CN104651262A - Lactobacillus plantarum freeze-drying preparation as well as preparation method and application thereof - Google Patents
Lactobacillus plantarum freeze-drying preparation as well as preparation method and application thereof Download PDFInfo
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- CN104651262A CN104651262A CN201410790557.XA CN201410790557A CN104651262A CN 104651262 A CN104651262 A CN 104651262A CN 201410790557 A CN201410790557 A CN 201410790557A CN 104651262 A CN104651262 A CN 104651262A
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- 240000006024 Lactobacillus plantarum Species 0.000 title claims abstract description 9
- 238000004108 freeze drying Methods 0.000 title abstract description 39
- 235000013965 Lactobacillus plantarum Nutrition 0.000 title abstract 5
- 229940072205 lactobacillus plantarum Drugs 0.000 title abstract 5
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- 229940039696 lactobacillus Drugs 0.000 claims description 86
- 239000002054 inoculum Substances 0.000 claims description 17
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- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 2
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- 201000010538 Lactose Intolerance Diseases 0.000 description 1
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- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- LPQOADBMXVRBNX-UHFFFAOYSA-N ac1ldcw0 Chemical compound Cl.C1CN(C)CCN1C1=C(F)C=C2C(=O)C(C(O)=O)=CN3CCSC1=C32 LPQOADBMXVRBNX-UHFFFAOYSA-N 0.000 description 1
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- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention discloses a lactobacillus plantarum freeze-drying preparation as well as a preparation method and an application of the lactobacillus plantarum freeze-drying preparation. The preparation method comprises the following steps: (1) inoculating lactobacillus plantarum into a tomato juice culture medium to be cultured for 6-12 hours at the temperature of 35-40 DEG C and collecting a fermentation liquid; and (2) regulating a pH value of the obtained fermentation liquid to be 6.5-7.5, and freezing and drying to obtain the freeze-drying preparation. The preparation method is simple in process; and the obtained lactobacillus plantarum freeze-drying preparation has the advantages of high survival rate of the included bacterial strains and high stability in storage.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of plant lactobacillus freeze-dried preparation and its preparation method and application.
Background technology
Milk-acid bacteria is the critical bacterial populations in human intestine, research shows, milk-acid bacteria can make the formation of intestinal microflora that useful change occurs, suppress the breeding of spoilage organism, recover colony balance in human intestinal, form antibacterial biological barrier, and clear up the toxin of spoilage organism generation, remove enteron aisle rubbish, safeguard HUMAN HEALTH.Secondly, the fermentating metabolism product of milk-acid bacteria can alleviate gastric acid secretion, and has good hormesis to intestines wall nerve, can promote the secretion of human digestive enzymes and the wriggling of enteron aisle, thus promote absorbing protein, monose and the nutritive substance such as calcium, magnesium, and prevention is just secreted.In addition, milk-acid bacteria improve lactose intolerance, reducing blood-fat, hypotensive, strengthen body immunity and resistibility, anti-ageing, antitumor etc. in all play active effect.
Vacuum Freezing & Drying Technology is frozen into solid-state at lower temperature (being generally-10 DEG C ~-50 DEG C) by wet stock or solution, then moisture is wherein made to be directly sublimed into gaseous state without liquid state under vacuum, finally make the dry technology of material dewatering, milk-acid bacteria keeps higher physiological property by this dry technology, and can directly in input food production.In order to strengthen the survival performance of bacterial strain in freeze-drying process, except to except the optimization of freeze-dry process, the selection of lyophilized vaccine is also the important external factor affecting cell stability in milk-acid bacteria drying process.At present; conventional lyophilized vaccine has trehalose, lactose, sucrose, D-glucitol, skim-milk, dextrin etc.; and; the formula involved by preparation of existing lactic acid bacteria freeze drying preparation is quite complicated; a formula often relates to multiple lyophilized vaccine; the complicated security of formula to lyophilized vaccine raw material is had higher requirement, and considerably increases the risk polluted in preparation process simultaneously, and then constitutes potential threat to human consumer.In addition; along with improving constantly of quality of life; the food-safety problem of the lyophilized vaccine (dextrin etc.) that the lactic acid bacteria formulation closely bound up with our everyone daily life adopts more and more is subject to the attention of human consumer, but very deficient natural, safe, healthy plant lactobacillus freeze-dried preparation in the market.Therefore, find that source is natural, easy and simple to handle, the preparation method of survival rate is high, storage stability is good novel lactic acid bacteria express freeze-dried preparation will be one of following lactic acid bacteria freeze drying preparation important research direction of preparing.
Summary of the invention
Therefore, the present invention lacks the problem of the plant lactobacillus freeze-dried preparation that source is natural, easy and simple to handle, probiotic bacterium survival rate is high, storage stability is good at present in order to solve, provide a kind of composite plant Bacterium lacticum freeze-dried preparation and its preparation method and application.
The present inventor finds, due to existing lactic acid bacteria freeze drying preparation preparation involved by formula quite complicated, just as described in the background section, comprise trehalose, lactose, sucrose, D-glucitol, skim-milk, dextrin etc., because the formula of freeze-dried preparation is complicated, the security of plant lactobacillus freeze-dried preparation raw material is had higher requirement, considerably increase the risk producing pollution in preparation process simultaneously, and then potential threat is constituted to human consumer, the Consciousness of food security that this present situation and current consumer strengthen day by day constitutes great contradiction and conflicts, in order to solve this contradiction, contriver is to the training method of milk-acid bacteria particularly plant lactobacillus, comprise the selection of substratum, the temperature of fermentation culture, a series of technical parameter such as time has carried out conscientious analysis and screening, the raw material sources finally obtaining technical solutions according to the invention and gained plant lactobacillus freeze-dried preparation are natural, its preparation method simple process, in gained plant lactobacillus freeze-dried preparation, Strain survival rate is high, the technique effect that storage stability is good.
Therefore, for solving the problems of the technologies described above, one of technical scheme that the present invention takes is: a kind of preparation method of plant lactobacillus freeze-dried preparation, and it comprises the following steps:
(1) be inoculated in tomato juice substratum by plant lactobacillus and cultivate, culture temperature is 35 ~ 40 DEG C, cultivates 6 ~ 12 hr collections fermented liquids;
(2) step (1) gained fermented liquid pH value is adjusted to 6.5 ~ 7.5, lyophilize and get final product.
Wherein step (1) is: be inoculated in by plant lactobacillus in tomato juice substratum and cultivate, and culture temperature is 35 ~ 40 DEG C, and the time is 6 ~ 12 hr collections fermented liquids.Wherein said plant lactobacillus is preferably plant lactobacillus (L.plantarum) ST-III, plant lactobacillus ATCC14917, plant lactobacillus WCFS1, plant lactobacillus P8.Above-mentioned several plant Bacterium lacticum is prior art, and its preparation method is this area customary preparation methods, or obtains by buying.Wherein said tomato juice substratum is the tomato juice substratum of this area routine, described tomato juice substratum is preferably prepared by comprising the method that is made up of following steps and obtain: clean mature tomato, peeling, squeezes the juice, boils after filtration, 4, the centrifugal 10min of 000-12,000g, gets supernatant, sterilizing, cools and get final product.Wherein said filtration is preferably employing 100 order filtered through gauze, and the time of boiling is preferably 1-10 minute, and described sterilising temp is preferably 110-135 DEG C, and sterilization time is preferably 10-30 minute.The culture temperature of wherein said lactobacillus plantarum ST-III is preferably 37 DEG C, and the time is preferably 8 hours.Wherein said culture temperature is preferably 37 DEG C, and the time is preferably 8 hours, and the inoculum size of described plant lactobacillus is preferably 0.5%-5%, and described per-cent is volume percent.
Wherein step (2) is: step (1) gained fermented liquid pH value is adjusted to 6.5 ~ 7.5, lyophilize and get final product.The inflation method of wherein said pH value is the pH adjustment method of this area routine, and preferably for adding food-grade alkali adjusted to ph, described food-grade alkali is preferably: Na
2cO
3, NaHCO
3with one or more in NaOH.Described pH value is preferably adjusted to 7.0.
For solving the problems of the technologies described above, two of the technical scheme that the present invention takes is: a kind of plant lactobacillus freeze-dried preparation, and it is prepared by the following method comprised the following steps and obtains:
(1) be inoculated in tomato juice substratum by plant lactobacillus and cultivate, culture temperature is 35 ~ 40 DEG C, and the time is 6 ~ 12 hr collections fermented liquids;
(2) step (1) gained fermented liquid pH value is adjusted to 6.5 ~ 7.5, lyophilize and get final product.
Plant lactobacillus total viable count>=10 in plant lactobacillus freeze-dried preparation of the present invention
10cfu/g.
For solving the problems of the technologies described above, three of the technical scheme that the present invention takes is: a kind of plant lactobacillus freeze-dried preparation described above is preparing the application in food or dietary supplement.
The application of the field routine that bases on practicality of the present invention, described application preferably comprises and utilizes composite plant Bacterium lacticum freeze-dried preparation of the present invention to prepare leavened food, purposes in milk-product, or using gained composite plant Bacterium lacticum freeze-dried preparation as the purposes such as dietary supplement or nutritional additive.
On the basis meeting this area general knowledge, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
Positive progressive effect of the present invention is:
1, the raw material sources of preparation method's employing of plant lactobacillus freeze-dried preparation of the present invention are natural, and compared with the compound prescription that existing freeze-dried preparation technology is used, the freeze-dried preparation prepared has higher food safety;
2, preparation method's simple process of plant lactobacillus freeze-dried preparation of the present invention, eliminates the step of microorganism collection in conventional freeze dry preparation preparation technology, the physical abuse caused when decreasing microorganism collection to the full extent on the one hand; On the other hand, eliminate the step that the composite of follow-up lyophilized vaccine is resuspended with thalline, greatly reduce the possibility polluted in freeze-dried preparation preparation process.
3, in the plant lactobacillus freeze-dried preparation adopting preparation method of the present invention to obtain, Strain survival rate is high, storage stability is good, it can be used as a kind of novel preparation method and is applied in plant lactobacillus freeze-dried preparation suitability for industrialized production and association area, and application prospect is very wide.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.Room temperature described in the present invention refers to the temperature of carrying out the operation room tested, and is generally 15-25 DEG C.
The mensuration of the preparation of embodiment 1 plant lactobacillus freeze-dried preparation and survival rate, viable count
1, materials and methods
The preparation of tomato juice substratum: mature tomato cleans, peeling, juice extractor squeezes, and 100 order filtered through gauze get juice, boil 5min, and 8000g is centrifugal, and 10min gets supernatant, and 121 DEG C of sterilizing 20min, are cooled to room temperature, obtain aseptic tomato juice substratum.
The preparation of seed (fermented bacterium): (deposit number of described plant lactobacillus is CGMCC No.084 by L.plantarum ST-III, the source of this bacterial strain refers to the Chinese patent that publication number is CN 102604833A) lyophilized powder dissolve with a small amount of sterile distilled water, getting a ring with transfering loop lines on MRS solid medium (Merck Co. Germany), 37 DEG C of Anaerobic culturel 24h take out, 1mL MRS liquid (Merck Co. Germany) is put into transfering loop picking list bacterium colony, vortex oscillator is used to be dispersed in liquid nutrient medium by bacterium colony, 37 DEG C of Anaerobic culturel 24h take out, 50mLMRS liquid is inoculated in 2% (v/v) inoculum size, after 37 DEG C of cultivation 24h, culture 9, centrifugal 10 minutes of 000rpm, supernatant discarded, after thalline sterile distilled water washs 2 times, suspend with the sterile distilled water of former volume of culture, obtain the seed fermented.
2, the preparation of plant lactobacillus freeze-dried preparation
(1) by L.plantarum ST-III normal condition ferment, namely with inoculum size 2% (v/v) aseptic inoculation in aseptic tomato juice substratum, 37 DEG C cultivate 8 hours obtain fermented liquid.
(2) fermented liquid is regulated pH to 7.0 with aseptic food grade alkali lye, after lyophilize, namely obtain the freeze-dried preparation S1 of ST-III.
3, the mensuration of plant lactobacillus freeze-dried preparation survival rate, viable count
In freeze-dried preparation S1 prepared by aforesaid method, add sterile distilled water, the volume before making volume be reduced to freeze-drying, adopt the plant lactobacillus viable count in MRS plate count method analytical unit volume (being generally every milliliter).Quality (g/mL) after lyophilized powder viable count (cfu/g)=unit volume fermented liquid viable count (cfu/mL) ÷ unit volume fermented liquid freeze-drying, theoretical viable count × 100 in the viable count ÷ freeze-drying primary fermentation liquid unit volume after survival rate (%)=freeze-dried preparation reduction volume in unit volume.After measured, the Strain survival rate of ST-III freeze-dried preparation S1 is 95%, and viable count logarithmic value is 10.48 (namely 3.05 × 10
10cfu/g).
The mensuration of the preparation of embodiment 2 plant lactobacillus freeze-dried preparation and survival rate, viable count
1, materials and methods
The preparation of tomato juice substratum: with embodiment 1; The preparation of seed (fermented bacterium): with embodiment 1.
2, the preparation of plant lactobacillus freeze-dried preparation
(1) seed of gained L.plantarum ST-III is fermented with normal condition, namely with inoculum size 0.5% (v/v) aseptic inoculation in aseptic tomato juice substratum, 35 DEG C cultivate 12 hours obtain fermented liquid.
(2) fermented liquid is regulated pH to 6.5 with aseptic food grade alkali lye, after lyophilize, namely obtain the freeze-dried preparation S2 of ST-III.
3, the mensuration of plant lactobacillus freeze-dried preparation survival rate, viable count
In freeze-dried preparation S2 prepared by aforesaid method, add sterile distilled water, the volume before making volume be reduced to freeze-drying, adopt the plant lactobacillus viable count in MRS plate count method analytical unit volume (being generally every milliliter).Quality (g/mL) after lyophilized powder viable count (cfu/g)=unit volume fermented liquid viable count (cfu/mL) ÷ unit volume fermented liquid freeze-drying, theoretical viable count × 100 in the viable count ÷ freeze-drying primary fermentation liquid unit volume after survival rate (%)=freeze-dried preparation reduction volume in unit volume.After measured, the Strain survival rate of ST-III freeze-dried preparation S2 is 93%, and viable count logarithmic value is 10.39 (namely 2.48 × 10
10cfu/g).
The mensuration of the preparation of embodiment 3 plant lactobacillus freeze-dried preparation and survival rate, viable count
1, materials and methods
The preparation of tomato juice substratum: with embodiment 1; The preparation of seed (fermented bacterium): with embodiment 1.
2, the preparation of plant lactobacillus freeze-dried preparation
(1) by L.plantarum ST-III normal condition ferment, namely with inoculum size 5% (v/v) aseptic inoculation in aseptic tomato juice substratum, 40 DEG C cultivate 6 hours obtain fermented liquid.
(2) fermented liquid is regulated pH to 7.5 with aseptic food grade alkali lye, after lyophilize, namely obtain the freeze-dried preparation S3 of ST-III.
3, the mensuration of plant lactobacillus freeze-dried preparation survival rate, viable count
In freeze-dried preparation S3 prepared by aforesaid method, add sterile distilled water, the volume before making volume be reduced to freeze-drying, adopt the plant lactobacillus viable count in MRS plate count method analytical unit volume (being generally every milliliter).Quality (g/mL) after lyophilized powder viable count (cfu/g)=unit volume fermented liquid viable count (cfu/mL) ÷ unit volume fermented liquid freeze-drying.Theoretical viable count × 100 in viable count ÷ freeze-drying primary fermentation liquid unit volume after survival rate (%)=freeze-dried preparation reduction volume in unit volume.After measured, the Strain survival rate in ST-III freeze-dried preparation S3 is 88%, and viable count logarithmic value is 10.14 (namely 1.37 × 10
10cfu/g).
The mensuration of the preparation of embodiment 4 plant lactobacillus freeze-dried preparation and survival rate, viable count
1, materials and methods
The preparation of tomato juice substratum: with embodiment 1.
The preparation of seed (fermented bacterium): by L.plantarum ATCC14917 (buying from ATCC), L.plantarum WCFS1 is (from TI Food and Nutrition, Wageningen, The Netherlands buys), the lyophilized powder of L.plantarum P8 (agricultural university provides by the Inner Mongol) dissolves with a small amount of sterile distilled water, getting a ring with transfering loop lines on MRS solid medium (Merck Co. Germany), 37 DEG C of Anaerobic culturel 24h take out, 1mL MRS liquid (Merck Co. Germany) is put into transfering loop picking list bacterium colony, vortex oscillator is used to be dispersed in liquid nutrient medium by bacterium colony, 37 DEG C of Anaerobic culturel 24h take out, 50mL MRS liquid is inoculated in 2% (v/v) inoculum size, after 37 DEG C of cultivation 24h, culture 9, centrifugal 10 minutes of 000rpm, supernatant discarded, after thalline sterile distilled water washs 2 times, suspend with the sterile distilled water of former volume of culture, obtain the seed fermented.
2, the preparation of plant lactobacillus freeze-dried preparation
(1) the seed normal condition of above-mentioned plant lactobacillus is fermented, namely with inoculum size 2% (v/v) aseptic inoculation in aseptic tomato juice substratum, 37 DEG C cultivate 8 hours obtain fermented liquid.
(2) fermented liquid is regulated pH to 7.0 with aseptic food grade alkali lye, after lyophilize, namely obtain the freeze-dried preparation corresponding to each plant lactobacillus.
3, the mensuration of plant lactobacillus freeze-dried preparation survival rate, viable count
In different plant lactobacillus freeze-dried preparation prepared by aforesaid method, add sterile distilled water, the volume before making volume be reduced to freeze-drying, adopt the plant lactobacillus viable count in MRS plate count method analytical unit volume (being generally every milliliter).Quality (g/mL) after lyophilized powder viable count (cfu/g)=unit volume fermented liquid viable count (cfu/mL) ÷ unit volume fermented liquid freeze-drying.Theoretical viable count × 100 in viable count ÷ freeze-drying primary fermentation liquid unit volume after survival rate (%)=freeze-dried preparation reduction volume in unit volume.Strain survival rate, the viable count of different plant lactobacillus freeze-dried preparation are as shown in table 1.
Strain survival rate, the viable count of the different plant lactobacillus freeze-dried preparation of table 1
As shown in Table 1, the different plant lactobacillus freeze-dried preparation prepared by the present invention are used generally to have very high survival rate and very high viable count characteristic.
The mensuration of the preparation of embodiment 5 plant lactobacillus freeze-dried preparation and survival rate, viable count
1, materials and methods
The preparation of tomato juice substratum: mature tomato cleans, peeling, juice extractor squeezes, and 100 order filtered through gauze get juice, boil 1min, and 4000g is centrifugal, and 10min gets supernatant, and 110 DEG C of sterilizing 30min, are cooled to room temperature, obtain aseptic tomato juice substratum.
The preparation of seed (fermented bacterium): (deposit number of described plant lactobacillus is CGMCC No.084 by L.plantarum ST-III, the source of this bacterial strain refers to the Chinese patent that publication number is CN 102604833A) lyophilized powder dissolve with a small amount of sterile distilled water, getting a ring with transfering loop lines on MRS solid medium (Merck Co. Germany), 37 DEG C of Anaerobic culturel 24h take out, 1mL MRS liquid (Merck Co. Germany) is put into transfering loop picking list bacterium colony, vortex oscillator is used to be dispersed in liquid nutrient medium by bacterium colony, 37 DEG C of Anaerobic culturel 24h take out, 50mLMRS liquid is inoculated in 2% (v/v) inoculum size, after 37 DEG C of cultivation 24h, culture 9, centrifugal 10 minutes of 000rpm, supernatant discarded, after thalline sterile distilled water washs 2 times, suspend with the sterile distilled water of former volume of culture, obtain the seed fermented.
2, the preparation of plant lactobacillus freeze-dried preparation
(1) by L.plantarum ST-III normal condition ferment, namely with inoculum size 0.5% (v/v) aseptic inoculation in aseptic tomato juice substratum, 37 DEG C cultivate 8 hours obtain fermented liquid.
(2) fermented liquid is regulated pH to 7.0 with aseptic food grade alkali lye, after lyophilize, namely obtain the freeze-dried preparation S4 of ST-III.
3, the mensuration of plant lactobacillus freeze-dried preparation survival rate, viable count
In freeze-dried preparation S1 prepared by aforesaid method, add sterile distilled water, the volume before making volume be reduced to freeze-drying, adopt the plant lactobacillus viable count in MRS plate count method analytical unit volume (being generally every milliliter).Quality (g/mL) after lyophilized powder viable count (cfu/g)=unit volume fermented liquid viable count (cfu/mL) ÷ unit volume fermented liquid freeze-drying, theoretical viable count × 100 in the viable count ÷ freeze-drying primary fermentation liquid unit volume after survival rate (%)=freeze-dried preparation reduction volume in unit volume.After measured, the Strain survival rate of ST-III freeze-dried preparation S4 is 91%, and viable count logarithmic value is 10.34 (namely 2.20 × 10
10cfu/g).
The mensuration of the preparation of embodiment 6 plant lactobacillus freeze-dried preparation and survival rate, viable count
1, materials and methods
The preparation of tomato juice substratum: mature tomato cleans, peeling, juice extractor squeezes, and 100 order filtered through gauze get juice, boil 10min, and 8000g is centrifugal, and 10min gets supernatant, and 135 DEG C of sterilizing 100min, are cooled to room temperature, obtain aseptic tomato juice substratum.
The preparation of seed (fermented bacterium): (deposit number of described plant lactobacillus is CGMCC No.084 by L.plantarum ST-III, the source of this bacterial strain refers to the Chinese patent that publication number is CN 102604833A) lyophilized powder dissolve with a small amount of sterile distilled water, getting a ring with transfering loop lines on MRS solid medium (Merck Co. Germany), 37 DEG C of Anaerobic culturel 24h take out, 1mL MRS liquid (Merck Co. Germany) is put into transfering loop picking list bacterium colony, vortex oscillator is used to be dispersed in liquid nutrient medium by bacterium colony, 37 DEG C of Anaerobic culturel 24h take out, 50mLMRS liquid is inoculated in 2% (v/v) inoculum size, after 37 DEG C of cultivation 24h, culture 9, centrifugal 10 minutes of 000rpm, supernatant discarded, after thalline sterile distilled water washs 2 times, suspend with the sterile distilled water of former volume of culture, obtain the seed fermented.
2, the preparation of plant lactobacillus freeze-dried preparation
(1) by L.plantarum ST-III normal condition ferment, namely with inoculum size 5% (v/v) aseptic inoculation in aseptic tomato juice substratum, 37 DEG C cultivate 8 hours obtain fermented liquid.
(2) fermented liquid is regulated pH to 7.0 with aseptic food grade alkali lye, after lyophilize, namely obtain the freeze-dried preparation S5 of ST-III.
3, the mensuration of plant lactobacillus freeze-dried preparation survival rate, viable count
In freeze-dried preparation S5 prepared by aforesaid method, add sterile distilled water, the volume before making volume be reduced to freeze-drying, adopt the plant lactobacillus viable count in MRS plate count method analytical unit volume (being generally every milliliter).Quality (g/mL) after lyophilized powder viable count (cfu/g)=unit volume fermented liquid viable count (cfu/mL) ÷ unit volume fermented liquid freeze-drying, theoretical viable count × 100 in the viable count ÷ freeze-drying primary fermentation liquid unit volume after survival rate (%)=freeze-dried preparation reduction volume in unit volume.After measured, the Strain survival rate of ST-III freeze-dried preparation S5 is 93%, and viable count logarithmic value is 10.39 (namely 2.44 × 10
10cfu/g).
Effect example 1 detects the stability of bacterial strain in gained plant lactobacillus freeze-dried preparation
Plant lactobacillus freeze-dried preparation S1, S2, S3 of being prepared by embodiment 1-3 are distributed into aseptic aluminium foil bag, and normal temperature (25 DEG C) is preserved after 6 months and 12 months, takes out bacterium powder, and adopt MRS colony counting method to measure viable count, result is as shown in table 2.
The Stability Determination that table 2 lactobacillus plantarum ST-III freeze-dried preparation normal temperature is preserved
As shown in Table 2, ST-III freeze-dried preparation of all tests is after normal temperature preserves 12 months, and viable count Absorbable organic halogens remains on 10
8above.
Comparative example 1 detects survival rate and the viable count of bacterial strain in conventional freeze dry preparation
The aseptic tomato juice substratum wherein related to and the preparation method of microorganism seed identical with embodiment 1.
1, traditional method is utilized to prepare freeze-dried preparation:
(1) the protectant preparation of traditional freeze-drying: 7.06% skimming milk, 6.46% maltose and 6.70% Sodium Glutamate are dissolved in distilled water, 115 DEG C of sterilizings 15 minutes and get final product.(Wang Shikuan, Hong Yucheng, Yuan Xianling. response phase method optimizes the research [J] of plant lactobacillus and Bacillus coagulans lyophilized vaccine. Sichuan University of Science & Engineering's journal (natural science edition), 2013,5:23-26.)
(2) L.plantarum ST-III normal condition is fermented, namely with inoculum size 2% (v/v) aseptic inoculation in aseptic tomato juice substratum, cultivate for 37 DEG C and obtain fermented liquid in 8 hours, its 8,000g centrifugal 10min is obtained ST-III bacterial sediment.
(3) ST-III bacterial sediment is resuspended in traditional lyophilized vaccine of same volume, after lyophilize, namely obtains the freeze-dried preparation P2 of ST-III.
2, the mensuration of Strain survival rate, viable count in conventional freeze dry preparation
Example 1 prepares gained plant lactobacillus freeze-dried preparation S1, by its called after P1, in contrast.In freeze-dried preparation P1, P2 prepared by aforesaid method, add sterile distilled water, the volume before making volume be reduced to freeze-drying, adopt the plant lactobacillus viable count in MRS plate count method analytical unit volume (being generally every milliliter).Quality (g/mL) after lyophilized powder viable count (cfu/g)=unit volume fermented liquid viable count (cfu/mL) ÷ unit volume fermented liquid freeze-drying.Theoretical viable count × 100 before viable count ÷ freeze-drying after survival rate (%)=freeze-dried preparation reduction volume in unit volume in bacteria suspension unit volume.The survival rate of plant lactobacillus in freeze-dried preparation of the present invention and conventional freeze dry preparation, viable count are as shown in table 3.
Survival rate, the viable count of plant lactobacillus in table 3 freeze-dried preparation of the present invention and conventional freeze dry preparation
As shown in Table 3, freeze-dried preparation prepared by the inventive method all increases than traditional method in survival rate with viable count.
Comparative example 2 detects survival rate and the viable count of bacterial strain in freeze-dried preparation prepared by normal fermentation liquid
The MRS substratum wherein related to and the preparation method of microorganism seed identical with embodiment 1.
1, normal fermentation liquid is utilized to prepare freeze-dried preparation:
(1) L.plantarum ST-III normal condition is fermented, namely with inoculum size 2% (v/v) aseptic inoculation in MRS substratum, cultivate for 37 DEG C and obtain fermented liquid in 8 hours, its 8,000g centrifugal 10min is obtained ST-III bacterial sediment.
(2) fermented liquid is regulated pH to 7.0 with aseptic food grade alkali lye, after lyophilize, namely obtain the freeze-dried preparation P3 of ST-III.
2, the mensuration of Strain survival rate, viable count in the freeze-dried preparation prepared of normal fermentation liquid
With freeze-dried preparation P1 and P2 described in comparative example 1 in contrast.In freeze-dried preparation P1, P2 and P3 prepared by aforesaid method, add sterile distilled water, the volume before making volume be reduced to freeze-drying, adopt the plant lactobacillus viable count in MRS plate count method analytical unit volume (being generally every milliliter).Quality (g/mL) after lyophilized powder viable count (cfu/g)=unit volume fermented liquid viable count (cfu/mL) ÷ unit volume fermented liquid freeze-drying.Theoretical viable count × 100 before viable count ÷ freeze-drying after survival rate (%)=freeze-dried preparation reduction volume in unit volume in bacteria suspension unit volume.The survival rate of plant lactobacillus in freeze-dried preparation prepared by freeze-dried preparation of the present invention, conventional freeze dry preparation and normal fermentation liquid, viable count are as shown in table 4.
Survival rate, the viable count of plant lactobacillus in freeze-dried preparation prepared by table 4 freeze-dried preparation of the present invention, conventional freeze dry preparation and normal fermentation liquid
Can draw from the result of table 4, the freeze-dried preparation prepared of normal fermentation liquid significantly reduces than the Strain survival rate of preparation method's gained dietary supplements of the present invention and viable count.
Comparative example 3 detects survival rate and the viable count of plant lactobacillus in freeze-dried preparation
The aseptic tomato juice substratum wherein related to and the preparation method of microorganism seed identical with embodiment 1.
By the inoculum size of plant lactobacillus L.plantarum ST-III in embodiment, the pH value of tomato juice substratum, culture temperature, the pH value of incubation time and gained fermented liquid adjusts one by one, obtain the plant lactobacillus freeze-dried preparation prepared with next group different methods, and add sterile distilled water, the volume before making volume be reduced to freeze-drying in the freeze-dried preparation prepared to aforesaid method, adopt the viable count in MRS plate count method analytical unit volume (being generally every milliliter).Quality (g/mL) after lyophilized powder viable count (cfu/g)=unit volume fermented liquid viable count (cfu/mL) ÷ unit volume fermented liquid freeze-drying, viable count × 100 in the fermented liquid unit volume before the viable count ÷ freeze-drying after survival rate (%)=freeze-dried preparation reduction volume in unit volume.Detection acquired results is as shown in table 5:
Table 5 different methods prepares survival rate and the viable count of plant lactobacillus in obtained freeze-drying preparation
Can draw from the result shown in his-and-hers watches 5, by the inoculum size of bacterial classification in the preparation method of described plant lactobacillus freeze-dried preparation, culture temperature, time the pH of incubation time and fermented liquid adjusts to outside the present invention, in obtained freeze-drying agent, the survival rate of microorganism and viable count create remarkable reduction.
Should be understood that those skilled in the art can make various changes or modifications the present invention after having read foregoing of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. a preparation method for plant lactobacillus freeze-dried preparation, is characterized in that, comprises the following steps:
(1) be inoculated in tomato juice substratum by plant lactobacillus and cultivate, culture temperature is 35 ~ 40 DEG C, cultivates 6 ~ 12 hr collections fermented liquids;
(2) step (1) gained fermented liquid pH value is adjusted to 6.5 ~ 7.5, lyophilize and get final product.
2. preparation method as claimed in claim 1, it is characterized in that, plant lactobacillus described in step (1) is plant lactobacillus (L.plantarum) ST-III, plant lactobacillus ATCC14917, plant lactobacillus WCFS1 or plant lactobacillus P8.
3. preparation method as claimed in claim 1, it is characterized in that, the tomato juice substratum described in step (1) is prepared by the method comprised the following steps and obtains: cleaning mature tomato, peeling, squeeze the juice, boil after filtration, 4,000-12, the centrifugal 10min of 000g, get supernatant, sterilizing, cool and get final product.
4. preparation method as claimed in claim 2, it is characterized in that, described filtration adopts 100 order filtered through gauze, and the time of boiling is 1-10 minute, and described sterilizing adopts 110-135 DEG C, sterilizing 10-30 minute.
5. preparation method as claimed in claim 1, it is characterized in that, the culture temperature described in step (1) is 37 DEG C, and the time is 8 hours.
6. preparation method as claimed in claim 1, it is characterized in that, the inoculum size of step (1) described plant lactobacillus is 0.5%-5%, and described per-cent is volume percent.
7. preparation method as claimed in claim 1, it is characterized in that, the pH value described in step (1) is adjusted to 7.0.
8. a plant lactobacillus freeze-dried preparation, is characterized in that, it is that the method comprised the following steps is prepared and obtains:
(1) be inoculated in tomato juice substratum by plant lactobacillus and cultivate, culture temperature is 35 ~ 40 DEG C, and the time is 6 ~ 12 hr collections fermented liquids;
(2) step (1) gained fermented liquid pH value is adjusted to 6.5 ~ 7.5, lyophilize and get final product.
9. plant lactobacillus freeze-dried preparation as claimed in claim 8, it is characterized in that, the culture temperature described in step (1) is 37 DEG C, and the time is 8 hours, and the inoculum size of described plant lactobacillus is 0.5%-5%, and described per-cent is volume percent.
10. one kind as described in claim 8 or 9 plant lactobacillus freeze-dried preparation preparing the application in food or dietary supplement.
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