CN100564514C - A kind of method of producing active lactic acid bacteria powder with atmosphere pressure desiccation - Google Patents
A kind of method of producing active lactic acid bacteria powder with atmosphere pressure desiccation Download PDFInfo
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- CN100564514C CN100564514C CNB2007100780215A CN200710078021A CN100564514C CN 100564514 C CN100564514 C CN 100564514C CN B2007100780215 A CNB2007100780215 A CN B2007100780215A CN 200710078021 A CN200710078021 A CN 200710078021A CN 100564514 C CN100564514 C CN 100564514C
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Abstract
The present invention relates to a kind of constant pressure and dry preparation method of living lactic acid bacteria bacterium powder, by living lactic acid bacteria is adopted high temperature acclimation, enlarged culturing, mixing, dry technology of preparing, solved a living lactic acid bacteria exsiccant survival difficult problem at high temperature, avoided living lactic acid bacteria 37 ℃-38 ℃ dry down are bacterial contamination easily and lyophilize needs advanced expensive drying facility problem.Present technique provides living lactic acid bacteria bacterium powder, can be directly ferment, overcome in present domestic most lactic acid fermentation product (as poor capsicum, pickles etc.) traditional processing technology and cause the technical barrier that living contaminants is serious, quality product is low as the strain excellent of lactic acid fermentation product; Simplified the yoghurt production process, can preserve at normal temperatures more than 6 months, can form commodity, be convenient to transportation, also be convenient to the suitability for industrialized production of lactic acid fermentation product with the biodiasmin powder that the present invention obtains.
Description
Technical field:
The invention belongs to the technical field that the human lives needs the preparation of class related food or handles, specifically isolate pure good lactobacillus strain combination, adopt the technological method of producing active lactic acid bacteria powder with atmosphere pressure desiccation from fermented products such as poor capsicum, pickles, sour milks.
Background technology:
Milk-acid bacteria has the various active enzyme, and can produce special antimicrobial substance, therefore lactobacillus product is widely used in food, each side such as healthcare products and medical article, the preparation method of milk-acid bacteria is also a lot, go out strain excellent according to different purposes screening and separating from different fermented products and make lactobacillus product, lactobacillus product production is generally through three steps: 1. screening and separating goes out strain excellent, and enlarged culturing becomes excellent species; 2. bacterial classification is left in and be processed into lactobacillus product on the carrier; 3. the preservation of lactobacillus product.
Because living lactic acid bacteria only exists in liquid foodstuff or the beverage, therefore, lactobacillus inoculation generally leaves in the skimmed milk, after vacuum lyophilization, preserve at low temperatures, and leave bacterial classification in the solid siccative few, that is to say with the solid siccative to be that the report of lactic acid bacteria vector is few, patent of invention " technology of preparing of the dry bacterium powder of living lactic acid bacteria ", application number 00119386.4, be one of report of lactic acid bacteria vector exactly with the solid siccative, but it preserves still under low temperature condition (below 10 ℃), and its production purpose is still and makes foodstuff additive, therefore to bacterial classification fermentation or production performance do not do to make excessive demands.
Summary of the invention:
The objective of the invention is to from the good natural lactic acid fermentation goods of quality screening and separating and go out pure strain excellent combination, become good pure strain through the special methods enlarged culturing, do not contain any assorted bacterium, pure strain is left in the solid siccative, through dry under 50 ℃ of-70 ℃ of temperature behind the high temperature acclimation, and can under the normal temperature state, do longer preservation.
For achieving the above object, the present invention develops the technical process of a complete producing active lactic acid bacteria powder with atmosphere pressure desiccation, and has proposed main technologic parameters according to test.
Figure of description is seen in technical process of the present invention, is followed successively by 1. bacterial classification extraction separation, 2. spawn culture, and 3. high temperature acclimation, 4. strain expanded culture behind the high temperature acclimation 5. mixes, 6. drying, 7. packing;
Existing combined process schema is described in detail the concrete operations and the processing condition of each step in the flow process.
(1) bacterial classification extraction separation: get the good natural lactic acid fermentation goods of quality, as poor capsicum or pickles or sour milk, get wherein 10g and be positioned in the distilled water dilution 1000-10000 doubly, dip in and get diluent MRS solid culture primary surface line on flat board under method of scoring and the spirit lamp, solid MRS substratum after the line is positioned over 37.5 ℃ of incubators cultivates 12h-36h, observe thalli morphology on the substratum, the thalline that form on the substratum is different is streak culture on the flat board that the MRS solid medium is arranged respectively again with aforesaid method once more.And the like, until the bacterial classification on the substratum be purebred till;
(2) spawn culture: pure strain in the aseptic processing room, under spirit lamp, is transferred in the MRS solid medium with method of scoring, be positioned under 36.5 ℃ of-38.5 ℃ of conditions of anaerobic incubator and cultivate 12h-36h;
(3) high temperature acclimation:
1.: strain expanded culture before the domestication: bacterial classification is transferred on the domestication substratum that contains MRS liquid nutrient medium, protective material and grain infusion liquid, be positioned under 35 ℃ of-40 ℃ of incubator oxygen free conditions and cultivate, incubation time is 12h-36h;
2. high temperature ladder domestication: well-grown bacterial classification transferring under the aseptic condition in blank domestication substratum, is put in 40 ℃ of incubator oxygen free conditions and cultivates 12h～36h.Then that growing way is good bacterial strain is transferred on another blank domestication substratum, is put in 45 ℃ of incubator oxygen free conditions and cultivates 12h～36h, increases progressively 5 ℃ of culture temperature successively, and final is the purpose bacterial classification in the 65 ℃ of bacterial classifications that still can grow under oxygen free condition;
(4) domestication back strain expanded culture: will in the grain infusion liquid after 65 ℃ are seeded to sterilization under still can the bacterial classification aseptic condition of normal growth, under 35 ℃ of-40 ℃ of temperature, cultivate 12h-36h;
(5) mix: solid food baking soda carrier is pressed 1: 1 mixed after the grain infusion liquid of above-mentioned domestication enlarged culturing and the slaking, and adds protective material trehalose and the mixture weight 2%-4% vehicle Na-alginate of mixture weight 2%-3%, stirring and evenly mixing;
(6) drying: said mixture is put in 50 ℃ of-70 ℃ of baking ovens or the drying room dries, time of drying 12h-36h;
(7) packing: the mixture after will drying, carry out vacuum packaging with laminated film, vacuum tightness is 0.085Mpa-0.095Mpa;
The MRS solid medium is general general substratum,
The method for making of MRS solid medium is: get beef protein powder 10g, beef extract 10g, yeast extract 5g, glucose 20g, sodium-acetate 5g, dibasic ammonium citrate 2g, tween 80 0.1ml, sal epsom 0.58g, manganous sulfate 0.28g, purified agar powder 15g, aquae destillata 1000ml mixing, with pressure kettle 121 ℃ the sterilization 20min-30min, regulate pH6.2～6.4, fall on flat board, after treating the MRS condensation cured, become the MRS solid medium.
In the raw material of preparation MRS solid medium, if do not add purifying agar-agar powder, the MRS substratum that then processes claims MRS liquid nutrient medium, and adding protective material and grain infusion liquid claim to tame substratum in the MRS liquid nutrient medium.
The method of scoring of indication of the present invention is meant to dip in gets bacterial classification at the S-shaped curve paddling of media surface, and the bacterium colony that newly grows is the S curve-like.
The domestication substratum of indication of the present invention is meant the substratum that is added with protective material trehalose and grain infusion liquid in the MRS liquid nutrient medium.
The invention solves a living lactic acid bacteria exsiccant survival difficult problem at high temperature, avoided living lactic acid bacteria 37 ℃-38 ℃ dry down are bacterial contamination easily and lyophilize needs advanced expensive drying facility problem.Technological adaptability is strong, promotes easily, can reduce production costs greatly.Present technique provides living lactic acid bacteria bacterium powder, not only be convenient to make the various solid foods that are rich in living lactic acid bacteria, but also can be directly ferment as the strain excellent of lactic acid fermentation product, overcome in present domestic most lactic acid fermentation product (as poor capsicum, bubble green pepper, fermentation sauce cayenne, pickles etc.) traditional processing technology and adopted raw material surface attachment milk-acid bacteria to carry out spontaneous fermentation, caused that living contaminants is serious, quality product is low and unsettled technical barrier; Simplified in the yoghurt production process and to have adopted each production behind the bacterial classification cryopreservation all will carry out the technology of strain expanded culture.
The present invention compared with prior art has following characteristics
1. the bacterial classification through prepared of the present invention makes up bacterial classification for the pure milk-acid bacteria that does not contain any assorted bacterium, goods of the present invention not only can be used as additive, be convenient to make the various solid foods that are rich in living lactic acid bacteria, the more important thing is that can be used as pure excellent species combination is used for fermented capsicum, the industrial production of fruits and vegetables, sour milk, and guarantee that product can meet the food sanitation requirement.
2. the solid siccative of the present invention after with slaking is lactic acid bacteria vector, and longer-term is preserved at normal temperatures, is convenient to transportation, is convenient to be developed to commodity.
3. the present invention adopts high temperature acclimation bacterial classification means especially, thereby makes bacterial classification in the normal temperature and pressure drying process, can guarantee high viability, and reduces assorted bacterium.
4. the substratum that enlarged culturing of the present invention adopts, compound method is simple, and price is low, can reduce production costs;
5. the present invention need not any large-scale or advanced drying plant, less investment, and cost is low, and technology is easily promoted.
6. the bacterial strain produced of the present invention can be directly used in the fermentation or the production of relevant lactic acid fermentation product after activated, has overcome the deficiency of traditional spontaneous fermentation.
Description of drawings:
Figure of description is a process flow sheet of the present invention; be followed successively by 1. bacterial classification extraction separation; 2 spawn culture, 3. high temperature acclimation will add protective material and grain infusion liquid in the high temperature acclimation substratum; 4. strain expanded culture behind the high temperature acclimation; 5. mix: with after carrier mixed by weight 1: 1, adding protective material and vehicle stir with the grain infusion liquid of strain expanded culture behind the high temperature acclimation; 6. dry, 7. pack.
Embodiment:
Embodiment 1, combined process schema are tested each the process condition in the flow process, be raw material with poor capsicum, pickling pepper, sauce cayenne, sour milk respectively, therefrom extract milk-acid bacteria, high, medium and low three train values of taking technique parameter value table are tested (as following table) respectively, embodiment 1 gets (high row) value, embodiment 2 gets (middle row) value, and embodiment 3 gets (low row) value.
The processing parameter list of values
Embodiment 1 concrete operations are as follows: (height) value during test operation in each step unified taking technique parameter value table.
1. bacterial classification extraction separation: get pickling pepper or sauce cayenne or poor capsicum or sour milk 10g, after grinding, be positioned in the doubly heavy distilled water of 1000 (high row), dilution is dipped in and is got diluent MRS solid culture primary surface line on flat board under method of scoring and the spirit lamp, substratum after the line is positioned over 37.5 ℃ of incubators, incubation time 36h, observe thalli morphology on the substratum, the thalline that form on the substratum is different is streak culture on the flat board that the MRS solid medium is arranged respectively again with aforesaid method once more, and the like, until the bacterial classification on the substratum be purebred till;
2 spawn culture: pure strain in the aseptic processing room, under spirit lamp, is transferred in the MRS solid medium with method of scoring, be positioned over the anaerobic incubator and under 36.5 ℃ of (high row) conditions, cultivate incubation time 36h (high row);
3. high temperature acclimation:
1.: strain expanded culture before the domestication: step 2 gained bacterial classification is transferred in the test tube of domestication substratum, be put in 35 ℃ of (high row) incubator oxygen free conditions and cultivate down, incubation time is 36h (high row);
2. high temperature ladder domestication: well-grown bacterial classification is being transferred under the aseptic condition on the domestication substratum, be put in 40 ℃ of incubator oxygen free conditions and cultivate 36h (high row), then that growing way is good bacterial strain is transferred on another blank domestication substratum, be put in 45 ℃ of incubator oxygen free conditions and cultivate 36h (high row), increase progressively 5 ℃ of culture temperature successively, final is the purpose bacterial classification in the 65 ℃ of bacterial classifications that still can grow under oxygen free condition;
4. domestication back strain expanded culture: will in rice infusion liquid after 65 ℃ are seeded to sterilization under still can the bacterial classification aseptic condition of normal growth or suspension liquid, cultivate 36h (high row) at 35 ℃ (high row);
5. mix:, and add protective material trehalose and 2% (high being listed as) the vehicle Na-alginate of mixture heavy 2% (high row), stirring and evenly mixing the bacterial classification and the mixed of cooked rice flour noodles carrier of suspension liquid enlarged culturing by 1: 1.
6. dry: as, to be put in 50 ℃ of (high row) baking ovens or the drying room and to dry, 36h time of drying (high row) with said mixture.
7. pack: the milk-acid bacteria after will drying, carry out vacuum packaging with laminated film, vacuum tightness is 0.085Mpa (high row).
In aforesaid operations, be marked with (height) word, the high limit data in the expression taking technique parameter value table, several own substratum method for makings of developing are described below respectively in the operation:
Domestication medium preparation: in the MRS of above-mentioned no purified agar powder substratum (claiming the MRS liquid nutrient medium), add trehalose 2%-3% (weight percent), rice infusion liquid or suspension liquid 10%-20% (weight percent), fully after the dissolving, regulate pH to 6.2～6.4, to tame the substratum test tube of packing into, every test tube is adorned 5ml-10ml respectively, clogs the mouth of pipe with tampon; 7-10 test tube is that a bundle encases the test tube mouth with newspaper, puts into pressure kettle at 121 ℃ of 20min-30min that sterilize down, cools off standby.
Preparation rice infusion liquid: be equipped with 10000g water with the 1000g rice, boil, behind the infusion 20min-40min, behind quick filter paper filtering, place pressure kettle in 121 ℃ of 20min-30min that sterilize down, cooling back gained liquid is rice infusion liquid and claims suspension liquid again.
Cooked rice flour noodles preparing carriers: the rice that takes by weighing 500g-1000g, according to 1: 3-1: 5 ratio adds entry, cook into meal, with rice grain in 50 ℃ of-70 ℃ of oven dry, with common powder beater it being ground into powdery granule 60～80 orders then sieves, also the filter residue that the filtration of preparation suspension liquid can be obtained uses the common flour flour broken 50 ℃～70 ℃ oven dry then, and 60～80 orders sieve.
All expression technology that (high row) are arranged of indicating is counted and is got high columns value in the parameter value table in the concrete operations, at the domestication substratum, and rice infusion liquid, the cooked rice flour noodles preparing carriers is all got high numerical value in embodiment 1.
All processing parameters high numerical value in the taking technique parameter value table all among the embodiment 1.
The bacterial classification survival rate is more than 75% in the bacterium powder as a result to implement 1, and the bacterial classification number average can reach 10
6～10
7More than/the g.
Embodiment 2, and concrete operations are with embodiment 1, and just processing condition numerical value of (the middle row) in the taking technique parameter value table is all got intermediate value in the processing condition of domestication substratum, rice infusion liquid, cooked rice flour noodles carrier.
The bacterial classification survival rate is more than 75% in the experimental result bacterium powder, and the bacterial classification number average can reach 10
7～10
8More than/the g.
Embodiment 3, and concrete operations are with embodiment 1, and just processing condition low columns value in the taking technique parameter value table is all got lower bound numerical value in the processing condition of domestication substratum, suspension liquid, cooked rice flour noodles carrier.
The bacterial classification survival rate is more than 75% in the experimental result bacterium powder, and bacterial classification all can reach 10
6～10
7More than/the g.
In the concrete implementing process of the present invention step 5 " mixing " technology, except that bacterial classification, trehalose, sodium alginate with the suspension liquid enlarged culturing directly join in proportion stir in the carrier, also trehalose, sodium alginate, carrier can be proportioned stir after, directly be sprayed onto on the mixed carrier with the bacterial classification of atomizer, then at 60 ℃ of oven dry 12h-36h rice suspension liquid enlarged culturing.
In specific embodiment of the invention processing step 6 " drying " technology, the mixture after the mixing and stirring is taked spraying drying 5S～15S under 130 ℃～160 ℃ temperature, lactic acid bacteria number can be higher.
" bacterial classification extraction separation step can without levigate, directly be diluted by the sampling of pickles solid-to-liquid ratio pickles, " is if solid sample is levigate before dilution in the concrete operations step.
The biodiasmin powder that obtains with the present invention is used for capsicum fermented product, pickles and other lactic acid fermentation product, the nutrition of product, local flavor, color and luster are good, sanitary index meets the food sanitation requirement fully, and the like product quality that the quality basically identical of different batches product, different manufacturers are made of this bacterium powder is basically identical also.
Can preserve more than 6 months at normal temperatures with the biodiasmin powder that the present invention obtains.
Time h representative hour in the specification sheets, s represent second.
The present invention also once replaced rice infusion liquid with other grain infusion liquid such as corn, potato, jowar etc., was not so good as rice infusion liquid though can implement effect, here enumerated data one by one not.
The present invention also once used other ripe grain powder such as corn, and potato, jowar etc. replace the cooked rice powder, but effect is not as the cooked rice flour noodles carrier.
Claims (4)
1. the method for a producing active lactic acid bacteria powder with atmosphere pressure desiccation, technical process is followed successively by: (1) bacterial classification extraction separation, (2) spawn culture, (3) high temperature acclimation, strain expanded culture behind (4) high temperature acclimation, mix (5), (6) drying, (7) packing;
Processing step and concrete operations are:
(1) bacterial classification extraction separation: get the good natural lactic acid fermentation goods of quality, poor capsicum or pickles or sour milk, get wherein 5～15g and be positioned in the distilled water dilution 1000-10000 doubly, dip in and get diluent usefulness method of scoring in MRS solid culture primary surface line on flat board under the spirit lamp, MRS solid medium after the line is positioned over 37.5 ℃ of incubators cultivates 12h-36h, observe thalli morphology on the substratum, the thalline that form on the substratum is different is streak culture on the flat board that the MRS solid medium is arranged respectively again with aforesaid method once more, and the like, until the bacterial classification on the substratum be purebred till;
(2) spawn culture: pure strain in the aseptic processing room, under spirit lamp, is transferred in the MRS solid medium with method of scoring, be positioned under 36.5 ℃ of-38.5 ℃ of conditions of anaerobic incubator and cultivate 12h-36h;
(3) high temperature acclimation:
1.: strain expanded culture before the domestication: bacterial classification is transferred on the domestication substratum that MRS liquid nutrient medium, protective material and grain infusion liquid are housed, be positioned under 35 ℃ of-40 ℃ of incubator oxygen free conditions and cultivate, incubation time is 12h-36h;
2. high temperature ladder domestication: well-grown bacterial classification is being transferred under the aseptic condition in the above-mentioned domestication substratum of blank, be put in 40 ℃ of incubator oxygen free conditions and cultivate 12h～36h, then that growing way is good bacterial strain is transferred on another blank above-mentioned domestication substratum, be put in 45 ℃ of incubator oxygen free conditions and cultivate 12h～36h, increase progressively 5 ℃ of culture temperature successively, final is the purpose bacterial classification in the 65 ℃ of bacterial classifications that still can grow under oxygen free condition;
(4) domestication back strain expanded culture: will in the grain infusion liquid after 65 ℃ are seeded to sterilization under still can the bacterial classification aseptic condition of normal growth, under 35 ℃～40 ℃ temperature, cultivate 12h-36h;
(5) mix: the grain infusion liquid of above-mentioned domestication enlarged culturing and cooked rice flour noodles carrier are pressed 1: 1 mixed, cooked rice flour noodles preparing carriers: the rice that takes by weighing 500g-1000g, according to 1: 3-1: 5 ratio adds entry, cook into meal, rice grain in 50 ℃ of-70 ℃ of oven dry, is ground into powdery granule 60～80 orders with common powder beater with it then and sieves; Also the filter residue that the well-done liquid filtration of preparation rice can be obtained is 50 ℃～70 ℃ oven dry, pulverize with common pulverizer then, 60～80 orders sieve and get the cooked rice flour noodles carrier, add protective material trehalose and the mixture weight 2%-4% vehicle sodium alginate of mixture weight 2%-3% at last, stirring and evenly mixing;
(6) drying: said mixture, be put in 50 ℃ of-70 ℃ of baking ovens or the drying room and dry, time of drying 12h-36h;
(7) packing: the mixture after will drying, carry out vacuum packaging with laminated film, vacuum tightness is 0.085Mpa-0.095Mpa.
2. the method for a kind of producing active lactic acid bacteria powder with atmosphere pressure desiccation according to claim 1 is characterized in that each process condition is:
(1) bacterial classification extraction separation: extracting lactic acid fermented product 10g, dilute 5000 times;
(2) spawn culture: pure strain in the aseptic processing room, under spirit lamp, is transferred in the MRS solid medium with method of scoring, be positioned under 37.5 ℃ of conditions of anaerobic incubator and cultivate 24h;
(3) high temperature acclimation:
1.: strain expanded culture before the domestication: 37.5 ℃ of culture temperature, incubation time are 24h, 2. the high temperature acclimation gradient: 5 ℃ of high temperature acclimation gradients, 40～65 ℃ of high temperature acclimation scopes, each temperature incubation time 24h;
(4) domestication back strain expanded culture: 37.5 ℃ of incubation time 24h of culture temperature;
(5) mix: the protective material trehalose is 2.5% of a mixture weight, and the vehicle sodium alginate is 3% of a mixture weight;
(6) drying: 60 ℃ of drying temperatures, time of drying 24h;
(7) packing: vacuum tightness is 0.090Mpa.
3. the method for a kind of producing active lactic acid bacteria powder with atmosphere pressure desiccation according to claim 1 and 2 is characterized in that carrier is the cooked rice flour noodles carrier, and the substratum of domestication back strain expanded culture is a rice infusion liquid, and the substratum of high temperature acclimation is the domestication substratum.
4. the method for a kind of producing active lactic acid bacteria powder with atmosphere pressure desiccation according to claim 3 is characterized in that the preparation method of above-mentioned substratum has:
The domestication medium preparation: the MRS substratum of no purified agar powder claims MRS liquid nutrient medium, in the MRS liquid nutrient medium, add the trehalose of weight percent 2%-3%, the rice infusion liquid of weight percent 10%-20%, fully after the dissolving, regulate pH to 6.2～6.4, to tame the substratum test tube of packing into, every test tube is adorned 5ml-10ml respectively, clogs the mouth of pipe with tampon; 7-10 test tube is that a bundle encases the test tube mouth with newspaper, puts into pressure kettle at 121 ℃ of 20min-30min that sterilize down, cools off standby;
The preparation of rice infusion liquid: be equipped with 10000g water with the 1000g rice, under the 1000W power electric furnace, behind the infusion 20min-40min, behind quick filter paper filtering, get filtrate and place pressure kettle in 121 ℃ of 20min-30min that sterilize down, cooling back gained liquid is rice infusion liquid.
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| CN102229904B (en) * | 2011-06-08 | 2012-08-29 | 东北农业大学 | Microbe starter culture for producing conjugated linoleic acid and preparation method thereof |
| CN102406104B (en) * | 2011-11-24 | 2013-05-22 | 西华大学 | Szechuan pickled tuber vegetable fermentation composite microbial agent and preparation method thereof |
| CN102492611B (en) * | 2011-12-31 | 2013-10-23 | 常州益菌加生物科技有限公司 | Fermentation tank, equipment system for preparing probiotics and method for preparing probiotics |
| CN104664300A (en) * | 2015-03-27 | 2015-06-03 | 四川东坡中国泡菜产业技术研究院 | Pickle fermentation promoter and preparation method thereof |
| CN105018379B (en) * | 2015-07-16 | 2018-12-28 | 山东凤凰生物有限公司 | One plant of active lactobacillus plantarum of tool high anti-oxidation and its application |
| CN105039235B (en) * | 2015-08-31 | 2018-09-25 | 广东省农业科学院农业资源与环境研究所 | A kind of domestication and culture method of grate sulfur thiobacillus applied to high temperature aerobic composting |
| CN108865922A (en) * | 2018-05-02 | 2018-11-23 | 山东出入境检验检疫局检验检疫技术中心 | listeria monocytogenes standard sample and preparation method thereof |
| CN115594308B (en) * | 2021-06-28 | 2024-07-09 | 中国石油化工股份有限公司 | High-temperature fungus domestication method and biochemical device |
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