermentor tank is cooled to leavening temperature.By the scale-up medium of lactobacillus paraceasi KL1-Liu bacterial strain by 4% (V/V, volume fraction) inoculum size culture transferring in fermentor tank, utilizing fermentor tank automatic temperature control system to maintain leavening temperature is 34 DEG C, add the sterilizing Na of 10% (W/V, mass volume ratio concentration) with stream in fermenting process
2cO
3solution controls fermented liquid pH6.5, and mixing speed is 90r/min, and fermentation 16h, obtains fermented liquid.
In aforesaid method, 5L fermentor tank is produced by Shanghai Gaoji Bioengineering Co., Ltd., and its model is BIOF6005GBN.
In described method, on above-mentioned test-results basis, utilize vertical and high-speed refrigerated centrifuge by 2L fermented liquid under 4 DEG C of conditions, the centrifugal 20min of 4000r/min, abandons supernatant liquor, collects and obtains bacterium mud.
In described method, vertical and high-speed refrigerated centrifuge is provided by Xiang Yi whizzer Instrument Ltd., and model is GL-21M.
In described method, on above-mentioned test-results basis, the bacterium mud of collection is on average added 2 and fill in the freeze-drying bottle of the lyophilized vaccine (14.5% maltodextrin+5.5% skim-milk) of 100mL, after mixing, obtain concentrated active bacteria formulation.
In aforesaid method; adopt single factor test multilevel test; determine that the more excellent combination formula of the lyophilized vaccine of lactobacillus paraceasi KL1-Liu is for (W/V, mass volume ratio concentration): 14.5% maltodextrin+5.5% skim-milk (Erie's board high protein high calcium skim-milk).
In described method, active bacteria formulation pre-freeze 13h in-30 DEG C of cryogenic refrigerators will be concentrated and, to fully charge state, obtain pre-freeze active bacteria formulation.
In described method, on above-mentioned test-results basis, utilize 6L LABCONCO vacuum freeze drier (U.S.) by pre-freeze active bacteria formulation under the condition of freeze temperature-55 DEG C, vacuum tightness 0.13mBar, freeze-drying 48h, to complete drying state, obtains freeze drying viable microorganism preparation.
In described method, on above-mentioned test-results basis, 50L fermentor tank is utilized to carry out pilot scale fermentation test, 25L 501 rapeseed protein peptone modified MRS culture medium is joined in 50L automatic fermenter, adopt fermentor tank sterilising system in 115 DEG C of sterilizing 20min, utilize cooling-water machine that the substratum in fermentor tank is cooled to leavening temperature.By the scale-up medium of lactobacillus paraceasi KL1-Liu bacterial strain by 4% (V/V, volume fraction) inoculum size culture transferring in fermentor tank, utilizing fermentor tank automatic temperature control system to maintain leavening temperature is 34 DEG C, add the sterilizing Na of 20% (W/V, mass volume ratio concentration) with stream in fermenting process
2cO
3solution controls fermented liquid pH6.5, and mixing speed is 140r/min, and fermentation 16h, obtains fermented liquid.
In aforesaid method, 50L fermentor tank is provided by Guoqiang Biochemical Engineering Equipment Co., Ltd., Shanghai, and model is BIOTECH.
In described method, on above-mentioned test-results basis, utilize high speed tubular-bowl centrifuge by 25L fermented liquid under 3 ~ 6 DEG C of conditions, the centrifugal 10 ~ 20min of 16000r/min, collect and obtain bacterium mud.
In aforesaid method, tubular-bowl centrifuge is provided by Shanghai Centrifuge Institute Co., Ltd., and model is GQ105G.
In described method, on above-mentioned test-results basis, the bacterium mud of collection is added in the Freeze Drying Equipment of lyophilized vaccine (14.5% maltodextrin+5.5% skim-milk) of 2500mL, after mixing, obtain concentrated active bacteria formulation.
In described method, utilize Fourth Ring vacuum freeze drier will concentrate active bacteria formulation in-55 DEG C of pre-freeze 17h to fully charge state, obtain pre-freeze active bacteria formulation.
In described method, on above-mentioned test-results basis, utilize Fourth Ring vacuum freeze drier by pre-freeze active bacteria formulation under the condition of freeze temperature-66 DEG C, vacuum tightness 0.08mBar, freeze-drying 72h, to complete drying state, obtains freeze drying viable microorganism preparation.
In aforesaid method, Fourth Ring vacuum freeze drier is provided by Fourth Ring scientific instrument Co., Ltd., Factory, and model is LGJ-25C.
In described method, the number of viable of the product bile salt hydrolase lactobacillus paraceasi KL1-Liu active bacteria formulation adopting lab scale and pilot scale fermentation production technology and freeze-dry process to obtain all can reach 10
10more than CFU/g, bacterial classification survival rate can reach more than 85%.This kind of active bacteria formulation adopts rapeseed protein peptone to instead of nitrogenous source in modified MRS culture medium completely due to substratum, and products production cost is reduced, and viable bacteria content is higher.
The 501 rapeseed protein peptone modified MRS culture medium formulas that aforesaid method obtains and the lab scale producing bile salt hydrolase lactobacillus paraceasi KL1-Liu active bacteria formulation and pilot scale fermentation production technology and freeze-dry process, and special preparing strain belongs to scope.
Embodiment
The invention will be further described and do not limit the scope of the invention for following embodiment.
Experimental technique in following embodiment, if no special instructions, is ordinary method.
Percentage composition in following embodiment, if no special instructions, is volume fraction.
The lab scale production technology of embodiment 1, product bile salt hydrolase lactobacillus paraceasi KL1-Liu active bacteria formulation
1, the activation of bacterial classification and enlarged culturing
The lactobacillus paraceasi KL1-Liu bacterial strain of MRS test tube conservation is cultivated 16h with modified MRS culture medium in 37 DEG C, so activated for 2 ~ 3 generations, refrigerate for subsequent use; By the bacterium liquid that activated with in inoculum size culture transferring to the 501 rapeseed protein peptone modified MRS culture medium of 3%, cultivate 16h in 37 DEG C, obtain scale-up medium.
2, the determination of fermention medium
(1) the rapeseed protein peptone of different mass quality substitutes the test of modified MRS partial nitrogen source
The Tryptones in modified MRS culture medium is replaced respectively with 501 rapeseed protein peptones, 502 rapeseed protein peptones and 503 rapeseed protein peptones, by the bacterial classification that activated by 4% inoculum size respectively in culture transferring to above-mentioned substratum, after 34 DEG C of fermentation 16h, MRS substratum is adopted to detect the number of viable in fermented liquid with pour plate culture method, each extent of dilution establishes 3 repetitions, determine more excellent rapeseed protein peptone nitrogenous source substitute, the results are shown in Table 1.
(2) rapeseed protein peptone substitutes the test of modified MRS different nitrogen sources
Above-mentioned test preferably rapeseed protein peptone substitute on the basis of Tryptones in modified MRS culture medium, substitute extractum carnis, yeast leaching powder, extractum carnis+yeast leaching powder more respectively, with above-mentioned similarity condition fermentation, counting, the com-parison and analysis number of viable of the fermented liquid in more excellent rapeseed protein peptone respectively alternative modified MRS during different nitrogen sources, the results are shown in Table 2.
(3) the rapeseed protein peptone of different amounts substitutes the whole nitrogenous source test of modified MRS
On above-mentioned test-results basis, more excellent rapeseed protein peptone with 1.5%, 2%, 2.5%, 3%, 3.5% substitutes the whole nitrogenous source of modified MRS respectively, with above-mentioned similarity condition fermentation, counting, the number of viable of fermented liquid when substituting whole nitrogenous source in modified MRS by comparing the more excellent rapeseed protein peptone of different amounts, determine the consumption of more excellent rapeseed protein peptone, the results are shown in Table 3.
The different rapeseed protein peptone of table 1 substitutes modified MRS partial nitrogen source fermention medium to the impact of lactobacillus paraceasi KL1-Liu number of viable
As shown in Table 1, during with 501 rapeseed protein peptone modified MRS culture medium fermentation, number of viable is higher, reaches 1.53 × 10
9cFU/mL, adopting number of viable during 502 rapeseed protein peptone modified MRS culture medium to take second place, is 1.45 × 10
9cFU/mL, adopts number of viable during 503 rapeseed protein peptone modified MRS culture medium to be 1.43 × 10
9cFU/mL, and adopt the number of viable of modified MRS culture medium minimum, therefore determine that more excellent rapeseed protein peptone nitrogenous source substitute is 501 rapeseed protein peptones.
Table 2 501 rapeseed protein peptone substitutes the impact of modified MRS different nitrogen sources on lactobacillus paraceasi KL1-Liu number of viable
As shown in Table 2, when substituting extractum carnis and the modified MRS culture medium fermentation of yeast leaching powder with 501 rapeseed protein peptones, number of viable is higher, reaches 5.61 × 10
9cFU/mL, the number of viable fermented when substituting extractum carnis modified MRS culture medium takes second place, and is 5.13 × 10
9cFU/mL, the number of viable fermented when substituting yeast leaching powder modified MRS culture medium is 4.21 × 10
9cFU/mL, therefore determine that 501 rapeseed protein peptones substitute the modified MRS culture medium of extractum carnis and yeast leaching powder for optimum.
The rapeseed protein peptone of table 3 different amounts substitutes the whole nitrogenous source of modified MRS to the impact of lactobacillus paraceasi KL1-Liu number of viable
As shown in Table 3, adopt 2.5%501 rapeseed protein peptone modified MRS culture medium to cultivate number of viable higher, reach 5.51 × 10
9cFU/mL, adopting 3%501 rapeseed protein peptone modified MRS culture medium to cultivate number of viable and take second place, is 5.03 × 10
9cFU/mL, adopting 2.0%501 rapeseed protein peptone modified MRS culture medium to cultivate number of viable is 4.42 × 10
9cFU/mL, therefore determine that 2.5%501 rapeseed protein peptone modified MRS culture medium are best medium.
3, optimization of orthogonal test lactobacillus paraceasi KL1-Liu lab scale fermentation condition
According to single factor test multilevel test result, select leavening temperature, fermentation time, fermentation inoculum size, fermentation pH to be influence factor, design four factor three level [L
9(3
4)] orthogonal test.In 5L Fermentation, with 2L 501 rapeseed protein peptone modified MRS for fermention medium, in batch fermentation process, add the sterilizing Na of 10% (W/V, mass volume ratio concentration) with stream
2cO
3solution controls fermented liquid pH6.5, and mixing speed is 90r/min, and adopt MRS substratum to detect the number of viable in fermented liquid with pour plate culture method, each extent of dilution establishes 3 repetitions, the results are shown in Table 4.
Table 4 lactobacillus paraceasi KL1-Liu fermentation condition optimization orthogonal experiments
From table 4 range analysis, to the order of the viable count influence factor of fermented liquid be: R
b> R
a> R
c> R
d, namely fermentation time has the greatest impact, and leavening temperature takes second place, and fermentation inoculum size, fermentation pH affect minimum; Show that the optimum combination of batch fermentation conditions is A by the intuitive analysis of K value
1b
3c
2d
2, namely optimization of fermentation conditions is: leavening temperature is 34 DEG C, and fermentation time is 16h, and inoculum size is 4%, pH is 6.5.
4, high density fermentation
2L 501 rapeseed protein peptone modified MRS culture medium is added in 5L Fermentation, utilizes fermentor tank sterilising system in 115 DEG C of sterilizing 15min, utilize cooling-water machine that the substratum in fermentor tank is cooled to leavening temperature.The scale-up medium of KL1-Liu bacterial strain is pressed the inoculum size culture transferring of 4% in fermentor tank, utilizing fermentor tank temperature controlling system to maintain leavening temperature is 34 DEG C, adds the sterilizing Na of 10% in fermenting process with stream
2cO
3the pH that solution controls fermented liquid is 6.5, and mixing speed is 90r/min, and fermentation 16h, obtains viable count 10
9the fermented liquid of more than CFU/mL.
5, centrifugal concentrating and add protective material
By the amount of above-mentioned centrifugal primary fermentation liquid 1/10 volume, be sub-packed in the freeze-drying bottle of 600mL by the lyophilized vaccine of various combination in table 5 respectively, the bottled amount of each freeze-drying is 100mL; Get 1L fermented liquid in 4 DEG C, the centrifugal 20min of 4000r/min, abandon supernatant liquor, collect bacterium mud, and mix with lyophilized vaccine, obtain concentrated active bacteria formulation.Adopt MRS substratum to detect the preparation number of viable before freeze-drying with pour plate culture method, each extent of dilution establishes 3 repetitions, the results are shown in Table 5, to determine the more excellent combination formula of lyophilized vaccine.
6, pre-freeze and freeze-drying
Active bacteria formulation will be concentrated in-30 DEG C of pre-freeze 13h to fully charge state, obtain pre-freeze active bacteria formulation.Utilize 6L LABCONCO vacuum freeze drier (U.S.) by pre-freeze active bacteria formulation in-55 DEG C, under the condition of vacuum tightness 0.13mBar, freeze-drying 48h, to complete drying state, obtains freeze drying viable microorganism preparation.Adopt MRS substratum to detect the preparation number of viable after freeze-drying with pour plate culture method, calculate bacterial classification survival rate, each extent of dilution establishes 3 repetitions, the results are shown in Table 5.
The lyophilized vaccine of table 5 various combination is on the impact of lactobacillus paraceasi KL1-Liu survival rate
From table 5, choose lyophilized vaccine and be combined as 14.5% maltodextrin+5.5% skim-milk, lactobacillus paraceasi KL1-Liu survival rate is the highest, is 85.27%; When 5.5% maltodextrin+5.5% skim-milk is lyophilized vaccine, bacterial classification survival rate is taken second place, and is 78.00%; During with 11% skim-milk for lyophilized vaccine, bacterial classification survival rate is minimum is 55.36%.Therefore determine that the more excellent combination formula of the lyophilized vaccine of lactobacillus paraceasi KL1-Liu is for (W/V, mass volume ratio concentration): 14.5% maltodextrin+5.5% skim-milk, its number of viable can reach 10
10more than CFU/g, bacterial classification survival rate can reach more than 85%.
The scale up test technology of embodiment 2, product bile salt hydrolase lactobacillus paraceasi KL1-Liu active bacteria formulation
1, pilot scale fermentation proof test
Pilot scale fermentation proof test is carried out according to optimization of orthogonal test result.In 50L Fermentation with 25L501 rapeseed protein peptone modified MRS for fermention medium, add the sterilizing Na of 20% in batch fermentation process with stream
2cO
3solution controls fermented liquid pH value, mixing speed control 140r/min.Take modified MRS culture medium as fermention medium, simultaneously not control fermented liquid pH (nature) as a control group.Adopt MRS substratum to detect the number of viable in fermented liquid with pour plate culture method, each extent of dilution establishes 3 repetitions, the results are shown in Table 6.
Table 6 pilot scale fermentation proof test result
As shown in Table 6, ferment with optimal conditions, fermented liquid viable count reaches 8.50 × 10
9cFU/mL, for optimize before (2.6 × 10
8cFU/mL) 32.69 times of viable count.
2, centrifugal concentrating and add protective material
Utilize high speed tubular-bowl centrifuge by 25L fermented liquid under 3 ~ 6 DEG C of conditions, the centrifugal 10 ~ 20min of 16000r/min, collect and obtain bacterium mud; The bacterium mud of collection is added in the Freeze Drying Equipment of lyophilized vaccine (14.5% maltodextrin+5.5% skim-milk) of 2500mL, after mixing, obtain concentrated active bacteria formulation.
3, pre-freeze and freeze-drying
Utilize Fourth Ring vacuum freeze drier will concentrate active bacteria formulation in-55 DEG C of pre-freeze 17h to fully charge state, obtain pre-freeze active bacteria formulation; Utilize Fourth Ring vacuum freeze drier by pre-freeze active bacteria formulation under the condition of freeze temperature-66 DEG C, vacuum tightness 0.08mBar, freeze-drying 72h, to complete drying state, obtains freeze drying viable microorganism preparation.Its number of viable can reach 10
10more than CFU/g, bacterial classification survival rate can reach more than 85%.
In sum, the lab scale production technology of bile salt hydrolase lactobacillus paraceasi KL1-Liu active bacteria formulation is produced: lactobacillus paraceasi KL1-Liu is cultivated 16h with modified MRS culture medium in 37 DEG C, so activated for 2 ~ 3 generations; By the bacterium liquid that activated with in inoculum size culture transferring to the 501 rapeseed protein peptone modified MRS culture medium of 3%, cultivate 16h in 37 DEG C, obtain scale-up medium; 501 rapeseed protein peptone modified MRS culture medium formula compositions: glucose 20g, 501 rapeseed protein peptone 25g, dibasic ammonium citrate 2g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, bitter salt 0.58g, four anhydrous manganese 0.25g, tween-80 1mL, distilled water 1000mL, regulate pH to be 7.0; Above-mentioned scale-up medium is pressed the inoculum size culture transferring of 4% in 2L 501 rapeseed protein peptone modified MRS fermention medium, utilize 5L Fermentation to carry out sterilizing and high density fermentation, control leavening temperature 34 DEG C, fermented liquid pH6.5, mixing speed 90r/min, fermentation time 16h, obtains fermented liquid; By fermented liquid in 4 DEG C, the centrifugal 20min of 4000r/min, collect and obtain bacterium mud; In 600mL sterilizing freeze-drying bottle, by the amount (200mL) of centrifugal primary fermentation liquid 1/10 volume, add the lyophilized vaccine of 14.5% maltodextrin+5.5% skim-milk and the bacterium mud of collection, after mixing, obtain starter culture concentrates; The starter culture concentrates by bottled for each freeze-drying amount being 100mL, in-30 DEG C of pre-freeze 13h to fully charge state, obtains pre-freeze starter; Utilize U.S. 6L LABCONCO vacuum freeze drier by pre-freeze starter in-55 DEG C, under the condition of vacuum tightness 0.13mBar, freeze-drying 48h, to complete drying state, obtains producing bile salt hydrolase lactobacillus paraceasi dry powder active bacteria formulation.
Produce the scale up test technology of bile salt hydrolase lactobacillus paraceasi KL1-Liu active bacteria formulation: lactobacillus paraceasi KL1-Liu is cultivated 16h with modified MRS culture medium in 37 DEG C, so activated for 2 ~ 3 generations; By the bacterium liquid that activated with in inoculum size culture transferring to the 501 rapeseed protein peptone modified MRS culture medium of 3%, cultivate 16h in 37 DEG C, obtain scale-up medium; 501 rapeseed protein peptone modified MRS culture medium formula compositions: glucose 20g, 501 rapeseed protein peptone 25g, dibasic ammonium citrate 2g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, bitter salt 0.58g, four anhydrous manganese 0.25g, tween-80 1mL, distilled water 1 000mL, regulate pH to be 7.0; Above-mentioned scale-up medium is pressed the inoculum size culture transferring of 4% in 25L 501 rapeseed protein peptone modified MRS fermention medium, 50L Fermentation is utilized to carry out sterilizing and high density fermentation, control leavening temperature 34 DEG C, fermented liquid pH6.5, mixing speed 140r/min, fermentation time 16h, obtains fermented liquid; By fermented liquid under 3 ~ 6 DEG C of conditions, the centrifugal 10 ~ 20min of 16000r/min, collects and obtains bacterium mud; By the amount (2500mL) of centrifugal primary fermentation liquid 1/10 volume in freeze-drying pallet, add the lyophilized vaccine of 14.5% maltodextrin+5.5% skim-milk and the bacterium mud of collection, after mixing, obtain starter culture concentrates; By starter culture concentrates in-55 DEG C of pre-freeze 17h to fully charge state, obtain pre-freeze active bacteria formulation; Utilize Fourth Ring, Beijing vacuum freeze drier by pre-freeze active bacteria formulation in-66 DEG C, under the condition of vacuum tightness 0.08mBar, freeze-drying 72h, to complete drying state, obtains producing bile salt hydrolase lactobacillus paraceasi dry powder active bacteria formulation.
The number of viable of the product bile salt hydrolase lactobacillus paraceasi KL1-Liu dry powder active bacteria formulation adopting above-mentioned lab scale and pilot scale fermentation production technology and freeze-dry process to obtain all can reach 10
10more than CFU/g, bacterial classification survival rate can reach more than 85%.This active bacteria formulation production technique is simple, and low in raw material price, cost is lower, and number of viable is higher, is suitable for suitability for industrialized production.The product bile salt hydrolase lactobacillus paraceasi dry powder active bacteria formulation that the present invention obtains, both can as the starter producing probiotics yogurt, not only the sticky fine and smooth children of mouthfeel is sliding to make product, sweet mild acidity, there is strong ester Flavor, and have and reduce effect of serum cholesterol, edible compound probiotic sheet can be produced again, and the leavened prod such as feed composite microecologic agent.
Project 1 belonging to this patent: Beijing's Natural Science Fund In The Light " Kefir grain probiotic bacterium produces the research of active substances and physiological function "
Item number: 5062006
The project beginning and ending time: 2006.01-2008.12
Project leader: Liu Hui
Project 2 belonging to this patent: Department of Science and Technology's Transformation of Agricultural Sci-Tech Achievements capital items " utilizes Kefir grain source to produce bile salt hydrolase milk-acid bacteria and produces functional yoghourt technical transform "
Item number: 2013GB2A000006
The project beginning and ending time: 2013.09-2015.08
Project leader: Bai Yongqiang
Project 3 belonging to this patent: Department of Science and Technology's national science and technology key special subjects " disease-resistant transgenic sheep rearing new variety " sub-problem " foundation that disease-resistant transgenic sheep expansion traditional font is/disease-resistant transgenic goat-anti disease, production performance and safety evaluation "
Item number: 2013ZX08008-005
The project beginning and ending time: 2013.01-2013.12
Project leader: Liu Hui.
Claims (3)
1. lactobacillus paraceasi (Lactobacillus paracasei) KL1-Liu CGMCC No.7029.
2. one kind utilizes the method for lactobacillus paraceasi (Lactobacillus paracasei) the KL1-Liu CGMCC No.7029 bacterial strain little trial production dry powder active bacteria formulation producing bile salt hydrolase, it is characterized in that: lactobacillus paraceasi KL1-Liu is cultivated 16h with modified MRS culture medium in 37 DEG C, so activated for 2 ~ 3 generations; By the bacterium liquid that activated with in inoculum size culture transferring to the 501 rapeseed protein peptone modified MRS culture medium of 3%, cultivate 16h in 37 DEG C, obtain scale-up medium; 501 rapeseed protein peptone modified MRS culture medium formula compositions: glucose 20g, 501 rapeseed protein peptone 25g, dibasic ammonium citrate 2g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, bitter salt 0.58g, four anhydrous manganese 0.25g, tween-80 1mL, distilled water 1000mL, regulate pH to be 7.0; Above-mentioned scale-up medium is pressed the inoculum size culture transferring of 4% in 2L 501 rapeseed protein peptone modified MRS fermention medium, utilize 5L Fermentation to carry out sterilizing and high density fermentation, control leavening temperature 34 DEG C, fermented liquid pH6.5, mixing speed 90r/min, fermentation time 16h, obtains fermented liquid; By fermented liquid in 4 DEG C, the centrifugal 20min of 4000r/min, collect and obtain bacterium mud; In 600mL sterilizing freeze-drying bottle, by the amount (200mL) of centrifugal primary fermentation liquid 1/10 volume, add the lyophilized vaccine of 14.5% maltodextrin+5.5% skim-milk and the bacterium mud of collection, after mixing, obtain starter culture concentrates; The starter culture concentrates by bottled for each freeze-drying amount being 100mL, in-30 DEG C of pre-freeze 13h to fully charge state, obtains pre-freeze starter; Utilize U.S. 6L LABCONCO vacuum freeze drier by pre-freeze starter in-55 DEG C, under the condition of vacuum tightness 0.13mBar, freeze-drying 48h, to complete drying state, obtains producing bile salt hydrolase lactobacillus paraceasi dry powder active bacteria formulation.
3. one kind utilizes the method for lactobacillus paraceasi (Lactobacillus paracasei) the KL1-Liu CGMCC No.7029 bacterial strain scale up test dry powder active bacteria formulation producing bile salt hydrolase, it is characterized in that: lactobacillus paraceasi KL1-Liu is cultivated 16h with modified MRS culture medium in 37 DEG C, so activated for 2 ~ 3 generations; By the bacterium liquid that activated with in inoculum size culture transferring to the 501 rapeseed protein peptone modified MRS culture medium of 3%, cultivate 16h in 37 DEG C, obtain scale-up medium; 501 rapeseed protein peptone modified MRS culture medium formula compositions: glucose 20g, 501 rapeseed protein peptone 25g, dibasic ammonium citrate 2g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, bitter salt 0.58g, four anhydrous manganese 0.25g, tween-80 1mL, distilled water 1000mL, regulate pH to be 7.0; Above-mentioned scale-up medium is pressed the inoculum size culture transferring of 4% in 25L 501 rapeseed protein peptone modified MRS fermention medium, 50L Fermentation is utilized to carry out sterilizing and high density fermentation, control leavening temperature 34 DEG C, fermented liquid pH6.5, mixing speed 140r/min, fermentation time 16h, obtains fermented liquid; By fermented liquid under 3 ~ 6 DEG C of conditions, the centrifugal 10 ~ 20min of 16000r/min, collects and obtains bacterium mud; By the amount (2500mL) of centrifugal primary fermentation liquid 1/10 volume in freeze-drying pallet, add the lyophilized vaccine of 14.5% maltodextrin+5.5% skim-milk and the bacterium mud of collection, after mixing, obtain starter culture concentrates; By starter culture concentrates in-55 DEG C of pre-freeze 17h to fully charge state, obtain pre-freeze active bacteria formulation; Utilize Fourth Ring, Beijing vacuum freeze drier by pre-freeze active bacteria formulation in-66 DEG C, under the condition of vacuum tightness 0.08mBar, freeze-drying 72h, to complete drying state, obtains producing bile salt hydrolase lactobacillus paraceasi dry powder active bacteria formulation.
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| CN104450562A (en) * | 2014-11-14 | 2015-03-25 | 北京农学院 | Preparation method of Chinese-date probiotics tablet of lactobacillus paracasei capable of producing bile salt hydrolase |
| CN111286469A (en) * | 2018-12-10 | 2020-06-16 | 东北农业大学 | Preparation method of lactobacillus paracasei freeze-dried powder |
| CN112608870A (en) * | 2020-12-31 | 2021-04-06 | 重庆第二师范学院 | Probiotics starter based on material source protein |
| CN113773999A (en) * | 2021-07-22 | 2021-12-10 | 华熙生物科技股份有限公司 | Lactobacillus paracasei fermentation filtrate, preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450562A (en) * | 2014-11-14 | 2015-03-25 | 北京农学院 | Preparation method of Chinese-date probiotics tablet of lactobacillus paracasei capable of producing bile salt hydrolase |
| CN111286469A (en) * | 2018-12-10 | 2020-06-16 | 东北农业大学 | Preparation method of lactobacillus paracasei freeze-dried powder |
| CN111286469B (en) * | 2018-12-10 | 2022-11-01 | 东北农业大学 | Preparation method of lactobacillus paracasei freeze-dried powder |
| CN112608870A (en) * | 2020-12-31 | 2021-04-06 | 重庆第二师范学院 | Probiotics starter based on material source protein |
| CN113773999A (en) * | 2021-07-22 | 2021-12-10 | 华熙生物科技股份有限公司 | Lactobacillus paracasei fermentation filtrate, preparation method and application thereof |
| CN113773999B (en) * | 2021-07-22 | 2023-08-29 | 华熙生物科技股份有限公司 | Lactobacillus paracasei fermentation filtrate, preparation method and application thereof |
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Application publication date: 20150218 |