CN107446841A - A kind of direct putting type Lactobacillus plantarum high-density cultivation method and obtained freeze-dried powder and application - Google Patents
A kind of direct putting type Lactobacillus plantarum high-density cultivation method and obtained freeze-dried powder and application Download PDFInfo
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- CN107446841A CN107446841A CN201710568704.2A CN201710568704A CN107446841A CN 107446841 A CN107446841 A CN 107446841A CN 201710568704 A CN201710568704 A CN 201710568704A CN 107446841 A CN107446841 A CN 107446841A
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- JYJIGFIDKWBXDU-MNNPPOADSA-N inulin Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@]1(OC[C@]2(OC[C@]3(OC[C@]4(OC[C@]5(OC[C@]6(OC[C@]7(OC[C@]8(OC[C@]9(OC[C@]%10(OC[C@]%11(OC[C@]%12(OC[C@]%13(OC[C@]%14(OC[C@]%15(OC[C@]%16(OC[C@]%17(OC[C@]%18(OC[C@]%19(OC[C@]%20(OC[C@]%21(OC[C@]%22(OC[C@]%23(OC[C@]%24(OC[C@]%25(OC[C@]%26(OC[C@]%27(OC[C@]%28(OC[C@]%29(OC[C@]%30(OC[C@]%31(OC[C@]%32(OC[C@]%33(OC[C@]%34(OC[C@]%35(OC[C@]%36(O[C@@H]%37[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O%37)O)[C@H]([C@H](O)[C@@H](CO)O%36)O)[C@H]([C@H](O)[C@@H](CO)O%35)O)[C@H]([C@H](O)[C@@H](CO)O%34)O)[C@H]([C@H](O)[C@@H](CO)O%33)O)[C@H]([C@H](O)[C@@H](CO)O%32)O)[C@H]([C@H](O)[C@@H](CO)O%31)O)[C@H]([C@H](O)[C@@H](CO)O%30)O)[C@H]([C@H](O)[C@@H](CO)O%29)O)[C@H]([C@H](O)[C@@H](CO)O%28)O)[C@H]([C@H](O)[C@@H](CO)O%27)O)[C@H]([C@H](O)[C@@H](CO)O%26)O)[C@H]([C@H](O)[C@@H](CO)O%25)O)[C@H]([C@H](O)[C@@H](CO)O%24)O)[C@H]([C@H](O)[C@@H](CO)O%23)O)[C@H]([C@H](O)[C@@H](CO)O%22)O)[C@H]([C@H](O)[C@@H](CO)O%21)O)[C@H]([C@H](O)[C@@H](CO)O%20)O)[C@H]([C@H](O)[C@@H](CO)O%19)O)[C@H]([C@H](O)[C@@H](CO)O%18)O)[C@H]([C@H](O)[C@@H](CO)O%17)O)[C@H]([C@H](O)[C@@H](CO)O%16)O)[C@H]([C@H](O)[C@@H](CO)O%15)O)[C@H]([C@H](O)[C@@H](CO)O%14)O)[C@H]([C@H](O)[C@@H](CO)O%13)O)[C@H]([C@H](O)[C@@H](CO)O%12)O)[C@H]([C@H](O)[C@@H](CO)O%11)O)[C@H]([C@H](O)[C@@H](CO)O%10)O)[C@H]([C@H](O)[C@@H](CO)O9)O)[C@H]([C@H](O)[C@@H](CO)O8)O)[C@H]([C@H](O)[C@@H](CO)O7)O)[C@H]([C@H](O)[C@@H](CO)O6)O)[C@H]([C@H](O)[C@@H](CO)O5)O)[C@H]([C@H](O)[C@@H](CO)O4)O)[C@H]([C@H](O)[C@@H](CO)O3)O)[C@H]([C@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O)[C@@H](CO)O1 JYJIGFIDKWBXDU-MNNPPOADSA-N 0.000 description 1
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- 229960003966 nicotinamide Drugs 0.000 description 1
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- 239000011570 nicotinamide Substances 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
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- 229920000573 polyethylene Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000011241 protective layer Substances 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
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G9/00—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor
- A23G9/32—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds
- A23G9/36—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins
- A23G9/363—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins containing microorganisms, enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/065—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
Abstract
The invention discloses a kind of direct putting type Lactobacillus plantarum high-density cultivation method and obtained freeze-dried powder and application, comprise the following steps：(1) freeze-dried powder of Lactobacillus plantarum is placed in MRS culture mediums after being activated twice respectively and obtains seed fermentation agent, the bacterial content of the seed fermentation agent is 108~109CFU/mL；(2) using volume ratio as 3~6:100 ratio, seed fermentation agent is accessed in high-density culture medium, fermented and cultured is carried out at 36~38 DEG C, zymophyte suspension is collected after 18~24h；(3) zymophyte suspension is centrifuged 25~35min, gives up supernatant, clean precipitation with the physiological saline after sterilizing, and collect bacterium mud in 6000~8000rpm；(4) it is 2~5 by the volume ratio of composite protectant and bacterium mud in bacterium mud:1 adds composite protectant, must contain protective agent bacteria suspension after well mixed；(5) protective agent bacteria suspension 1.5~6.0h of pre-freeze at 20~65 DEG C will be contained and obtain freeze-dried powder after dry 18~24h in vacuum freeze drier.Freeze-dried powder viable count prepared by the present invention is up to 1010~1011Cfu/g, the freeze-dried powder moisture are less than 3%, prevented from caking, rehydration is good, have extensive use.
Description
Technical field
The present invention relates to direct putting type Lactobacillus plantarum high-density cultivation method, in particular to a kind of Lactobacillus plantarum high density
Cultural method and obtained freeze-dried powder and application.
Background technology
The initial substrate that the High Density Cultivation technology of lactic acid bacteria avoids higher concentration may suppress the growth of microbial body
So as to reduce ultimate density.At present, the domestic research for the High Density Cultivation of Lactobacillus plantarum is developing, and its is industrial
Application then also in the elementary step.Scholar's research buffer salt and addition chemical neutralizer are to promote the side of lactobacter growth
Method, but turn out that to carry out the concentration of bacteria suspension be still 109Cfu/mL level.Other scholars by study the cell cycle culture with thoroughly
Analysis culture, to which substantial amounts of lactic acid bacteria can be obtained.Hayakawa K et al. in《Journal of Fermentation and
Bioengineering》" the High density culture of lactobacillus that phase magazine nineteen ninety the 6th delivers
casei,by a Cross-Flow culture method based on kinetic properties of the
Microorganism " is mentioned in consumption water 0.2m3In the case of/kg, acquisition density is 40g/L bacteria suspensions, obtains bacteria suspension
Speed be 13g/L/h.The cell concentration and speed of production obtained using cell cycle and dialysis process is higher than using buffering
The cell concentration and speed of production that salt and chemical method obtain, but cell cycle and dialysis machine and technical elements is costly
It is high.
Liu winter plum et al. in《South China Science ＆ Engineering University's journal (natural science edition)》3rd phase in 2014 delivers " newborn by L-
Lactobacillus is identified in the upstream and downstream DNA sequence dna identification Lactobacillus sp.DMDL 9010 " of acidohydrogenase 1
sp. DMDL 9010.Wang Pan et al. in《Modern food science and technology》" the Lactobacillus plantarum DMDL 9010 that 6th phase in 2015 delivers
The engineering bacteria that report is obtained using gene engineering method in gene cloning, expression and the purifying of nitrite reductase " can have
Degrading nitrite is imitated, and the NIR of high-purity can be obtained using affinity chromatography.Number of patent application is CN201510493977.6
Chinese patent disclose a kind of pickles and preparation method containing mixing lactic acid bacteria, gained fermentation pickled vegetable is practically free of nitrous acid
Salt, with short production cycle, product quality is stable, can be instant containing probiotics living.
Lin Qiao et al. in《China brewages》" the response phase method optimization Lactobacillus plantarum culture medium that 7th phase in 2012 delivers
Report in research " and the culture medium of the plant Lactobacillus brevis JCBY-1 with higher vigor optimized using response phase method,
The horizontal progress Box-Behnken Optimal Experimentals of four factor three are have selected, understand to add albumen into basal medium by experiment
When peptone 1.01mg/mL, dusty yeast 0.73mg/mL, glucose 1.99mg/mL, sodium acetate 0.51mg/mL, the cell concentration of acquisition
Highest.Zhou Jianzhong et al. in《Jiangsu's agriculture science》" the Response Surface Method optimization Lactobacillus plantarum vegetable that the fifth phase in 2008 delivers
To the optimization for the Lactobacillus plantarum progress culture medium that be used to produce fermentation vegetable juice in the research of vegetable juice culture medium ", using just
Hand over experiment or Box-Behnken Optimal Experimentals to carry out the optimization of High Density Cultivation to lactic acid bacteria, can both obtain higher concentration bacterium
Body, the cost of culture can be reduced again, be relatively good method.
Number of patent application is that CN201510038278.2 Chinese invention patent discloses a kind of norcholesterol plant breast bar
The method of bacterium bacteria preparation fermentation pickled vegetable, by norcholesterol Lactobacillus plantarum actication of culture, culture, lyophilized 24h, norcholesterol is made
Lactobacillus plantarum bacterium powder, the ratio progress pickles preparation for cleaning the carrot dried or other vegetables to be fermented is added, adds and is made
Norcholesterol Lactobacillus plantarum bacterium powder.The bacterial strain, which is not known, carries out manner of packing explanation and preliminary secure estimate.
It is indicated above it is domestic dry the research of microbial inoculum also in the elementary step to preparing lactic acid bacteria, the scholar studied compared with
It is few, and be to applying more streptococcus thermophilus and lactobacillus acidophilus to be dried in microbial inoculum preparation technology in lactic acid bacteria mostly
Optimization.Mainly due to China in the application of lactic acid bacteria, based on the preparation of Yoghourt, the application of other strains need to be expanded
Exhibition.The conditions such as high-density culture medium, centrifugation, harvest cell age, protective agent, pre-freeze selected by above-mentioned technology are not suitable for, and institute
The bacterial strain of acquisition does not carry out safety evaluatio, and so as to cause viable count not high, the security and drug resistance of Metabolite are not
The problems such as specifying.
The content of the invention
The invention aims to solve above-mentioned the shortcomings of the prior art, there is provided a kind of viable count content is 1010
~1011Cfu/g, for produce fermentation time is short, rehydration is good, safe direct putting type Lactobacillus plantarum High Density Cultivation and
The preparation method of its freeze-dried powder.The direct putting type Lactobacillus plantarum freeze-dried powder can be used for fermenting and producing pickles, Juice, ice cream,
Jelly, milk piece, stirred yoghurt, solidification type yoghourt, boruga, sausage, corn flour, dregs of beans, peanut meal etc..
The purpose of the present invention is achieved through the following technical solutions：
A kind of direct putting type Lactobacillus plantarum high-density cultivation method, comprises the following steps：
(1) freeze-dried powder of Lactobacillus plantarum is placed in MRS culture mediums after being activated twice respectively and obtains seed fermentation agent,
The bacterial content of the seed fermentation agent is 108~109CFU/mL；
(2) using volume ratio as 3~6:100 ratio, seed fermentation agent is accessed in high-density culture medium, in 36~38 DEG C
Lower carry out fermented and cultured, zymophyte suspension is collected after 18~24h；
(3) zymophyte suspension is centrifuged 25~35min, gives up supernatant, with the life after sterilizing in 6000~8000rpm
Salt solution cleaning precipitation is managed, and collects bacterium mud；
(4) it is 2~5 by the volume ratio of composite protectant and bacterium mud in bacterium mud:1 adds composite protectant, is well mixed
After must contain protective agent bacteria suspension；
(5) protective agent bacteria suspension will be contained to be placed in vacuum freeze drier after pre-freeze 1.5h~6.0h at -20~-65 DEG C
Dry 18~24h and obtain freeze-dried powder.
In terms of parts by weight, the formula of the high-density culture medium is：0.8~1.0 part of casein digest, yeast extract powder
0.3~0.5 part, 1.5~2.0 parts of glucose, 0.1~0.2 part of Triammonium citrate, 0.048~0.058 part of magnesium sulfate, beef extract
0.5~1.0 part, 0.1~0.2 part of dipotassium hydrogen phosphate, 0.4~0.5 part of sodium acetate, 0.1~0.3 part of Tween 80, Soyprotein peptide
0.8~1.2 part, 0.01~0.02 part of ascorbic acid, 0.05~0.25 part of manganese sulfate.
The high-density culture medium is：Above-mentioned raw materials are settled to 100 parts with distilled water, adjust pH to 6.8, stirring and dissolving
Uniformly after 0.08~0.10MPa sterilize 15~20min, be cooled to 36~38 DEG C it is standby.
In terms of parts by weight, the formula of described composite protectant is：10~14 parts of skimmed milk powder, bacteriological peptone
7.8~8.2 parts, 13~17 parts of trehalose, 0.9 part of sodium chloride.
Described composite protectant is：Above-mentioned raw materials are settled to 100 parts with distilled water, stirred after dissolving, in
0.08~0.10MPa, sterilize 15~20min, and it is standby to be cooled to normal temperature.
Described centrifugal rotational speed is 8000rpm, centrifugation time 30min.
Step (5) obtained freeze-drying powder, its temperature is first returned into normal temperature with the constant humidity air that humidity is 50~60%, then used
Air-flow packing machine carries out aluminum-plastic packaged.
The moisture of the freeze-dried powder is less than 3%, and viable count reaches 1010~1011cfu/g。
The freeze-dried powder is in fermentation pickled vegetable, Juice, ice cream, jelly, milk piece, stirred yoghurt, solidification type yoghourt, acid
Applied in milk drink, sausage, corn flour, dregs of beans or peanut meal.
Compared with prior art, the invention has the advantages that and beneficial effect：
(1) working stock culture of the invention is cultivated successively with two kinds of culture mediums, Lactobacillus plantarum is activated as far as possible, and be in
Synchronous growth state.
(2) Soyprotein peptide, ascorbic acid, the manganese sulfate of the high-density culture medium use used in the present invention can ensure bacterium
Growth factor required in body growth course, promote thalline raised growth.
(3) freeze drying protectant used in the present invention be containing skimmed milk powder 10%~14%, bacteriological peptone 7.8%~
8.2%, the normal saline solution of trehalose 13%~17%.Culture medium used is edible, protective agent skimmed milk powder
And trehalose is also edible food ingredient, while to be preserved in China Committee for Culture Collection of Microorganisms common for the bacterial strain
Microorganism center.
(4) freeze-dried powder viable count prepared by the present invention is up to 1010~1011Cfu/g, it is prevented from caking, rehydration is good.The jelly
Dry powder moisture is less than 3%.
(5) freeze-dried powder for preparing of the present invention has that rehydration is good, color and luster is good, good fluidity, preserves convenient, is easy for technique
Production, reduce inoculation, the link that spreads cultivation and reduce pollution, ensure the stability of product quality, shorten fermentation period, solution is given birth to
The safety problem of food is produced, meets the needs of large-scale production, has a wide range of application simultaneously.
Lactobacillus plantarum of the present invention is DMDL 9010, is preserved in Chinese microorganism strain preservation within 19th in August in 2011
Administration committee's common micro-organisms center, abbreviation CGMCC, deposit number are CGMCC NO.5172, address：Chaoyang District, Beijing City
The institute 3 of North Star West Road 1, Institute of Microorganism, Academia Sinica.The bacterial strain is public in Chinese patent CN102978134A
Open, belong to prior art.According to 16SrDNA oligonucleotide sequences, physio-biochemical characteristics and encoding lactate dehydrogenase gene
The Sequence Identification DMDL 9010 of LDH1 upstream and downstream segments is Lactobacillus plantarum.
Brief description of the drawings
The nitroreductase testing result figure of Fig. 1 embodiments 1；Three, the right test tube is blank test, and middle three test tubes are
The experimental group of Quality-control strains staphylococcus aureus is added, three, left side test tube is the experiment for adding Lactobacillus plantarum DMDL9010
Group.
The hemolytic activity testing result figure of Fig. 2 embodiments 1；(a) it is Lactobacillus plantarum DMDL9010 streak inoculations, (b) is plant
Thing lactobacillus DMDL9010 percutaneous puncture-inoculations, (c) are Quality-control strains Escherichia coli streak inoculation, and (d) is Quality-control strains Escherichia coli
Percutaneous puncture-inoculation.
The indole test testing result figure of Fig. 3 embodiments 1, left side conical flask are blank test, and middle conical flask is planted to add
Thing lactobacillus DMDL9010 experimental group, the right conical flask are the experimental group for adding Quality-control strains Escherichia coli.
The growth curve charts of 2 Lactobacillus plantarum DMDL of Fig. 4 embodiments 9010.
Fig. 5 embodiment 2Box-Behnken Optimal Experimentals are fitted response surface design interaction influence figure；(a)A:Soybean protein
Peptide and B:Vitamin C interaction influence contour map, (b) A:Soyprotein peptide and B:Vitamin C interaction influence 3D is bent
Face figure, (c) A:Soyprotein peptide and C:MnSO4·H2O interaction influence contour maps, (d) A:Soyprotein peptide and C:
MnSO4·H2O interaction influence 3D surface charts, (e) B:Vitamin C and C:MnSO4·H2O interaction influence contour maps,
(f)B:Vitamin C and C:MnSO4·H2O interaction influence 3D surface charts.
Fig. 6 embodiment 3Box-Behnken Optimal Experimentals are fitted response surface design interaction influence figure；(a)A:Skimmed milk powder
With B:Peptone interaction influence contour map, (b) A:Skimmed milk powder and B:Peptone interaction influence 3D surface charts,
(c)A:Skimmed milk powder and C:Trehalose interaction influence contour map, (d) A:Skimmed milk powder and C:Trehalose reciprocation
Influence 3D surface charts, (e) B:Peptone and C:Trehalose interaction influence contour map, (f) B:Peptone and C:Trehalose
Interaction influence 3D surface charts.
Embodiment
To be best understood from the present invention, the present invention is described in further details with reference to embodiment, but application claims
The scope of protection is not limited to the scope represented by embodiment.
Embodiment 1：Freeze-dried powder safety evaluatio is studied
(1) bacteria suspension in logarithmic phase growing plants lactobacillus DMDL9010 is continuously entered in seed culture medium
2~3 activation cultures of row, obtain seed culture fluid, and the bacterial content of the seed fermentation agent is 109CFU/mL。
(2) using volume ratio as 3:Seed fermentation agent is accessed high-density culture medium by 100 ratio, and high density is carried out in 37 DEG C
18h is cultivated, collects zymophyte suspension；Described high-density culture medium is：By mass, 1.0 parts of casein digest, yeast
0.5 part of cream powder, 2.0 parts of glucose, 0.1 part of Triammonium citrate, 0.054 part of magnesium sulfate, 0.8 part of beef extract, dipotassium hydrogen phosphate 0.1
Part, 0.5 part of sodium acetate, 0.18 part of Tween 80,1 part of Soyprotein peptide, 0.02 part of ascorbic acid, 0.15 part of manganese sulfate, with distillation
Water is settled to 100 parts, adjusts pH to 6.8, is cooled down after sterilizing standby.
(3) zymophyte suspension is cleaned 2 times after 4000~10000rpm with continuous normal saline, centrifuges 10~30min
Bacterium mud is collected, gained centrifuge results are as shown in table 1.The combination of each centrifugal rotational speed and centrifugation time is contrasted, with centrifugal rotational speed
Rise, supernatant viable count substantially reduces, and it is then first to rise to reduce afterwards to precipitate viable count, and this shows too low centrifugal rotational speed
So that thalline can not be precipitated fully, the bacterium mud amount deficiency of acquisition, though and too high centrifugal rotational speed can effectively make bacterial sediment,
Also larger to the degree of injury of viable bacteria, a large amount of viable bacterias damage in centrifugal process, are dead.As centrifugation time extends, supernatant
Viable count, which substantially reduces, precipitates viable count substantially increases, and illustrating the centrifugation of enough time can be such that viable bacteria fully precipitates.But centrifuge
Time, which is not suitable for long, long centrifugation time, to reduce efficiency, be unfavorable for mass producing.By protective agent and wet bacterium mud body
Product is than being 5:1, after addition strain protective agent is well mixed, packing is laid in tray, is carried out vacuum freeze drying and is obtained bacterium powder.
The protection agent compounding method is (being calculated according to mass ratio)：10.0 parts of skimmed milk powder, with physiological saline solution, and
100 parts are settled to, is configured to 10% (w/v) skimmed milk powder solution.
1 Lactobacillus plantarum DMDL9010 safety evaluatios are studied
1.1 nitroreductases detect
The Lactobacillus plantarum DMDL9010 activated and Quality-control strains staphylococcus aureus are connect by 3% inoculum concentration
Kind is into standby nitroreductase detection culture medium, incubated 72h at 37 DEG C, while does blank test.Into culture medium
Alpha-naphthylamine solution and p-aminophenyl methanesulfonic acid solution are sequentially added, is gently mixed, culture medium color change is observed and photographs to record,
Culture medium reddens, and shows the nitroreductase testing result positive, is otherwise feminine gender.As a result as shown in figure 1, Lactobacillus plantarum
DMDL9010 nitroreductase testing result is feminine gender, i.e., inactive nitro reduction in Lactobacillus plantarum DMDL9010 thalline
Enzyme, can not be nitrite by nitrate reduction.
1.2 hemolytic activities detect
The collarium line that connects that the Lactobacillus plantarum DMDL9010 activated and Quality-control strains Escherichia coli are sterilized connects
Kind cultivates 48h in Columbia Blood Agar flat board, in 37 DEG C, while does blank test, and observation periphery of bacterial colonies whether there is haemolysis circle
Occur and photograph to record.As a result as shown in Fig. 2 Fig. 2 (a) and Fig. 2 (b) are Lactobacillus plantarum DMDL9010.The present embodiment selects
Quality-control strains Escherichia coli there is hemolytic, from Fig. 2 (c), occur haemolysis circle around Quality-control strains E. coli clones.
Formed and compareed with Quality-control strains, Lactobacillus plantarum DMDL9010 periphery of bacterial colonies is irised out now without haemolysis, i.e. Lactobacillus plantarum
DMDL9010 will not make human body haemolysis occur, be safe.
1.3 indoles are tested
The Lactobacillus plantarum DMDL9010 activated and Quality-control strains staphylococcus aureus are distinguished by 3% bacterium amount that connects
It is linked into peptone water medium, after 37 DEG C of culture 72h, adds indole reagent 8 to drip, viewing test result, while do blank examination
Test, two layers of liquid interface red ring occurs for the positive after indole reagent is added dropwise, and feminine gender is not changed color as after reagent is added dropwise.By Fig. 3
Experimental result is understood, after indole reagent is added, the friendship between two layers of liquid of the culture medium for being inoculated with staphylococcus aureus
There is red ring in interface, and indole test result is the positive；And the culture medium for being inoculated with Lactobacillus plantarum DMDL9010 is not obvious
Color change, indole test testing result for feminine gender.
1.4K-B scraps of paper diffusion tests
Take 1mL that the Lactobacillus plantarum DMDL9010 activated is added in culture dish, add 15mL MRS agar cultures
Base, shake up, after culture medium solidifying, the scraps of paper immersed with tetracycline, erythromycin, streptomysin, ampicillin are placed in solidification
On culture medium, in 37 DEG C of constant incubators, quiescent culture 24h.The upgrowth situation of bacterium colony to be tried in plate is observed, if in antibiosis
There is transparent circle around the plain scraps of paper, the diameter of transparent circle is measured with ruler, judges whether it has drug sensitivity.Simultaneously with golden yellow
Staphylococcus carries out control experiment as Quality-control strains.Strains tested is to the resistance to the action of a drug of antibiotic with reference to U.S. clinical and experiment
The standard that room Association for Standardization (Clinical and Laboratory Standards Institute, CLSI) formulates (is shown in Table
1) judged.
The drug sensitive test paper content of dispersion of table 1 and resistance criterion
The embodiment 1K-B scraps of paper diffusion test results of table 2
As a result as shown in table 2, K-B scraps of paper diffusion tests result understands that Lactobacillus plantarum DMDL9010 is to tetracycline and ammonia
The sensitiveness in benzyl XiLin is relatively low, medium to the sensitiveness of erythromycin and streptomysin.I.e. tetracycline and ampicillin are to plant breast bar
Bacterium DMDL9010 has stronger inhibitory action, and erythromycin and streptomysin are in Lactobacillus plantarum DMDL9010 inhibitory action
Deng.Compared with staphylococcus aureus, Lactobacillus plantarum DMDL9010 is low to the sensitiveness of tetracycline, to the sensitiveness of streptomysin
It is high.Sensitivity levels one of the Lactobacillus plantarum DMDL9010 to the sensitiveness and staphylococcus aureus of erythromycin and ampicillin
Cause.I.e. tetracycline is less than staphylococcus aureus to Lactobacillus plantarum DMDL9010 inhibitory action, and streptomysin is to plant breast bar
Bacterium DMDL9010 inhibitory action is higher than staphylococcus aureus.And erythromycin and ampicillin are to Lactobacillus plantarum DMDL9010
It is suitable with the suppression level of staphylococcus aureus.
By analyzing Lactobacillus plantarum DMDL9010 to the drug sensitivity of these four antibiotic and and staphylococcus aureus
Contrasted, it is known that common antibiotic has stronger inhibitory action, Lactobacillus plantarum to Lactobacillus plantarum DMDL9010
DMDL9010 is safe.
Described peptone water medium, by mass, 1 part of bacteriological peptone, 0.5 part of sodium chloride, addition distillation
Water is stirred to 100 parts, and regulation Medium's PH Value is dispensed into conical flask, 121 DEG C of autoclaving 15min to 7.6.
Described nitroreductase detection culture medium, by mass, 1 part of bacteriological peptone, 1 part of sodium chloride, nitric acid
0.5 part of potassium, addition distilled water are stirred to 100 parts, and regulation Medium's PH Value is dispensed into conical flask, 121 to 6.8
DEG C autoclaving 15min.
Embodiment 2：Lactobacillus plantarum DMDL9010 High Density Cultivation researchs
The first step collects fermentation culture after Lactobacillus plantarum DMDL9010 bacterium powders are placed in into 2 activation of MRS culture mediums；With
Volume ratio is 3:100 ratio, fermentation culture is accessed in high-density culture medium, fermented and cultured, 18h are carried out at 37 DEG C
After collect zymophyte suspension；In terms of parts by weight, described high-density culture medium is：1.0 parts of casein digest, yeast extract powder
0.5 part, 2.0 parts of glucose, 0.2 part of Triammonium citrate, 0.058 part of magnesium sulfate, 1.0 parts of beef extract, 0.1 part of dipotassium hydrogen phosphate,
0.5 part of sodium acetate, 0.18 part of Tween 80,1 part of Soyprotein peptide, 0.02 part of ascorbic acid, 0.1 part of manganese sulfate, determined with distilled water
Hold to 100 parts, adjust pH to 6.8.Stirring and dissolving uniformly after 0.10MPa sterilize 15min, be cooled to 30 DEG C it is standby；
Zymophyte suspension in 8000rpm, is centrifuged 30min, gives up supernatant by second step, clear with the physiological saline after sterilizing
Wash after bacterium mud and collect；
3rd step is 3 by the normal saline solution of complex protection agent solution and the volume ratio of bacterium mud in bacterium mud:1 adds
Complex protection agent solution, it is well mixed as protective agent bacteria suspension；In terms of parts by weight, described composite protectant solution formula group
Turn into：12 parts of skimmed milk powder, 7.95 parts of bacteriological peptone, 15.2 parts of trehalose, 0.9 part of sodium chloride, it is settled to distilled water
100 parts.Stirred after dissolving, in 0.10MPa, sterilize 15min, and it is standby to be cooled to normal temperature；
Protective agent bacteria suspension is placed in drier by the 4th step, and vacuum freeze drying is placed in after pre-freeze 6.0h at -20 DEG C
24h is dried in machine and obtains freeze-dried powder, the temperature of freeze-dried powder is returned into normal temperature with constant humidity air (humidity 60%), packed with air-flow
Machine progress is aluminum-plastic packaged, and freeze-dried powder moisture is less than 3%, and viable count reaches 1011cfu/g。
By the further investigation to Lactobacillus plantarum DMDL9010 condition of culture, with fermentation time to Lactobacillus plantarum
The logarithm value of DMDL9010 viable counts is curve such as Fig. 4, from fig. 4, it can be seen that 16h viable counts reach peak, now viable bacteria
Number is (2.93 ± 0.13) × 109cfu/mL。
Described MRS basal medium formulations：1.0 parts of casein digest, 0.4 part of yeast extract powder, 2.0 parts of glucose,
0.2 part of Triammonium citrate, 0.02 part of magnesium sulfate, 1.0 parts of beef extract, 0.2 part of dipotassium hydrogen phosphate, 0.5 part of sodium acetate, Tween 80
0.108 part, 0.005 part of manganese sulfate.
The production medium of the high-density culture medium of the second step and the 3rd step optimized by following single factor test,
Plackett-Burmen test and Box-Behnken Optimal Experimentals and obtain, it is specific as follows：
2.1. High Density Cultivation single factor test optimizes
2.1.1 nitrogen source optimizes
(1) bacteriological peptone optimizes
1.0,1.5,2.0,2.5,3.0 times of bacteriological peptones are separately added into basic MRS culture mediums.Can be with from table 3
Find out, when 2.5 times of peptone content in MRS culture mediums based on bacteriological peptone content, obtain viable bacteria in bacteria suspension
Number at most (P<0.05), now content suitable for plant lactobacillus DMDL9010 growth.Viable count reduces after 2.5 times, explanation
Excessive nitrogen source can produce inhibitory action, be unfavorable for the growth of bacterium.
The influence that the various concentrations bacteriological peptone of table 3 grows to Lactobacillus plantarum DMDL9010
Note：Unit refers to (again)：Based on peptone addition in culture medium casein digest content multiple, that is, distinguish
Add 1.0 parts, 1.5 parts, 2.0 parts, 2.5 parts, 3.0 parts of peptones.
(2) Soyprotein peptide optimizes
0.5,1.0,1.5,2.0,2.5,3.0 times of Soyprotein peptide is added into basic MRS culture mediums, can from table 4
Go out, when based on adding the content of Soyprotein peptide 0.5 times of peptone content in MRS culture mediums, obtain bacteria suspension viable bacteria
Number at most (P<0.05), now Lactobacillus plantarum DMDL9010 upgrowth situation is better than the group of no added Soyprotein peptide.
With the rising of the content of Soyprotein peptide in culture medium, obtain bacteria suspension viable count and decline, illustrate to add Soyprotein peptide mistake
The propagation for suppressing Lactobacillus plantarum DMDL9010, it is unfavorable for obtaining high-concentration bacterial suspension more.
The influence that the Different adding amount Soyprotein peptide of table 4 grows to Lactobacillus plantarum DMDL9010
Note：Unit refers to (again)：Based on Soyprotein peptide addition in culture medium casein digest content multiple, i.e.,
0.0 part, 0.5 part, 1.0 parts, 1.5 parts, 2.0 parts, 2.5 parts, 3.0 parts of Soyprotein peptide are added respectively.
2.1.2 carbon source optimizing
As can be seen from Table 5, do not add glucose additionally or additionally add a small amount of glucose, to Lactobacillus plantarum DMDL9010
Propagation is favourable, but its bacteria suspension viable count change unobvious.As extra addition glucose content gradually increases, obtain
Bacteria suspension viable count gradually reduces (P<0.05).Illustrate when glucose amount is 1.0 times~2.0 times, Lactobacillus plantarum
DMDL9010 can make full use of glucose, and when concentration is more than 2.0 times, adding excessive glucose on the contrary can be to the propagation of bacterium
There is certain inhibitory action.
The influence that the Different adding amount glucose solution of table 5 grows to Lactobacillus plantarum DMDL9010
Note：Unit refers to (again)：Based on glucose addition in culture medium glucose content multiple, that is, add 2.0 parts,
3.0 parts, 4.0 parts, 5.0 parts, 6.0 parts, 7.0 parts, 8.0 parts of glucose.
2.1.3 growth factor optimizes
(1) ascorbic acid optimizes
Table 6 can be seen that, when adding 0.01% ascorbic acid, the Lactobacillus plantarum DMDL9010 bacteria suspension viable counts of acquisition
At most (P<0.05), now bacteria growing situation is better than blank group.But with ascorbic acid content rising in culture medium, obtain
Bacteria suspension viable count tends towards stability.
The influence that the various concentrations ascorbic acid of table 6 grows to Lactobacillus plantarum DMDL9010
(2) vitamin B compound optimizes
Table 7 obtains viable count at most (P as can be seen that when adding 2 vitamin B complex tablets<0.05), now plant
Lactobacillus DMDL9010 growths are preferable.As the vitamin B complex tablet quantity added in culture medium rises, the plant breast bar of acquisition
Bacterium DMDL9010 bacteria suspension viable counts tend towards stability.
The influence that the various concentrations vitamin B compound of table 7 grows to Lactobacillus plantarum DMDL9010
Note：Vitamin B complex tablet main component is：Every contains 3mgVB1, 1.5mgVB2, 0.2mgVB6, 10mg niacinamide,
1mg calcium pantothenates, auxiliary material are starch, dextrin, magnesium stearate.
(3) manganese sulfate optimizes
Table 8 can be seen that, when 3.0 times of sulfuric acid manganese content in MRS culture mediums based on the content of manganese sulfate in culture medium,
Viable count significantly increases (P in Lactobacillus plantarum DMDL9010 bacteria suspensions<0.05), with the sulfuric acid manganese content increase of addition, obtain
The viable count of the Lactobacillus plantarum DMDL9010 bacteria suspensions obtained gradually reduces (P<0.05), or even will be less than the basis being not optimised
MRS culture mediums, illustrate to add excessive manganese sulfate, have significant inhibitory action to Lactobacillus plantarum DMDL9010 propagation.
The influence that the Different adding amount manganese sulfate of table 8 grows to Lactobacillus plantarum DMDL9010
Note：Unit refers to (again)：Based on manganese sulfate addition in culture medium sulfuric acid manganese content multiple, that is, add 0.005
Part, 0.01 part, 0.015 part, 0.02 part, 0.025 part, 0.030 part, 0.035 part of manganese sulfate.
(4) magnesium sulfate optimizes
Table 9 can be seen that, when based on content 2.0 times of sulfuric acid content of magnesium in MRS culture mediums, bacteria suspension bacterium number significantly increases
(P<0.05) rising of the content, and with the magnesium sulfate of addition, the viable count of acquisition gradually reduce, or even to be less than and be not optimised
Basic MRS culture mediums, illustrate to add excessive magnesium sulfate have to Lactobacillus plantarum DMDL9010 propagation and significant suppress to make
With.In the present embodiment, adding growth of the magnesium sulfate to Lactobacillus plantarum DMDL9010 has significant facilitation.With other researchs
It is probably strain difference that the result of person is different, or its culture medium provides Mg from other approach2+, therefore need not additionally add
Magnesium sulfate is as growth factor.The Mg contained in basic MRS culture mediums selected by the present embodiment2+The sulphur for being all from and being added
Sour magnesium, therefore growth of the addition to Lactobacillus plantarum DMDL9010 of magnesium sulfate has facilitation in the present embodiment.
The influence that the Different adding amount magnesium sulfate of table 9 grows to Lactobacillus plantarum DMDL9010
Note：Unit refers to (again)：Based on magnesium sulfate addition in culture medium sulfuric acid content of magnesium multiple, that is, add 0.02
Part, 0.04 part, 0.06 part, 0.08 part, 0.1 part, 0.12 part of magnesium sulfate.
2.2 culture medium prescription optimization designs
Medium optimization is carried out using Design Expert8.0.5 softwares.Above when carrying out single factor analysis, selection
Bacteriological peptone, Soyprotein peptide, glucose, ascorbic acid, vitamin B complex tablet, manganese sulfate, magnesium sulfate this seven kinds of lists
Factor is analyzed, and is found to work as and is contained 2.5 times of bacteriological peptones, 0.5 (compared with basic MRS culture mediums) in culture medium respectively
Times Soyprotein peptide, 1.5 times of glucose, 0.01% ascorbic acid, 2 vitamin B complex tablets, 3.0% manganese sulfate, 2.0% sulphur
During sour magnesium, it is viable count content highest in respective single factor test Optimal Experimental group to obtain bacteria suspension.Therefore select these dense
Degree is used as benchmark, carries out Plackett-Burmen experiments and Box-Behnken Optimal Experimentals.
The Plackett-Burmen experimental result tables of table 10
The data in upper table are analyzed using Design Expert 8.0.5 softwares, it is as a result as follows：
The Plackett-Burmen experimental analysis tables of table 11
As known from Table 11, in the growth for promoting Lactobacillus plantarum DMDL9010, ascorbic acid, Soyprotein peptide, sulfuric acid
The contribution degree of manganese is higher, respectively 15.09%, 13.64% and 6.99%, and the contribution margin of other several factors is not higher than 5%.
As a result show in this seven kinds of promotive factors, ascorbic acid, Soyprotein peptide, manganese sulfate are added, for Lactobacillus plantarum
The facilitation of DMDL9010 growth is compared with other four kinds obvious, and the contribution margin of ascorbic acid, Soyprotein peptide is much
Higher than other factors, although the contribution margin of manganese sulfate is far below ascorbic acid and Soyprotein peptide, but still higher than others
Several factors.
The result tested according to Plackett-Burman, ascorbic acid, Soyprotein peptide, manganese sulfate are selected as research
Object, carry out the Box-Behnken Optimal Experimentals of next step.
2.3 Box-Behnken Optimal Experimentals
The Box-Behnken experimental result tables of table 12
Analyzed using Box-Benhken center combination design experimental data, then utilize Design Expert
8.0.5 software is analyzed.
The Box-Behnken experimental analysis tables (b) of table 13
As shown in Table 13, the p=0.0012 ＜ 0.05 of model of fit, coefficient of determination R2=0.9462, illustrate model and reality
Border situation fitting is good.Analysis learns viable count Y to independent variable Soyprotein peptide (A), ascorbic acid (B), sulfuric acid more than
The multiple regression equation of manganese (C)：
Y=61.75000-35.45000A-2698.75000B-5.48750C+715.00000AB-2. 35000AC+
625.00000BC+36. 50000A2+44500.00000B2-0.55000C2
The equation expresses the linear relationship between the viable count of Lactobacillus plantarum DMDL9010 nutrient solutions and 3 independents variable
It is significant, i.e., this method is reliable.The Monomial coefficient of regression equation is bigger, AB, BC friendship in interaction term coefficient
Mutual coefficient ratio is larger, illustrates that the interaction between Soyprotein peptide and ascorbic acid, ascorbic acid and manganese sulfate is big, and AC
Interaction coefficient it is smaller, illustrate that the interaction between Soyprotein peptide and manganese sulfate is small.
In order to examine the validity of equation, to the mathematics of the viable count in the Lactobacillus plantarum DMDL9010 nutrient solutions of measure
Model carries out variance analysis, and the partial regression coefficient of each factor is detected.B regression coefficient highly significant in first order,
P=0.0033, illustrate that ascorbic acid concentrations have highly significant effect to Lactobacillus plantarum DMDL9010 growths.C regression coefficient
B is only second to, illustrates that manganese sulfate various concentrations are only second to ascorbic acid to Lactobacillus plantarum DMDL9010 growth effects.In quadratic term
A partial regression coefficient reaches the pole level of signifiance.Interaction item BC regression coefficients are notable compared with other factors, illustrate ascorbic acid and sulfuric acid
Growth of the interaction item of manganese to Lactobacillus plantarum DMDL9010 is more notable.Degree of fitting R2=0.9462, illustrate the equation to reality
It is good to test degree of fitting.
In order to obtain the Lactobacillus plantarum DMDL9010 increments of maximum, Soyprotein peptide and manganese sulfate are horizontal in culture medium
Need proper ratio.Therefore, learnt according to Fig. 5 and variance analysis, to Lactobacillus plantarum DMDL9010 growth effect size orders
For AB ＞ BC ＞ AC.
Single order local derviation is sought quadratic regression equation, and when response Y (viable count) maximum, each factor level is soybean egg
White peptide (A) is 1.0 times, ascorbic acid (B) is 0.02%, manganese sulfate (C) is 4.00 times.Now theoretical prediction Lactobacillus plantarum
DMDL9010 prediction cell concentration is 5.20 × 109cfu/mL
Checking test is carried out to the culture medium prescription obtained, actual cell concentration is 4.84 × 109cfu/mL。
Adding 6.0% skimmed milk powder into bacterium mud, the viable count of the bacterium powder obtained after lyophilized can reach 1.353 ×
1010Cfu/g, skimmed milk powder is good organic nitrogen source, carbon source, and the lactalbumin contained in skimmed milk powder is in freeze-drying
During have certain protective effect to Lactobacillus plantarum DMDL9010.Compared with other alternative protective agents, skimmed milk powder conduct
Every gram of viable count highest of protectant group, and skimmed milk powder is readily available, cost is relatively low, therefore selects skimmed milk powder conduct
The freeze drying protectant subsequently optimized.And macromolecular protective agent skimmed milk powder is selected as dried medium, because：(1) skimmed milk powder
Cellular damage can be prevented by the composition of stabilizing cell membrane；(2) loose structure is produced in freezing dry process so that de-
Water is more prone to；(3) skimmed milk powder plays the role of protein in itself can provide a protective layer for cell.
The freeze-dried powder of embodiment 3 prepares optimizing research
The first step：Lactobacillus plantarum DMDL9010 bacterium powders collect fermentation culture after being placed in 2 activation of MRS culture mediums；
Second step：Using volume ratio as 3:100 ratio, fermentation culture is accessed in high-density culture medium, at 37 DEG C
Fermented and cultured is carried out, zymophyte suspension is collected after 18h；In terms of parts by weight, described high-density culture medium is：Casein digest
1.0 parts of thing, 0.5 part of yeast extract powder, 2.0 parts of glucose, 0.2 part of Triammonium citrate, 0.058 part of magnesium sulfate, 1.0 parts of beef extract,
0.1 part of dipotassium hydrogen phosphate, 0.5 part of sodium acetate, 0.18 part of Tween 80,1 part of Soyprotein peptide, 0.02 part of ascorbic acid, sulfuric acid
0.1 part of manganese, 100 parts are settled to distilled water, adjusts pH to 6.8.Stirring and dissolving is cold uniformly after 0.10MPa sterilizings 15min
But it is standby to 30 DEG C；
3rd step：Using volume ratio as 5:100 ratio, fermentation culture is accessed in production medium, entered at 37 DEG C
Row fermented and cultured, zymophyte suspension is collected after 18h；In terms of parts by weight, described productive culture based formulas composition and high density
Culture medium is identical, adjusts pH to 6.8.Stirring and dissolving uniformly after 0.10MPa sterilize 20min, be cooled to 37 DEG C it is standby；
4th step：Bacterium mud will be collected after High Density Cultivation, with continuous normal saline clean 2 times after 4000~
10000rpm, 10~30min of centrifugation collect bacterium mud, and gained centrifuge results are as shown in table 14.Contrast each centrifugal rotational speed and centrifugation
The combination of time, with the rise of centrifugal rotational speed, supernatant viable count substantially reduces, and it is then first to rise to drop afterwards to precipitate viable count
Low, this shows too low centrifugal rotational speed so that thalline can not be precipitated fully, and the bacterium mud amount of acquisition is insufficient, and too high centrifugal rotational speed
Though can effectively make bacterial sediment, also larger to the degree of injury of viable bacteria, a large amount of viable bacterias damage in centrifugal process, are dead.
As centrifugation time extends, supernatant viable count, which substantially reduces, precipitates viable count substantially increases, and illustrates that the centrifugation of enough time can
So that viable bacteria is fully precipitated.Long centrifugation time can reduce efficiency, be unfavorable for mass producing.
The Lactobacillus plantarum DMDL9010 centrefuge experiment results of 14 embodiment of table 3
5th step：It is 3 by the normal saline solution of complex protection agent solution and the volume ratio of bacterium mud in bacterium mud:1 adds
Complex protection agent solution, it is well mixed as protective agent bacteria suspension；In terms of parts by weight, described composite protectant solution formula group
Turn into：12 parts of skimmed milk powder, 7.95 parts of bacteriological peptone, 15.2 parts of trehalose, 0.9 part of sodium chloride, it is settled to distilled water
100 parts.Stirred after dissolving, in 0.10MPa, sterilize 15min, and it is standby to be cooled to normal temperature；
Protective agent bacteria suspension is placed in drier by the 6th step, and vacuum freeze drying is placed in after pre-freeze 6.0h at -20 DEG C
24h is dried in machine and obtains freeze-dried powder, the temperature of freeze-dried powder is returned into normal temperature with constant humidity air (humidity 60%), packed with air-flow
Machine progress is aluminum-plastic packaged, and freeze-dried powder moisture is less than 3%, and viable count reaches 1011cfu/g。
By the optimization of screening single factor test and Box- that freeze drying protectant is carried out to Lactobacillus plantarum DMDL9010 lyophilisation conditions
Behnken Optimal Experimentals design.
3.1. the screening of the freeze drying protectant of Lactobacillus plantarum
3.1.1 the single factor test screening of freeze drying protectant
Prepare sufficient amount of 10mL centrifuge tubes, weigh the weight of each centrifuge tube, and mark on centrifuge tube.Prepare
Enough MRS basal mediums, it is 3% toward MRS inoculation of medium Lactobacillus plantarum DMDL9010 by inoculum concentration, cultivates 24h,
By the centrifugal rotational speed and centrifugation time of optimization gained：Rotating speed 8000rpm centrifugations 30min is centrifuged, and it is standby to collect lower floor's bacterium mud.
The alternative frozen-dried protective agent solution of various concentrations is prepared, by the bacterium mud after centrifugation and the frozen-dried protective agent solution prepared
Volume is 1:3 ratio is well mixed.It is put into 10mL centrifuge tubes, is positioned in vacuum freeze drier and is freeze-dried.Completely
After lyophilized, weigh the gross weight of 10mL centrifuge tubes and obtained bacterium powder, calculate the weight of obtained bacterium powder, add into bacterium powder
Enter sterilized water constant volume to redissolve to 10mL, dilution plate counts, and obtains under more different alternative freeze drying protectants and each concentration
The viable count (cfu/g) of every gram of bacterium powder.
(1) selection of skimmed milk powder concentration
Influence of the skimmed milk powder concentration of table 15 to Lactobacillus plantarum DMDL9010 bacterium powders
Protective effect of the skimmed milk powder to Lactobacillus plantarum DMDL9010 is notable, and 6.0% skimmed milk is added into bacterium mud
Powder, the viable count of the bacterium powder obtained after lyophilized can reach 1.353 × 1010Cfu/g, it is good organic nitrogen to illustrate skimmed milk powder
Source, carbon source, and the lactalbumin contained in skimmed milk powder has one during freeze-drying to Lactobacillus plantarum DMDL9010
Fixed protective effect.Skimmed milk powder is readily available, and cost is relatively low, therefore selects skimmed milk powder as the frozen-dried protective subsequently optimized
Agent.
(2) selection of concentration of glucose
Influence of the concentration of glucose of table 16 to Lactobacillus plantarum DMDL9010 bacterium powders
Glucose has protective effect to Lactobacillus plantarum DMDL9010,8.0% glucose is added into bacterium mud, after freezing
The viable count highest of the bacterium powder of acquisition.Reach 5.06 × 109cfu/g；The glucose of other concentration is added, the viable count of acquisition
Change unobvious, add excessive glucose, the viable count contained by every gram of bacterium powder is reduced on the contrary.With other alternative protective agents
Compare, protective effect of the glucose to Lactobacillus plantarum DMDL9010 will be less than skimmed milk powder, trehalose and bacteriological peptone.
(3) selection of bacteriological peptone concentration
Influence of the bacteriological peptone concentration of table 17 to Lactobacillus plantarum DMDL9010 bacterium powders
Bacteriological peptone has preferable protective effect to Lactobacillus plantarum DMDL9010, and 8.0% is added into bacterium mud
Bacteriological peptone, the viable count highest of the bacterium powder obtained after lyophilized.Reach 9.48 × 108cfu/g；Add the Portugal of other concentration
Grape sugar, obtains that the change of viable count is obvious, adds the bacterium powder of the acquisition of 8.0% bacteriological peptone, the viable count in every gram
Bacteriological peptone to add 2.0% obtains 3 times of bacterium powder.But excessive bacteriological peptone is added, the bacterium powder of acquisition
Every gram of viable count reduces.
(4) selection of soluble starch concentration
Influence of the soluble starch concentration of table 18 to Lactobacillus plantarum DMDL9010 bacterium powders
Soluble starch has protective effect to Lactobacillus plantarum DMDL9010, and 16.0% soluble shallow lake is added into bacterium mud
Powder, the bacterium powder viable count highest obtained after lyophilized.Reach 1.49 × 108cfu/g.In 10.0% to 16.0% stage, with adding
The soluble starch amount entered increases, and viable count contained by every gram of bacterium powder is in the trend risen.And after 16.0%, addition can
Soluble starch is more, and viable count contained by every gram of bacterium powder is fewer.It is the optium concentration for adding soluble starch to add 16.0%.
(5) selection of trehalose concentration
Influence of the trehalose concentration of table 19 to Lactobacillus plantarum DMDL9010 bacterium powders
Trehalose has preferable protective effect to Lactobacillus plantarum DMDL9010, and 14.0% marine alga is added into bacterium mud
Sugar, the bacterium powder viable count highest obtained after lyophilized, reaches 8.99 × 1010cfu/g.Compared with other alternative protective agents, trehalose
Higher as protectant group viable count, every gram of viable count of acquisition is only second to group of the skimmed milk powder as freeze drying protectant
Not.Therefore selection trehalose is as the freeze drying protectant subsequently optimized.3.2Box-Behnken Optimal Experimentals design
The data in upper table are analyzed using Design Expert 8.0.5 softwares, it is as a result as follows：
The Box-Behnken experimental results of table 20
Analyzed using the center combination design experimental data of response surface, then utilize Design Expert 8.0.5
Software is analyzed.
The Box-Behnken experimental analysis tables (b) of table 21
As shown in Table 25, the p=0.0002 ＜ 0.05 of model of fit, coefficient of determination R2=0.9696, illustrate model and reality
Border situation fitting is good.Analysis learns viable count Y to independent variable skimmed milk powder (A), bacteriological peptone (B), sea more than
The multiple regression equation of algae sugar (C)：
Y=672.09375+27.37708A+39.95500B+61.33063C-0.88750AB-0.40 458AC-
2.86375BC-0.70625 A2+0.87906B2-1.35781C2
The equation expresses the linear pass between the viable count of every gram of bacterium powder of Lactobacillus plantarum DMDL9010 and 3 independents variable
System is significant, i.e., this method is reliable.The Monomial coefficient of regression equation is bigger, BC interaction in interaction term coefficient
Coefficient ratio is larger, illustrates that the interaction between bacteriological peptone and trehalose is big, and AB, AC interaction coefficient are smaller,
Illustrate that the interaction between skimmed milk powder and bacteriological peptone, skimmed milk powder and trehalose is small.
In order to examine the validity of equation, to the number of the viable count in every gram of bacterium powder of Lactobacillus plantarum DMDL9010 of measure
Learn model and carry out variance analysis, and the partial regression coefficient of each factor is detected.A regression coefficient is highly aobvious in first order
Write, p ＜ 0.0001, illustrate that skimmed milk powder concentration has highly significant effect to Lactobacillus plantarum DMDL9010 growths.C recurrence
Coefficient is only second to A, illustrates that various concentrations are only second to skimmed milk powder to Lactobacillus plantarum DMDL9010 growth effects.Interaction item BC
Regression coefficient is notable compared with other factors, illustrates the interaction item between bacteriological peptone and trehalose to Lactobacillus plantarum
DMDL9010 growth is more notable.Degree of fitting R2=0.9696, illustrate that the equation is good to experimental fit degree.
According to above-mentioned regression analysis, the character of Box-Behnken Optimal Experimentals fitting response surface design is investigated in the face that responds,
Skimmed milk powder (A), bacteriological peptone (B), the various concentrations of trehalose (C) are analyzed to combine to Lactobacillus plantarum DMDL9010
The influence for the viable count situation that every gram of bacterium powder obtains.As a result it is as shown in Figure 6.In order to obtain the Lactobacillus plantarum of maximum
The viable count of every gram of bacterium powder of DMDL9010, skimmed milk powder and trehalose level need proper ratio in culture medium.Therefore, according to attached
Fig. 6 and variance analysis learn that the influence size order to the viable count of every gram of bacterium powder of Lactobacillus plantarum DMDL9010 is BC ＞ AB
＞ AC.
Single order local derviation is sought quadratic regression equation, each factor level when response Y (every gram of bacterium powder viable count) maximum
For skimmed milk powder (A) addition 12.0%, bacteriological peptone (B) addition 7.95%, trehalose (C) addition 15.20%.Now manage
Viable count by prediction Lactobacillus plantarum DMDL9010 is 4.195 × 1011cfu/g。
Embodiment 4：Lactobacillus plantarum DMDL9010 is applied to the production of fermentation pickled vegetable
Vegetables clear water wash clean, and drain the moisture on surface, it is cut into appropriately sized and uses scalding.By plant breast bar
Bacterium DMDL9010, freeze-dried powder are placed in MRS culture mediums, and after 30 DEG C~40 DEG C static gas wave refrigerator 18h~36h, seed liquor is made,
Its viable count is set to respectively reach 108More than CFU/mL；With 1%~10% percent by volume, by Lactobacillus plantarum
DMDL9010 seed liquors are respectively placed in fermentation medium, and after 30 DEG C~40 DEG C static gas wave refrigerator 18h~36h, zymotic fluid is made,
Its viable count is set to respectively reach 108More than CFU/mL；The fermentative medium formula is：80~90 parts of fresh vegetables juice, dimension
Raw plain 0.5 part~3.0 parts of C, 0.5~3.0 part of NaCl, 100 parts is settled to sterilized water, after being cooled down after sterilizing, is as fermented
Culture medium；By weight, 20~40 parts of vegetables, according to (5~1):1 volume ratio access Lactobacillus plantarum DMDL9010 hair
Totally 3~10 parts of zymotic fluid, 0.1~2.0 part of vitamin C, each 0.2~5.0 part of NaCl are added wherein, is settled to sterilized water
100 parts, make vegetables submergence in a liquid.Anaerobic fermentation is carried out under air-proof condition, 20~40 DEG C of 24~72h of fermentation of temperature control are
Can.Described fresh vegetables juice is more than one or both of mustard, cabbage juice, Tomato juice and carrot juice.The vegetable
Dish be leaf mustard, root-mustard, Chinese cabbage, cabbage, cucumber, fresh kidney beans, carrot, ternip, lettuce and capsicum in it is one or two kinds of with
On.Vegetables load in polyethylene plastic bag or aluminium plastic bag after fermentation, after vacuumizing, heat sealing.Prepared by gained mixes containing living
Lactobacillus-fermented pickles are closed, by vacuum packaging, are preserved 6 months at normal temperatures, fermentation pickled vegetable contains Lactobacillus plantarum
DMDL9010 total viable counts reach 105~106cfu/g。
Embodiment 5:Lactobacillus plantarum DMDL9010 is applied to the production of fermented fruits and vegetables juice
Lactobacillus plantarum DMDL9010 is inoculated in MRS broth bouillons, and through 1~2 Secondary Culture, pH value reaches 6.0
± 0.5, strain is stored in 0~4 DEG C when reaching normal vital, and the pawpaw after stripping and slicing and carrot are pressed into 2~4:1 ratio mixing
And after crushing, then mixed fruit and vegetable juice beverage is obtained after adding sucrose and water, by 90~96 DEG C of heating 7min~15min and cool down
Afterwards, it is the activating solutions of 6%~8% access Lactobacillus plantarum DMDL 9010 according to percent by volume, ferment 12h at 28 DEG C~40 DEG C
~22h, that is, fermentation mixed fruit and vegetable juice beverage is made, by sterile filling, refrigeration, viable type fermentation mixed fruit and vegetable juice drink is made
Material.
Embodiment 6：Lactobacillus plantarum DMDL9010 is applied to the production of fermentation ice cream
Lactobacillus plantarum DMDL9010 freeze-dried powders are placed in MRS culture mediums, in 30 DEG C~40 DEG C static gas wave refrigerator 18h~36
After h, seed liquor is made, its viable count is respectively reached 108More than CFU/mL；With 1%~10% percent by volume, will plant
Thing lactobacillus DMDL9010 seed liquors are respectively placed in fermentation medium, after the h of 30 DEG C~40 DEG C static gas wave refrigerator 18h~36, system
Into zymotic fluid, its viable count is set to respectively reach 108More than CFU/mL；By milk, cream, whole milk powder, condensed milk, butter,
White granulated sugar, yolk powder are well mixed using rotating speed as 300r/min stirrings 10min obtains mixture, is heated to 75 DEG C, is incubated 25min
Sterilization, by the mixture homogenization 30min after sterilization, 3%~6% (v/v) Lactobacillus plantarum DMDL9010 is added, stands 6-
10h completes fermentation, obtains ice river in Henan Province and insults base-material；The salt of ice cube weight 1/3rd is added in 0 DEG C of ice cube, i.e., salt is in ice cube
Mass ratio 1:The temperature of water is down to -20 DEG C by 3, obtains coolant, and being stirred ice cream 10min with 40r/min can obtain
Fermentation ice cream rich in Lactobacillus plantarum DMDL9010, viable count 107More than CFU/mL, acidity are 75.0~80.0 ° of T,
Protein is 3.0~3.2g/100g, and fat content is 3.1~3.4g/100g, and nonfat milk solids content is 8.1~9.0g/
100g。
Embodiment 7：Lactobacillus plantarum DMDL9010 is applied to the production of fermentation jelly
Lactobacillus plantarum DMDL9010 freeze-dried powders are placed in MRS culture mediums, in 30 DEG C~40 DEG C static gas wave refrigerator 18h~36
After h, seed liquor is made, its viable count is respectively reached 108More than CFU/mL；With 1%~10% percent by volume, will plant
Thing lactobacillus DMDL9010 seed liquors are respectively placed in fermentation medium, after the h of 30 DEG C~40 DEG C static gas wave refrigerator 18h~36, system
Into zymotic fluid, its viable count is set to respectively reach 108More than CFU/mL；By 1:Coixlacrymajobi powder, the inulin of 1 volume ratio are stirred
Add compounding konjaku flour and carragheen (4 after even：3), it is dissolved in 80 DEG C of hot water, heats 8-10 min while stirring, then adds lemon
Lemon acid sodium adjusts pH runny carbohydrate gum body, to access 3%~6% (v/v) Lactobacillus plantarum DMDL9010 to clear viscous is obtained,
Stand 6-10h complete after fermentation with the screen filtrations of 100 mesh clean and foam after be placed in die for molding after pack.
Embodiment 8：Lactobacillus plantarum DMDL9010 is applied to the production of cultured milk's piece
Lactobacillus plantarum DMDL9010, freeze-dried powder are placed in MRS culture mediums, in 30 DEG C~40 DEG C static gas wave refrigerator 18h~
After 36 h, seed liquor is made, its viable count is respectively reached 108More than CFU/mL；With 1%~10% percent by volume,
Lactobacillus plantarum DMDL9010 seed liquors are respectively placed in fermentation medium, in the h of 30 DEG C~40 DEG C static gas wave refrigerator 18h~36
Afterwards, zymotic fluid is made, its viable count is respectively reached 108More than CFU/mL；Lactobacillus plantarum is obtained after lyophilized
DMDL9010 freeze-dried powders viable count is up to 1010More than CFU/g；By glucose 6%, white granulated sugar 6.8%, amylum pregelatinisatum 5%, hard
Fatty acid magnesium 1.6%, the grinding and sieving of dextrin 2.7% obtain original washing powder, add Lactobacillus plantarum DMDL9010 freeze-dried powders 6%, full-cream
After milk powder 12.5%, Fruit powder are mixed evenly, carry out tabletting and can be prepared by the milk rich in Lactobacillus plantarum DMDL9010
Piece, viable count is up to 1010CFU/g。
Embodiment 9：Lactobacillus plantarum DMDL9010 is applied to coagulating type and stirred type acidified milk produces
Lactobacillus plantarum freeze-dried powder is resuspended with 1mL sterilized waters, is then all forwarded in MRS fluid nutrient mediums, in 37 DEG C
Lower culture 16h~20h, then be forwarded in fresh MRS fluid nutrient mediums and activated 3 times with 5%~10% inoculum concentration, in 4000
R/min low-temperature centrifugations collect thalline, and its whole is forwarded into activation culture 2~3 times at 35 DEG C of fermentation milk, produce plant
Thing lactobacillus leavening agent；The whole-fat milk powder, 7.5% sucrose, 0.0005% gluconic acid that the activation milk is 10.0%
It is sterilized to form after the raw materials such as manganese, 2.0% soya-bean polypeptides are well mixed.By fresh milk, 8.5% sucrose, 0.1% stabilizer powder,
After being well mixed by mass volume ratio, 60 DEG C of homogeneous under 18Mpa are preheated to, and 37 DEG C are cooled to after sterilizing 5min in 95 DEG C,
It is standby；Inoculation and fermentation：5% lactobacillus plantarum ferment is added in fermentation milk by volume, is fermented in 37 DEG C,
Fermentation is terminated after titratable acidity reaches 75 °, obtains acidified milk, pH, retention ability and the viable count of fermentation termination be respectively 3.85,
99.03% and 9.30 × 1010CFU/mL, mouthfeel quality meet the requirements, and acidity is 75.0~80.0 ° of T, and protein is 3.0~
3.2g/100g, fat content are 3.1~3.4g/100g, and nonfat milk solids content is 8.1~9.0g/100g.If it will pass through upper
The solidified-type fermented milk that step obtains is stated, is demulsified by peristaltic pump, as stirred type acidified milk.
Embodiment 10：Lactobacillus plantarum DMDL9010 is applied to the production of fermented bean dregs and peanut meal
By weight, by 1:1 dregs of beans (or peanut meal) mixes with water, in 0.10MPa sterilizing 20min, is cooled to 37 DEG C
Access the freeze-dried powder of embodiment 2, make by weight viable count up to 107Individual/g, ferment 36h under 30 DEG C of closed environments, fermentation knot
PH is 5.0~6.0 after beam, and tunning is carried out into hot-air spray drying, dries EAT and controls 250 DEG C, mixing temperature control
150 DEG C of system, drop temperature control 75 DEG C.Dried material moisture control less than 12%.Due to having used the freeze-dried powder of high viable count,
So substantially reducing fermentation time, improve fermentation efficiency and improve protein content, reduce the molecular weight of protein
Be advantageous to digest and assimilate.
Embodiment 11：Lactobacillus plantarum DMDL9010 is applied to the production of ferment sausage
After raw meat is picked a bone, shred and stir, by weight, add 3.0% sucrose, 2.0% salt and 1.5%
Cooking wine, freeze-dried powder prepared by embodiment 2 is inoculated with after being well mixed, make by weight viable count up to 107Individual/g, after being well mixed
Bowel lavage, tie up and fermented in the environment of 20 DEG C, relative humidity is 95%, terminate fermentation when fermentation to pH is 5.0, be put into 15
DEG C, relative humidity carries out afterripening fermentation 7d in the environment of being 80%, enters altar and pickles into various flavors.Because viable count content is high,
Dominant microflora fermentation is carried out, without adding nitrite, the ferment sausage smell produced is very pure, and color is naturally, be free of
Nitrite.
Embodiment 12：Lactobacillus plantarum DMDL9010 is applied to the production of boruga
Lactobacillus plantarum DMDL9010, freeze-dried powder are placed in MRS culture mediums, in 30 DEG C~40 DEG C static gas wave refrigerator 18h~
After 36 h, seed liquor is made, its viable count is respectively reached 108More than CFU/mL；With 1%~10% percent by volume,
Lactobacillus plantarum DMDL9010 seed liquors are respectively placed in fermentation medium, in the h of 30 DEG C~40 DEG C static gas wave refrigerator 18h~36
Afterwards, zymotic fluid is made, its viable count is respectively reached 108More than CFU/mL；Lactobacillus plantarum is obtained after lyophilized
DMDL9010 freeze-dried powders viable count is up to 1010More than CFU/g；By weight, by milk 80%, white granulated sugar 6%, Lactobacillus plantarum
DMDL9010 freeze-dried powders 0.04%, fermented 6 hours at 37 DEG C, acid can be obtained after carrying out cooling aseptic canning after homogeneous sterilization
Milk drink finished product.The fruit yoghurt drinks beverage acidity obtained reaches 75, and mouthfeel acid is refreshing suitable, is adapted to industrialized production.
Claims (10)
1. a kind of direct putting type Lactobacillus plantarum high-density cultivation method, it is characterised in that comprise the following steps：
(1) freeze-dried powder of Lactobacillus plantarum is placed in MRS culture mediums after being activated twice respectively and obtains seed fermentation agent, this kind
The bacterial content of sub- leavening is 108~109CFU/mL；
(2) using volume ratio as 3~6:100 ratio, seed fermentation agent is accessed in high-density culture medium, entered at 36~38 DEG C
Row fermented and cultured, zymophyte suspension is collected after 18~24h；
(3) zymophyte suspension is centrifuged 25~35min, gives up supernatant, with the physiology salt after sterilizing in 6000~8000rpm
Water cleaning precipitation, and collect bacterium mud；
(4) it is 2~5 by the volume ratio of composite protectant and bacterium mud in bacterium mud:1 adds composite protectant, after being well mixed
Bacteria suspension containing protective agent；
(5) protective agent bacteria suspension will be contained and be placed in drying in vacuum freeze drier after 1.5~6.0h of pre-freeze at -20~-65 DEG C
18~24h obtains freeze-dried powder.
2. according to the method for claim 1, it is characterised in that in terms of parts by weight, the formula of the high-density culture medium
For：0.8~1.0 part of casein digest, 0.3~0.5 part of yeast extract powder, 1.5~2.0 parts of glucose, Triammonium citrate 0.1~
0.2 part, 0.048~0.058 part of magnesium sulfate, 0.5~1.0 part of beef extract, 0.1~0.2 part of dipotassium hydrogen phosphate, sodium acetate 0.4~
0.5 part, 0.1~0.3 part of Tween 80,0.8~1.2 part of Soyprotein peptide, 0.01~0.02 part of ascorbic acid, manganese sulfate 0.05
~0.25 part.
3. according to the method for claim 2, it is characterised in that the high-density culture medium is：Above-mentioned raw materials are distilled
Water is settled to 100 parts, adjusts pH to 6.8, and stirring and dissolving uniformly after 0.08~0.10MPa, 15~20min of sterilizing, is cooled to 36
~38 DEG C standby.
4. according to the method described in claim 1 or 2 or 3, it is characterised in that in terms of parts by weight, described composite protectant
Formula be：10~14 parts of skimmed milk powder, 7.8~8.2 parts of bacteriological peptone, 13~17 parts of trehalose, 0.9 part of sodium chloride.
5. according to the method for claim 4, it is characterised in that described composite protectant is：Above-mentioned raw materials are distilled
Water is settled to 100 parts, is stirred after dissolving, and in 0.08~0.10MPa, sterilize 15~20min, and it is standby to be cooled to normal temperature.
6. according to the method described in claim 1 or 2 or 3 or 4 or 5, it is characterised in that described centrifugal rotational speed is 8000rpm,
Centrifugation time is 30min.
7. according to the method described in claim 1 or 2 or 3 or 4 or 5 or 6, it is characterised in that step (5) obtained freeze-drying powder, first
Its temperature is returned into normal temperature with the constant humidity air that humidity is 50~60%, then carried out with air-flow packing machine aluminum-plastic packaged.
8. according to the method described in claim 1 or 2 or 3, it is characterised in that the Lactobacillus plantarum is DMDL9010, preservation
Numbering is CGMCC NO.5172.
9. freeze-dried powder prepared by claim 1~8 any one method, it is characterised in that the moisture of the freeze-dried powder is low
In 3%, viable count reaches 1010~1011cfu/g。
10. freeze-dried powder described in claim 9 is in fermentation pickled vegetable, Juice, ice cream, jelly, milk piece, stirred yoghurt, solidification
Applied in type Yoghourt, boruga, sausage, corn flour, dregs of beans or peanut meal.
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