CN104651262B - A kind of lactobacillus plantarum lyophilized preparation and its preparation method and application - Google Patents
A kind of lactobacillus plantarum lyophilized preparation and its preparation method and application Download PDFInfo
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- CN104651262B CN104651262B CN201410790557.XA CN201410790557A CN104651262B CN 104651262 B CN104651262 B CN 104651262B CN 201410790557 A CN201410790557 A CN 201410790557A CN 104651262 B CN104651262 B CN 104651262B
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- 238000002360 preparation method Methods 0.000 title claims abstract description 174
- 240000006024 Lactobacillus plantarum Species 0.000 title claims abstract description 98
- 235000013965 Lactobacillus plantarum Nutrition 0.000 title claims abstract description 88
- 229940072205 lactobacillus plantarum Drugs 0.000 title claims abstract description 87
- 239000007788 liquid Substances 0.000 claims abstract description 60
- 238000000855 fermentation Methods 0.000 claims abstract description 55
- 230000004151 fermentation Effects 0.000 claims abstract description 55
- 238000000034 method Methods 0.000 claims abstract description 42
- 239000001963 growth medium Substances 0.000 claims abstract description 30
- 235000015193 tomato juice Nutrition 0.000 claims abstract description 29
- 239000012467 final product Substances 0.000 claims abstract description 9
- 241000894006 Bacteria Species 0.000 claims description 28
- 239000002054 inoculum Substances 0.000 claims description 18
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- 238000004140 cleaning Methods 0.000 claims description 5
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- 244000185256 Lactobacillus plantarum WCFS1 Species 0.000 claims description 3
- 235000011227 Lactobacillus plantarum WCFS1 Nutrition 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 241000227653 Lycopersicon Species 0.000 claims 1
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- 230000004083 survival effect Effects 0.000 abstract description 43
- 230000008569 process Effects 0.000 abstract description 4
- 238000003860 storage Methods 0.000 abstract description 3
- 238000004108 freeze drying Methods 0.000 description 52
- 239000012153 distilled water Substances 0.000 description 22
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 22
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- 238000005259 measurement Methods 0.000 description 14
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- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products 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-WSWWMNSNSA-N 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 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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- 241000193749 Bacillus coagulans Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 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
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- 102000038379 digestive enzymes Human genes 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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- 238000007710 freezing Methods 0.000 description 1
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- 210000000936 intestine Anatomy 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
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- 150000002632 lipids Chemical class 0.000 description 1
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- 229910052749 magnesium Inorganic materials 0.000 description 1
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- 239000006041 probiotic Substances 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
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- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
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- 229910000029 sodium carbonate Inorganic materials 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
Landscapes
- 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 kind of lactobacillus plantarum lyophilized preparations and its preparation method and application.The preparation method comprises the following steps: (1) lactobacillus plantarum being inoculated in tomato juice culture medium and cultivated, cultivation temperature is 35~40 DEG C, cultivates 6~12 hours collection fermentation liquids；(2) gained fermentation liquid pH value is adjusted to 6.5~7.5, be freeze-dried to obtain the final product.The preparation method simple process, gained lactobacillus plantarum lyophilized preparation have comprising Strain survival rate it is high, and the advantage that its storage stability is splendid.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of lactobacillus plantarum lyophilized preparation and preparation method thereof and answer
With.
Background technique
Lactic acid bacteria is the critical bacterial populations in human intestine, studies have shown that lactic acid bacteria can be such that the composition of intestinal flora occurs
Beneficial to variation, inhibit the breeding of spoilage organisms, restore colony balance in human body intestinal canal, forms antibacterial biological barrier, and clear up corruption
The toxin that bacterium generates removes enteron aisle rubbish, safeguards human health.Secondly, the fermentating metabolism product of lactic acid bacteria can mitigate gastric acid point
It secretes, and has good stimulation to intestinal wall nerve, the secretion of human digestive enzymes and the wriggling of enteron aisle can be promoted, to promote
The nutriments such as protein, monosaccharide and calcium, magnesium are absorbed and utilized, and prevent just to secrete.In addition, lactic acid bacteria is improving lactose not
Resistance to, reducing blood lipid, blood pressure lowering enhance human immunity and resistance, anti-aging, antitumor etc. all play positive effect.
Vacuum Freezing & Drying Technology be by wet stock or solution under lower temperature (generally -10 DEG C~-50 DEG C)
It is frozen into solid-state, then moisture therein is made to be directly sublimed into gaseous state without liquid under vacuum conditions, finally makes material dewatering
Dry technology, lactic acid bacteria can keep higher physiological property by this dry technology, and can direct plunge into food production.
In order to enhance survival performance of the bacterial strain in freeze-drying process, other than the optimization to lyophilized technique, the selection of freeze drying protectant
It is the important external factor for influencing cell stability in lactic acid bacteria drying process.Currently, common freeze drying protectant have trehalose,
Lactose, sucrose, D-glucitol, skimmed milk power, dextrin etc., also, formula involved in the preparation of existing lactic acid bacteria freeze drying preparation
Considerably complicated, a formula is often related to a variety of freeze drying protectants, safety of the complicated formula to freeze drying protectant raw material
More stringent requirements are proposed, while considerably increasing the risk polluted during the preparation process, and then constitutes to consumer potential
Threat.In addition, with the continuous improvement of quality of life, the closely bound up lactobacillus preparation institute with each of us daily life
The food-safety problem of the freeze drying protectant (dextrin etc.) of use is however non-currently on the market increasingly by the attention of consumer
Often deficient natural, safe and healthy lactobacillus plantarum lyophilized preparation.Therefore, searching source is natural, easy to operate, survival rate is high,
The preparation method of the good novel lactic acid bacteria lyophilized preparation of storage stability by be the preparation of following lactic acid bacteria freeze drying preparation important grinding
Study carefully one of direction.
Summary of the invention
Therefore, source is natural, easy to operate, probiotics survival rate is high, saves and stablizes in order to solve to lack at present by the present invention
Property good lactobacillus plantarum lyophilized preparation the problem of, provide a kind of composite plant lactobacillus lyophilized preparation and preparation method thereof and
Using.
The inventors discovered that the formula as involved in the preparation of existing lactic acid bacteria freeze drying preparation is considerably complicated, as back
Described in scape part, including trehalose, lactose, sucrose, D-glucitol, skimmed milk power, dextrin etc., due to the formula of lyophilized preparation
Complexity, to the safety of lactobacillus plantarum lyophilized preparation raw material, more stringent requirements are proposed, while considerably increasing and preparing
The risk of pollution is generated in journey, and then potential threat is constituted to consumer, and this status is increasingly enhanced with current consumer
Consciousness of food security constitute great contradiction and conflict, in order to solve this contradiction, inventor especially plants lactic acid bacteria
The training method of object lactobacillus, the selection including culture medium, temperature, time of fermented and cultured etc., a series of technical parameters carried out
Conscientious analysis and screening has finally obtained the original of technical solutions according to the invention and gained lactobacillus plantarum lyophilized preparation
Material source is natural, preparation method simple process, Strain survival rate height, storage stability in gained lactobacillus plantarum lyophilized preparation
Good technical effect.
Therefore, in order to solve the above technical problems, the technical solution that the present invention takes first is that: a kind of freeze-drying of lactobacillus plantarum
The preparation method of preparation comprising following steps:
(1) lactobacillus plantarum is inoculated in tomato juice culture medium and is cultivated, cultivation temperature is 35~40 DEG C, culture 6
~12 hours collection fermentation liquids；
(2) fermentation liquid pH value obtained by step (1) is adjusted to 6.5~7.5, be freeze-dried to obtain the final product.
Wherein step (1) are as follows: lactobacillus plantarum is inoculated in tomato juice culture medium and is cultivated, cultivation temperature be 35~
40 DEG C, the time is 6~12 hours collection fermentation liquids.Wherein the lactobacillus plantarum is preferably lactobacillus plantarum
(L.plantarum) ST- III, lactobacillus plantarum ATCC14917, lactobacillus plantarum WCFS1, lactobacillus plantarum P8.It is above-mentioned several
Lactobacillus plantarum is the prior art, and preparation method is this field customary preparation methods, or is obtained by purchase.Wherein institute
The tomato juice culture medium that tomato juice culture medium is this field routine is stated, the tomato juice culture medium is preferably by including by following
The method of step composition is prepared: cleaning mature tomato is removed the peel, and juicing is boiled after filtering, 4,000-12,000g centrifugation
10min takes supernatant, and sterilizing, cooling to obtain the final product.Wherein the filtering preferably uses 100 mesh filtered through gauze, the time boiled
Preferably 1-10 minutes, the sterilising temp was preferably 110-135 DEG C, and sterilization time is preferably 10-30 minutes.Its
Described in the cultivation temperature of lactobacillus plantarum ST-III be preferably 37 DEG C, the time is preferably 8 hours.The wherein culture
Temperature is preferably 37 DEG C, and the time is preferably 8 hours, and the inoculum concentration of the lactobacillus plantarum is preferably 0.5%-5%, institute
Stating percentage is percent by volume.
Wherein step (2) are as follows: fermentation liquid pH value obtained by step (1) is adjusted to 6.5~7.5, is freeze-dried to obtain the final product.Wherein
The method of adjustment of the pH value is the pH adjustment method of this field routine, and preferably addition food-grade alkali adjusts pH value, institute
It states food-grade alkali to be preferably: Na2CO3、NaHCO3With one of NaOH or a variety of.The pH value is preferably adjusted to 7.0.
In order to solve the above technical problems, the technical solution that the present invention takes second is that: a kind of lactobacillus plantarum lyophilized preparation,
It is prepared by the method for following steps included below:
(1) lactobacillus plantarum is inoculated in tomato juice culture medium and is cultivated, cultivation temperature is 35~40 DEG C, and the time is
6~12 hours collection fermentation liquids；
(2) fermentation liquid pH value obtained by step (1) is adjusted to 6.5~7.5, be freeze-dried to obtain the final product.
Lactobacillus plantarum total viable count >=10 in lactobacillus plantarum lyophilized preparation of the present invention10cfu/g。
In order to solve the above technical problems, the technical solution that the present invention takes third is that: a kind of lactobacillus plantarum as described above
Lyophilized preparation is preparing the application in food or dietary supplement.
The application of the field routine of the present invention that bases on practicality, the application are preferably comprised using of the present invention multiple
It closes lactobacillus plantarum lyophilized preparation and prepares fermented food, the purposes in dairy products, or gained composite plant lactobacillus is lyophilized
Preparation is as purposes such as dietary supplement or nutritional additives.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention
Example.
The reagents and materials used in the present invention are commercially available.
The positive effect of the present invention is that:
1, the raw material sources that the preparation method of lactobacillus plantarum lyophilized preparation of the present invention uses are natural, with existing jelly
Compound prescription used in dry preparation technology is compared, and the lyophilized preparation prepared has higher foodsafety；
2, the preparation method simple process of lactobacillus plantarum lyophilized preparation of the present invention, is on the one hand omitted traditional freeze-drying
In preparation process thereof the step of microorganism collection, when reducing microorganism collection to the full extent caused by mechanical damage；Another party
The step of face, the compounding and thallus for eliminating subsequent freeze drying protectant are resuspended, greatly reduces dirty in lyophilized preparation preparation process
A possibility that dye.
3, Strain survival rate is high in the lactobacillus plantarum lyophilized preparation obtained using preparation method of the invention, saves and stablizes
Property it is good, can be used as a kind of novel preparation method and be applied to lactobacillus plantarum lyophilized preparation industrialized production and related fields
In, application prospect is very wide.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
The selection of product specification.Heretofore described room temperature refers to the temperature for the operation room tested, generally 15-25 DEG C.
The preparation of 1 lactobacillus plantarum lyophilized preparation of embodiment and the measurement of survival rate, viable count
1, materials and methods
The preparation of tomato juice culture medium: mature tomato cleaning, peeling, juice extractor squeezing, 100 mesh filtered through gauze take juice, boil
5min is boiled, 8000g centrifugation 10min takes supernatant, and 121 DEG C of sterilizing 20min are cooled to room temperature to get sterile tomato juice culture medium.
The preparation of seed (fermenting microbe): by L.plantarum ST- III, (deposit number of the lactobacillus plantarum is
CGMCC No.084, the source of the bacterial strain refer to the Chinese patent of Publication No. CN 102604833A) freeze-dried powder with a small amount of
Sterile distilled water dissolution takes a ring to line in MRS solid medium (Merck Co. Germany), 37 DEG C of anaerobism trainings with oese
It supports and takes out for 24 hours, be put into 1mL MRS liquid (Merck Co. Germany) with oese picking single colonie, with vortex oscillator by bacterium
It falls and is dispersed in fluid nutrient medium, 37 DEG C of Anaerobic culturels take out for 24 hours, are inoculated in 50mL MRS liquid with 2% (v/v) inoculum concentration
Body, after 37 DEG C of cultures for 24 hours, culture 9,000rpm is centrifuged 10 minutes, is discarded supernatant, after thallus washs 2 times with sterile distilled water,
It is suspended with the sterile distilled water of former volume of culture, obtains the seed of fermentation.
2, the preparation of lactobacillus plantarum lyophilized preparation
(1) III normal condition of L.plantarum ST- is fermented, i.e., with inoculum concentration 2% (v/v) aseptic inoculation in sterile
Tomato juice culture medium, 37 DEG C of cultures, 8 hours acquisition fermentation liquids.
(2) fermentation liquid is adjusted into pH to 7.0 with sterile food grade lye, up to the freeze-drying system of ST- III after freeze-drying
Agent S1.
3, the measurement of lactobacillus plantarum lyophilized preparation survival rate, viable count
Sterile distilled water is added in the lyophilized preparation S1 prepared to the above method, volume is made to be reduced to the volume before freeze-drying,
Using the lactobacillus plantarum viable count in MRS plate count method analytical unit volume (usually every milliliter).Freeze-dried powder viable bacteria
Quality (g/mL) after number (cfu/g)=unit volume zymotic fluid viable count (cfu/mL) ÷ unit volume fermentation liquid freeze-drying, is deposited
The theory in viable count ÷ freeze-drying primary fermentation liquid unit volume after motility rate (%)=lyophilized preparation reduction volume in unit volume
Viable count × 100.After measured, the Strain survival rate of III lyophilized preparation S1 of ST- is 95%, and viable count logarithm is 10.48 (i.e.
3.05×1010cfu/g)。
The preparation of 2 lactobacillus plantarum lyophilized preparation of embodiment and the measurement of survival rate, viable count
1, materials and methods
The preparation of tomato juice culture medium: with embodiment 1；The preparation of seed (fermenting microbe): with embodiment 1.
2, the preparation of lactobacillus plantarum lyophilized preparation
(1) seed of gained L.plantarum ST- III is fermented with normal condition, i.e., with inoculum concentration 0.5% (v/v) nothing
Bacterium is inoculated in sterile tomato juice culture medium, 35 DEG C of cultures, 12 hours acquisition fermentation liquids.
(2) fermentation liquid is adjusted into pH to 6.5 with sterile food grade lye, up to the freeze-drying system of ST- III after freeze-drying
Agent S2.
3, the measurement of lactobacillus plantarum lyophilized preparation survival rate, viable count
Sterile distilled water is added in the lyophilized preparation S2 prepared to the above method, volume is made to be reduced to the volume before freeze-drying,
Using the lactobacillus plantarum viable count in MRS plate count method analytical unit volume (usually every milliliter).Freeze-dried powder viable bacteria
Quality (g/mL) after number (cfu/g)=unit volume zymotic fluid viable count (cfu/mL) ÷ unit volume fermentation liquid freeze-drying, is deposited
The theory in viable count ÷ freeze-drying primary fermentation liquid unit volume after motility rate (%)=lyophilized preparation reduction volume in unit volume
Viable count × 100.After measured, the Strain survival rate of III lyophilized preparation S2 of ST- is 93%, and viable count logarithm is 10.39 (i.e.
2.48×1010cfu/g)。
The preparation of 3 lactobacillus plantarum lyophilized preparation of embodiment and the measurement of survival rate, viable count
1, materials and methods
The preparation of tomato juice culture medium: with embodiment 1；The preparation of seed (fermenting microbe): with embodiment 1.
2, the preparation of lactobacillus plantarum lyophilized preparation
(1) III normal condition of L.plantarum ST- is fermented, i.e., with inoculum concentration 5% (v/v) aseptic inoculation in sterile
Tomato juice culture medium, 40 DEG C of cultures, 6 hours acquisition fermentation liquids.
(2) fermentation liquid is adjusted into pH to 7.5 with sterile food grade lye, up to the freeze-drying system of ST- III after freeze-drying
Agent S3.
3, the measurement of lactobacillus plantarum lyophilized preparation survival rate, viable count
Sterile distilled water is added in the lyophilized preparation S3 prepared to the above method, volume is made to be reduced to the volume before freeze-drying,
Using the lactobacillus plantarum viable count in MRS plate count method analytical unit volume (usually every milliliter).Freeze-dried powder viable bacteria
Quality (g/mL) after number (cfu/g)=unit volume zymotic fluid viable count (cfu/mL) ÷ unit volume fermentation liquid freeze-drying.It deposits
The theory in viable count ÷ freeze-drying primary fermentation liquid unit volume after motility rate (%)=lyophilized preparation reduction volume in unit volume
Viable count × 100.After measured, the Strain survival rate in III lyophilized preparation S3 of ST- is 88%, and viable count logarithm is 10.14 (i.e.
1.37×1010cfu/g)。
The preparation of 4 lactobacillus plantarum lyophilized preparation of embodiment and the measurement of survival rate, viable count
1, materials and methods
The preparation of tomato juice culture medium: with embodiment 1.
The preparation of seed (fermenting microbe): by L.plantarum ATCC14917 (being bought from ATCC), L.plantarum
WCFS1 (being bought from TI Food and Nutrition, Wageningen, The Netherlands), L.plantarum P8
The freeze-dried powder of (agricultural university provides by the Inner Mongol) is dissolved with a small amount of sterile distilled water, takes a ring to line the training of MRS solid with oese
It supports in base (Merck Co. Germany), 37 DEG C of Anaerobic culturels take out for 24 hours, are put into 1mL MRS liquid with oese picking single colonie
Bacterium colony, is dispersed in fluid nutrient medium, 37 DEG C of Anaerobic culturels take for 24 hours by (Merck Co. Germany) with vortex oscillator
Out, 50mL MRS liquid being inoculated in 2% (v/v) inoculum concentration, after 37 DEG C of cultures for 24 hours, culture 9,000rpm is centrifuged 10 minutes,
It discards supernatant, after thallus washs 2 times with sterile distilled water, is suspended with the sterile distilled water of former volume of culture, obtain fermentation
Seed.
2, the preparation of lactobacillus plantarum lyophilized preparation
(1) the seed normal condition of above-mentioned lactobacillus plantarum is fermented, i.e., with inoculum concentration 2% (v/v) aseptic inoculation in nothing
The tomato juice culture medium of bacterium, 37 DEG C of cultures, 8 hours acquisition fermentation liquids.
(2) fermentation liquid is adjusted into pH to 7.0 with sterile food grade lye, up to each lactobacillus plantarum institute after freeze-drying
Corresponding lyophilized preparation.
3, the measurement of lactobacillus plantarum lyophilized preparation survival rate, viable count
Sterile distilled water is added in the different lactobacillus plantarum lyophilized preparations prepared to the above method, volume is made to be reduced to freeze
Volume before dry, using the lactobacillus plantarum viable count in MRS plate count method analytical unit volume (usually every milliliter).
Quality after freeze-dried powder viable count (cfu/g)=unit volume zymotic fluid viable count (cfu/mL) ÷ unit volume fermentation liquid freeze-drying
(g/mL).Primary fermentation liquid unit volume is lyophilized in viable count ÷ after survival rate (%)=lyophilized preparation reduction volume in unit volume
Interior theoretical viable count × 100.The Strain survival rate of different lactobacillus plantarum lyophilized preparations, viable count are as shown in table 1.
The Strain survival rate of the different lactobacillus plantarum lyophilized preparations of table 1, viable count
As shown in Table 1, generally there is very high survival with different lactobacillus plantarums lyophilized preparation prepared by the present invention
Rate and very high viable count characteristic.
The preparation of 5 lactobacillus plantarum lyophilized preparation of embodiment and the measurement of survival rate, viable count
1, materials and methods
The preparation of tomato juice culture medium: mature tomato cleaning, peeling, juice extractor squeezing, 100 mesh filtered through gauze take juice, boil
1min is boiled, 4000g centrifugation 10min takes supernatant, and 110 DEG C of sterilizing 30min are cooled to room temperature to get sterile tomato juice culture medium.
The preparation of seed (fermenting microbe): by L.plantarum ST- III, (deposit number of the lactobacillus plantarum is
CGMCC No.084, the source of the bacterial strain refer to the Chinese patent of Publication No. CN 102604833A) freeze-dried powder with a small amount of
Sterile distilled water dissolution takes a ring to line in MRS solid medium (Merck Co. Germany), 37 DEG C of anaerobism trainings with oese
It supports and takes out for 24 hours, be put into 1mL MRS liquid (Merck Co. Germany) with oese picking single colonie, with vortex oscillator by bacterium
It falls and is dispersed in fluid nutrient medium, 37 DEG C of Anaerobic culturels take out for 24 hours, are inoculated in 50mL MRS liquid with 2% (v/v) inoculum concentration
Body, after 37 DEG C of cultures for 24 hours, culture 9,000rpm is centrifuged 10 minutes, is discarded supernatant, after thallus washs 2 times with sterile distilled water,
It is suspended with the sterile distilled water of former volume of culture, obtains the seed of fermentation.
2, the preparation of lactobacillus plantarum lyophilized preparation
(1) III normal condition of L.plantarum ST- is fermented, i.e., with inoculum concentration 0.5% (v/v) aseptic inoculation in sterile
Tomato juice culture medium, 37 DEG C of cultures, 8 hours acquisition fermentation liquids.
(2) fermentation liquid is adjusted into pH to 7.0 with sterile food grade lye, up to the freeze-drying system of ST- III after freeze-drying
Agent S4.
3, the measurement of lactobacillus plantarum lyophilized preparation survival rate, viable count
Sterile distilled water is added in the lyophilized preparation S1 prepared to the above method, volume is made to be reduced to the volume before freeze-drying,
Using the lactobacillus plantarum viable count in MRS plate count method analytical unit volume (usually every milliliter).Freeze-dried powder viable bacteria
Quality (g/mL) after number (cfu/g)=unit volume zymotic fluid viable count (cfu/mL) ÷ unit volume fermentation liquid freeze-drying, is deposited
The theory in viable count ÷ freeze-drying primary fermentation liquid unit volume after motility rate (%)=lyophilized preparation reduction volume in unit volume
Viable count × 100.After measured, the Strain survival rate of III lyophilized preparation S4 of ST- is 91%, and viable count logarithm is 10.34 (i.e.
2.20×1010cfu/g)。
The preparation of 6 lactobacillus plantarum lyophilized preparation of embodiment and the measurement of survival rate, viable count
1, materials and methods
The preparation of tomato juice culture medium: mature tomato cleaning, peeling, juice extractor squeezing, 100 mesh filtered through gauze take juice, boil
10min is boiled, 8000g centrifugation 10min takes supernatant, and 135 DEG C of sterilizing 100min are cooled to room temperature to get sterile tomato juice culture
Base.
The preparation of seed (fermenting microbe): by L.plantarum ST- III, (deposit number of the lactobacillus plantarum is
CGMCC No.084, the source of the bacterial strain refer to the Chinese patent of Publication No. CN 102604833A) freeze-dried powder with a small amount of
Sterile distilled water dissolution takes a ring to line in MRS solid medium (Merck Co. Germany), 37 DEG C of anaerobism trainings with oese
It supports and takes out for 24 hours, be put into 1mL MRS liquid (Merck Co. Germany) with oese picking single colonie, with vortex oscillator by bacterium
It falls and is dispersed in fluid nutrient medium, 37 DEG C of Anaerobic culturels take out for 24 hours, are inoculated in 50mL MRS liquid with 2% (v/v) inoculum concentration
Body, after 37 DEG C of cultures for 24 hours, culture 9,000rpm is centrifuged 10 minutes, is discarded supernatant, after thallus washs 2 times with sterile distilled water,
It is suspended with the sterile distilled water of former volume of culture, obtains the seed of fermentation.
2, the preparation of lactobacillus plantarum lyophilized preparation
(1) III normal condition of L.plantarum ST- is fermented, i.e., with inoculum concentration 5% (v/v) aseptic inoculation in sterile
Tomato juice culture medium, 37 DEG C of cultures, 8 hours acquisition fermentation liquids.
(2) fermentation liquid is adjusted into pH to 7.0 with sterile food grade lye, up to the freeze-drying system of ST- III after freeze-drying
Agent S5.
3, the measurement of lactobacillus plantarum lyophilized preparation survival rate, viable count
Sterile distilled water is added in the lyophilized preparation S5 prepared to the above method, volume is made to be reduced to the volume before freeze-drying,
Using the lactobacillus plantarum viable count in MRS plate count method analytical unit volume (usually every milliliter).Freeze-dried powder viable bacteria
Quality (g/mL) after number (cfu/g)=unit volume zymotic fluid viable count (cfu/mL) ÷ unit volume fermentation liquid freeze-drying, is deposited
The theory in viable count ÷ freeze-drying primary fermentation liquid unit volume after motility rate (%)=lyophilized preparation reduction volume in unit volume
Viable count × 100.After measured, the Strain survival rate of III lyophilized preparation S5 of ST- is 93%, and viable count logarithm is 10.39 (i.e.
2.44×1010cfu/g)。
The stability of bacterial strain in the detection gained lactobacillus plantarum lyophilized preparation of effect example 1
Lactobacillus plantarum lyophilized preparation S1, S2, S3 prepared by embodiment 1-3 are distributed into sterile aluminium foil bag, room temperature (25
DEG C) after preservation 6 months and 12 months, take out bacterium powder, viable count is measured using MRS colony counting method, the results are shown in Table 2.
The Stability Determination that 2 lactobacillus plantarum ST-III lyophilized preparation of table is stored at room temperature
As shown in Table 2, for III lyophilized preparation of ST- of all tests after being stored at room temperature 12 months, viable count can stablize holding
108More than.
Comparative example 1 detects the survival rate and viable count of bacterial strain in traditional lyophilized preparation
The preparation method of the aseptic tomato juice culture medium and microorganism seed that are directed to is same as Example 1.
1, lyophilized preparation is prepared using conventional method:
(1) protectant preparation is lyophilized in tradition: 7.06% skimmed milk, 6.46% maltose and 6.70% sodium glutamate is molten
For solution in distilled water, 115 DEG C sterilize 15 minutes to obtain the final product.(Wang Shikuan, Hong Yucheng, Yuan elder generation bell response phase method optimize lactobacillus plantarum
With research [J] Sichuan University of Science & Engineering's journal (natural science edition) of bacillus coagulans freeze drying protectant, 2013,5:23-26.)
(2) III normal condition of L.plantarum ST- is fermented, i.e., with inoculum concentration 2% (v/v) aseptic inoculation in sterile
Its 8,000g centrifugation 10min is obtained III bacterial sediment of ST- by tomato juice culture medium, 37 DEG C of cultures, 8 hours acquisition fermentation liquids.
(3) III bacterial sediment of ST- is resuspended in traditional freeze drying protectant of same volume, it is freeze-dried rear up to ST-
III lyophilized preparation P2.
2, in traditional lyophilized preparation Strain survival rate, viable count measurement
The preparation gained lactobacillus plantarum lyophilized preparation S1 of Example 1, is named as P1, as control.To above-mentioned side
Sterile distilled water is added in lyophilized preparation P1, P2 of method preparation, so that volume is reduced to the volume before freeze-drying, using MRS plate meter
Lactobacillus plantarum viable count in counting method analytical unit volume (usually every milliliter).Freeze-dried powder viable count (cfu/g)=mono-
Quality (g/mL) after volume zymotic fluid viable count (cfu/mL) the ÷ unit volume fermentation liquid freeze-drying of position.Survival rate (%)=freeze-drying
Theoretical viable count × 100 before viable count ÷ after preparation reduction volume in unit volume is lyophilized in bacteria suspension unit volume.This
The survival rate of lactobacillus plantarum, viable count are as shown in table 3 in invention lyophilized preparation and traditional lyophilized preparation.
The survival rate of lactobacillus plantarum, viable count in the lyophilized preparation of the present invention of table 3 and traditional lyophilized preparation
As shown in Table 3, lyophilized preparation prepared by the method for the present invention has than conventional method in terms of survival rate with viable count
It is improved.
Comparative example 2 detects the survival rate and viable count of bacterial strain in the lyophilized preparation of normal fermentation liquid preparation
The preparation method of the MRS culture medium and microorganism seed that are directed to is same as Example 1.
1, lyophilized preparation is prepared using normal fermentation liquid:
(1) III normal condition of L.plantarum ST- is fermented, i.e., is trained with (v/v) aseptic inoculation of inoculum concentration 2% in MRS
Base is supported, its 8,000g centrifugation 10min is obtained III bacterial sediment of ST- by 37 DEG C of cultures, 8 hours acquisition fermentation liquids.
(2) fermentation liquid is adjusted into pH to 7.0 with sterile food grade lye, up to the freeze-drying system of ST- III after freeze-drying
Agent P3.
2, normal fermentation liquid preparation lyophilized preparation in Strain survival rate, viable count measurement
Using lyophilized preparation P1 and P2 described in comparative example 1 as control.To the above method prepare lyophilized preparation P1, P2 and
Sterile distilled water is added in P3, so that volume is reduced to the volume before freeze-drying, using MRS plate count method analytical unit volume
Lactobacillus plantarum viable count in (usually every milliliter).Freeze-dried powder viable count (cfu/g)=unit volume zymotic fluid viable count
(cfu/mL) quality (g/mL) after the freeze-drying of ÷ unit volume fermentation liquid.Unit after survival rate (%)=lyophilized preparation reduction volume
Theoretical viable count × 100 before viable count ÷ in volume is lyophilized in bacteria suspension unit volume.Lyophilized preparation of the present invention, conventional freeze
The survival rate of lactobacillus plantarum, viable count are as shown in table 4 in dry preparation and the lyophilized preparation of normal fermentation liquid preparation.
Lactobacillus plantarum in the lyophilized preparation of the lyophilized preparation of the present invention of table 4, traditional lyophilized preparation and the preparation of normal fermentation liquid
Survival rate, viable count
It can be concluded that, the lyophilized preparation of normal fermentation liquid preparation is than preparation method institute of the present invention from the result of table 4
The Strain survival rate and viable count for obtaining dietary supplements significantly reduce.
Comparative example 3 detects the survival rate and viable count of lactobacillus plantarum in lyophilized preparation
The preparation method of the aseptic tomato juice culture medium and microorganism seed that are directed to is same as Example 1.
By the inoculum concentration of lactobacillus plantarum L.plantarum ST- III in embodiment, the pH value of tomato juice culture medium, culture
The pH value of temperature, incubation time and gained fermentation liquid is adjusted one by one, obtains the plant of following set of distinct methods preparation
Object lactobacillus lyophilized preparation, and sterile distilled water is added in the lyophilized preparation prepared to the above method, make volume be reduced to be lyophilized
Preceding volume, using the viable count in MRS plate count method analytical unit volume (usually every milliliter).Freeze-dried powder viable count
(cfu/g)=unit volume zymotic fluid viable count (cfu/mL) quality (g/mL) after the freeze-drying of ÷ unit volume fermentation liquid, survival
The viable bacteria in the fermentation liquid unit volume before viable count ÷ freeze-drying after rate (%)=lyophilized preparation reduction volume in unit volume
Number × 100.It is as shown in table 5 to detect acquired results:
5 distinct methods of table prepare the survival rate and viable count of lactobacillus plantarum in obtained freeze-drying preparation
From to result shown in table 5 it can be concluded that, by strain in the preparation method of the lactobacillus plantarum lyophilized preparation
Inoculum concentration, it is micro- in obtained freeze-drying agent when the pH of cultivation temperature, incubation time and fermentation liquid is adjusted to except the present invention
The survival rate and viable count of biology produce significant decrease.
It should be understood that those skilled in the art can make the present invention various after having read above content of the invention
Change or modification, these equivalent forms also fall within the scope of the appended claims of the present application.
Claims (9)
1. a kind of preparation method of lactobacillus plantarum lyophilized preparation, which comprises the following steps:
(1) lactobacillus plantarum is inoculated in tomato juice culture medium and is cultivated, cultivation temperature is 35~40 DEG C, culture 8~12
Hour collects fermentation liquid；The inoculum concentration of the lactobacillus plantarum is 0.5%-5%, and the percentage is percent by volume；
(2) fermentation liquid pH value obtained by step (1) is adjusted to 6.5~7.5, be freeze-dried to obtain the final product.
2. preparation method as described in claim 1, which is characterized in that lactobacillus plantarum described in step (1) is plant cream bar
Bacterium (L.plantarum) ST- III, lactobacillus plantarum ATCC14917, lactobacillus plantarum WCFS1 or lactobacillus plantarum P8.
3. preparation method as described in claim 1, which is characterized in that tomato juice culture medium described in step (1) by include with
The method of lower step is prepared: cleaning mature tomato is removed the peel, and juicing is boiled after filtering, 4,000-12,000g centrifugation
10min takes supernatant, and sterilizing, cooling to obtain the final product.
4. preparation method as claimed in claim 3, which is characterized in that the filtering uses 100 mesh filtered through gauze, boils
Time is 1-10 minutes, and the sterilizing uses 110-135 DEG C, is sterilized 10-30 minutes.
5. preparation method as described in claim 1, which is characterized in that cultivation temperature described in step (1) is 37 DEG C, and the time is
8 hours.
6. preparation method as described in claim 1, which is characterized in that pH value described in step (1) is adjusted to 7.0.
7. a kind of lactobacillus plantarum lyophilized preparation, which is characterized in that it is that method comprising the following steps are prepared:
(1) lactobacillus plantarum is inoculated in tomato juice culture medium and is cultivated, cultivation temperature be 35~40 DEG C, the time be 8~
12 hours collection fermentation liquids；The inoculum concentration of the lactobacillus plantarum is 0.5%-5%, and the percentage is percent by volume；
(2) fermentation liquid pH value obtained by step (1) is adjusted to 6.5~7.5, be freeze-dried to obtain the final product.
8. lactobacillus plantarum lyophilized preparation as claimed in claim 7, which is characterized in that cultivation temperature described in step (1) is
37 DEG C, the time is 8 hours, and the inoculum concentration of the lactobacillus plantarum is 0.5%-5%, and the percentage is percent by volume.
9. a kind of lactobacillus plantarum lyophilized preparation as described in claim 7 or 8 is in the application prepared in food or dietary supplement.
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| CN102220260A (en) * | 2011-04-25 | 2011-10-19 | 北京市农林科学院 | Lyophilization process of lactobacillus plantarum |
| CN102604833A (en) * | 2012-03-23 | 2012-07-25 | 光明乳业股份有限公司 | Preparation method and product of multiple lactic acid bacteria fermentation agent for fermented milk |
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| CN102220260A (en) * | 2011-04-25 | 2011-10-19 | 北京市农林科学院 | Lyophilization process of lactobacillus plantarum |
| CN102604833A (en) * | 2012-03-23 | 2012-07-25 | 光明乳业股份有限公司 | Preparation method and product of multiple lactic acid bacteria fermentation agent for fermented milk |
| CN102732470A (en) * | 2012-07-20 | 2012-10-17 | 光明乳业股份有限公司 | Purely natural and low-cost lactobacillus (Lb.) plantarum ST-III culture method and products thereof, and application of products |
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