CN102272287A - Freeze-dried microbial cell powder and method for producing same - Google Patents
Freeze-dried microbial cell powder and method for producing same Download PDFInfo
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- CN102272287A CN102272287A CN2010800041726A CN201080004172A CN102272287A CN 102272287 A CN102272287 A CN 102272287A CN 2010800041726 A CN2010800041726 A CN 2010800041726A CN 201080004172 A CN201080004172 A CN 201080004172A CN 102272287 A CN102272287 A CN 102272287A
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- 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
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Abstract
Provided is a freeze-dried microbial cell powder suitable for prolonged storage which can be obtained by a convenient and effective freeze-drying method. A method for producing a freeze-dried microbial cell powder comprising freeze-drying a suspension, in which cells of a lactic acid bacterium and/or a bifidobacterium are suspended in a solution of sugars, to give a freeze-dried microbial cell powder, wherein the sugars are trehalose and sucrose and the trehalose and sucrose concentrations in the suspension before the freeze-drying are each greater than or equal to 4.5 wt%. The weight ratio of trehalose to sucrose is preferably 5:1 to 1:5.
Description
Technical field
The present invention relates to Powdered thalline of lyophilize and manufacture method thereof, though particularly relate under specific hot conditions also can prolonged preservation Powdered thalline of lyophilize and manufacture method thereof.
Background technology
In recent years, have beneficial bacterial strain of giving birth to function and caused people's attention.And, based on these bacterium carry out the improvement/normalizing in the digestive tube and the preventive medicine of the immune Accommodation of accompanying therewith is in vogue by absorbing.Wherein, particularly milk-acid bacteria, lactobacillus bifidus etc. are also referred to as probiotic bacterium, just absorb with the form of various food since ancient times.Recently, its functional development is than more flouring in the past, and the exploitation of having carried out making up functional commercial form that novelty is arranged.
For above-mentioned purposes such as improvement/normalizing of carrying out in the digestive tube, developed a large amount of processed food groups that contains these mushrooms.And be used for easy and absorbing effectively aspect the work of these mushrooms still under study for action.For example, can enumerate sour milk, but, also make these mushrooms Powdered in order further to be easy to picked-up as a common example of these food group.This Powdered thalline also can be used as and is used for making easily the novel raw material of using processed food, its utility value height.
But, these mushrooms are being made under pulverous situation, if these mushrooms are failed viable bacteria more than the remaining amount of being necessary when when picked-up or food mfg, then can't obtain the effect of these mushrooms.Thereby, wish to develop effective mushroom store method and be used for making easily pulverous method.For example, hope is developed in the method for carrying out improving under the cryodesiccated situation its viability.
In order to reach above-mentioned purpose, various researchs have been carried out so far.
For example, the purpose of patent documentation 1 is, by under acidity or the granulation that is covered of useful bacterium such as the milk-acid bacteria that can be killed under the high temperature, lactobacillus bifidus preserve such intention and make it that dissolving can not take place in gastric juice and solubility in intestines is improved.Specifically, physiologically active substance is made the granulation thing by 3 layers of lining, use carbohydrate in one deck therein.
In addition, proposed in substratum to add the viability activator that the contains carbohydrate method with the viability of improving bifidobacterium bacteria in patent documentation 2, described carbohydrate is selected from glycerine, Xylitol, ribitol, arabitol, N.F,USP MANNITOL.The purpose of this method is, when preserving under aerobic conditions or under the low pH condition, keeps viability and bacterium number.Particularly the main purpose of this technology is to improve the survival rate in culture medium system.
In addition, in patent documentation 3, provide the enteric solubility lactic bacteria composition of a kind of permanent stability excellence of in lactic acid thalline powder, sneaking into polyglycerol fatty acid ester and acid resistance, enteric solubility excellence.Above-mentioned technology mainly is conceived to keeping quality and patience under given conditions.
On the other hand, various technology have also been developed in the aspect of the viability when preserving about the freezing preservation, lyophilize of improvement mushroom.
For example, in patent documentation 4, for provide a kind of in freezing or lyophilize operation thalline damage or dead less and the high manufacture method of survival rate, disclose a kind of so cryodesiccated method: the polyose partial hydrolystate that adds from Rhizoma amorphophalli powder in the thalline dispersion liquid of lactic acid bacteria class carries out lyophilize.The polyose partial hydrolystate is to decompose the material with specified molecular weight that obtains by enzyme, and making this hydrolyzate needs numerous and diverse operation.In addition, this technology is a main purpose with the survival rate that improves freezing when fusing, does not wherein hint fully for the survival rate of the Powdered thalline of the lyophilize that utilizes this technology manufacturing after preservation or mentions.
In patent documentation 5, proposed at back bacteria living rate and active still good preservation liquid that frozen bacteria is thawed.This bacterium contains trehalose and/or polyoxyethylene glycol as effective constituent with freezing preservation liquid.This preservation liquid is characterised in that thus, the infringement during freezing-inhibiting, can use immediately after thawing.This technology is the system that contains moisture, particularly for the technology that solves from freezing problem to this process of thawing.
In addition, in patent documentation 6, utilize protective membrane that lactobacillus fermentum is covered and make the spraying drying powder with starchiness, saccharic.This technology is the technology of the viability of bacterium when emphasis is put in the raising spraying drying.
Further, in patent documentation 7, by dried microorganisms such as dry milk-acid bacteria and L-arginine acidic amino acid salt being coexisted improve the storage stability under the drying regime.The main purpose of this technology is, improves the storage stability of microorganism with microorganism (bacterium) preparation the time.In addition, wherein show the lactobacillus bifidus preparation that to make with this technology 40 ℃ of results that preserved for 2 weeks, but its survival rate is about 25% only, is not for tolerating the technology of the prolonged preservation that extends to several months.
In addition, following result of study has been shown in non-patent literature 1: according to the Analysis on Mechanism in this lyophilize, the damage when finding out microbial freezing drying is mainly to be caused by the variation and the proteinic structural changes of the physical condition of the phospholipid that constitutes cytolemma; Based on this discovery, in the presence of trehalose or sucrose, carry out lyophilize for intestinal bacteria etc.But, wherein do not hint fully or mention for trehalose being mixed with sucrose use.Further, do not carry out any corresponding research for the prolonged preservation of concrete lyophilize thing and in the preservation of specific hot conditions yet.
Further, in non-patent literature 2, for above-mentioned same purpose, carried out about the lyophilize of lactobacillus salivarius (lactobacillus salivarius) and the research of preservation.In the document, with 4% trehalose; 4% sucrose; 18% skim-milk; 4% trehalose and 18% skim-milk; 4% sucrose and 18% skim-milk; 4% trehalose and 4% sucrose; And 4% trehalose and 4% sucrose and 18% skim-milk as freezing drying protective agent, the Powdered thalline of lyophilize is kept at-85 ℃, its survival rate is studied.In addition, the sugared concentration in the thallus suspension liquid is that about 3.2%, skim-milk concentration is 14.4% when lyophilize.According to the disclosed result of the document, recorded and narrated and utilized 4% sucrose can not fully improve the Powdered thalline of lyophilize, but utilized 4% sucrose and 18% skim-milk can improve the Powdered thalline of lyophilize at-85 ℃ keeping quality at-85 ℃ keeping quality.Further recorded and narrated and utilized 4% trehalose to improve lyophilize Powdered thalline separately-85 ℃ conservatory result.Show the mixing solutions that to utilize 4% trehalose, 4% sucrose and 18% skim-milk in addition and carry out the resulting Powdered thalline of lyophilize preserved for 7 weeks down in room temperature (temperature is not put down in writing) result.Mention at this,, can keep high keeping quality when the following time of environment that is kept at specific humidity condition (humidity 2.8～5.6%, in water activity 0.028～0.056).But, under the environment of humidity 0% and humidity 8.8%, can not keep high keeping quality (survival rate is respectively about 40% and 10%), but the condition restriction of room temperature preservation is very narrow in extremely low humidity and humidity range, do not have practicality.So, shown in the technology of non-patent literature 2 is the preservation test-results that departs under the envrionment conditions of common living environment, further, the preservation under the hot conditions is not hinted fully or mentions, also is difficult to this technology is applied in the common food.
The prior art document
Patent documentation
Patent documentation 1: Japanese kokai publication hei 5-186337 communique
Patent documentation 2: Japanese kokai publication hei 11-137172 communique
Patent documentation 3: TOHKEMY 2001-64189 communique
Patent documentation 4: Japanese kokai publication hei 7-313140 communique
Patent documentation 5: TOHKEMY 2001-327280 communique
Patent documentation 6: TOHKEMY 2005-52100 communique
Patent documentation 7: international open WO2006/106806 communique
Non-patent literature
Non-patent literature 1:Samuel B.Leslie etc.; " utilize trehalose and sucrose when dry " to the two the protection (Trehalose and Sucrose Protect Both Membranes andProteins in IntactBacteria during Drying) of the cytolemma of non-damage bacterium and protein, application and environmental microorganism (Applied andEnvironmental Microbiology); the U.S.; October nineteen ninety-five, the 3592-3597 page or leaf
Non-patent literature 2:Gaber Zayed etc., " influence (Influence of trehalose and moisture content on survival ofLactobacillus salivarius subjected to freeze-drying and storage) of the viability of trehalose and moisture content lactobacillus salivarius " for lyophilize with when preserving, Process Biochemistry, 2004, the 39th volume, the 1081-1086 page or leaf
Summary of the invention
Invent problem to be solved
As mentioned above, up to now, various researchs have been carried out about preservation and effective form of useful mushroom.Particularly, carried out various trials, but kept in the function of mushroom, very big research leeway still arranged aspect the survival rate after the prolonged preservation for the simplicity of its goods form for pulverous lyophilize thalline or dry thalline etc.For example, in specific trial,, thereby we can say the still untapped industrial effective means that go out even only for example exist effectively or effectively but preservation condition has significantly restriction such as the very low temperature of needs and low humidity to multiple mushroom to specific mushroom.Particularly for as can be applicable to the Powdered thalline in the food with long preservation period under the normal temperature, need be under the high temperature in the scope that is assumed to be common living environment temperature (30～40 ℃) condition and be not limited to have high survival probability under the special preservation condition.Its quality of Powdered thalline that does not have such character can't be guaranteed, and can not be sure of can be used as the material of food.
Thereby, the object of the present invention is to provide a kind of Powdered thalline of lyophilize and manufacture method thereof that is suitable for prolonged preservation, obtains by easy and effective lyophilize.Even particularly provide and after lyophilize, carry out prolonged preservation, even and this prolonged preservation be not for very low temperature, it is believed that the life temperature band (particularly phase in summer) that has a significant impact for keeping quality carries out, preserve under also can the be high state in the survival rate of useful bacterium such as milk-acid bacteria and lactobacillus bifidus, after preservation, also can directly absorb with form of powder easily or can be effectively as the Powdered thalline of lyophilize and the manufacture method thereof of the raw material in the food mfg.The food compositions that contains the Powdered thalline of resulting lyophilize also is provided further.
The means of dealing with problems and being adopted
In view of above-mentioned existing problem, the inventor furthers investigate, found that, by the suspension that milk-acid bacteria and/or lactobacillus bifidus are suspended in the saccharic solution is carried out lyophilize, survival rate when the viability of the Powdered thalline of useful bacterium (milk-acid bacteria and/or lactobacillus bifidus), particularly high temperature are preserved is significantly improved, to such an extent as to finished the present invention.
According to the present invention, provide the Powdered thalline of lyophilize that is suitable for prolonged preservation, obtains by easy and effective lyophilize.
Promptly, one aspect of the invention is the manufacture method of the Powdered thalline of lyophilize, it is for carrying out the manufacture method that lyophilize obtains the Powdered thalline of lyophilize of the Powdered thalline of lyophilize to the suspension that milk-acid bacteria and/or lactobacillus bifidus are suspended in the saccharic solution, wherein, above-mentioned saccharic is trehalose and sucrose, and trehalose and sucrose concentration separately in the above-mentioned suspension before the lyophilize are more than the 4.5 weight %.
In the present invention, trehalose and sucrose concentration separately is preferably 4.5 weight %～15 weight %, 8 weight %～12 weight % more preferably.
In the present invention, the weight ratio of trehalose and sucrose be preferably 5: 1～1: 5.
In the present invention, as the preference of above-mentioned milk-acid bacteria and/or lactobacillus bifidus, can enumerate and belong to mushroom that bifidus bacillus (Bifidobacterium) belongs to etc.
As the preference of the mushroom that belongs to bifidus bacillus (Bifidobacterium) genus, can enumerate bifidumbacterium bifidum (Bifidobacterium bifidum) strain, bifidus longum bb (Bifidobacterium longum) strain etc.
As the preference of bifidumbacterium bifidum (Bifidobacterium bifidum) strain, can enumerate bifidumbacterium bifidum (Bifidobacterium bifidum) OLB6378 strain etc.
As the preference of bifidus longum bb (Bifidobacterium longum) strain, can enumerate bifidus longum bb (Bifidobacterium longum) OLB6001 strain etc.
In addition, others of the present invention relate to the Powdered thalline of lyophilize that the manufacture method of the Powdered thalline of lyophilize that utilizes above-mentioned any record produces.
The further others of the present invention relate to the food compositions of the Powdered thalline of above-mentioned lyophilize that contains significant quantity.
The invention effect
Particularly under guaranteeing the situation of prolonged preservation etc., needs such as solid matter/powder food product can demonstrate its effect especially based on the manufacture method of the Powdered thalline of lyophilize with long preservation period of the present invention.That is, because the survival rate of powder lyophilize powder thalline when high temperature is preserved of utilizing manufacture method of the present invention to produce significantly improved, even thereby after preservation, use and also can obtain the useful milk-acid bacteria and/or the effect of lactobacillus bifidus.
In addition, because manufacture method of the present invention is easy method, thereby do not need special device and complicated step, the cost that also can not cause accompanying therewith increases.
In addition, because the Powdered thalline of lyophilize that utilizes manufacture method of the present invention to produce can directly use, thereby after preservation, also can directly absorb with form of powder easily or can be effectively as the raw material in the food mfg.
Description of drawings
Fig. 1 shows trehalose concentration in the suspension before lyophilize and is respectively 6.6 weight % with sucrose concentration and (as the sugar soln before mixing with thalline, uses and contain the trehalose of 20 weight % and the sugar soln of sucrose respectively.) condition under the Powdered thalline of lyophilize of the prepared bifidumbacterium bifidum that goes out at the graphic representation of 20 ℃, 30 ℃, 40 ℃ preservation test-results at each temperature.
Fig. 2 shows trehalose concentration in the suspension before lyophilize and is respectively 10 weight % with sucrose concentration and (as the sugar soln before mixing with thalline, uses and contain the trehalose of 30 weight % and the sugar soln of sucrose respectively.) condition under the Powdered thalline of lyophilize of the prepared bifidumbacterium bifidum that goes out at the graphic representation of 20 ℃, 30 ℃, 40 ℃ preservation test-results at each temperature.
Fig. 3 shows trehalose concentration in the suspension before lyophilize and is respectively 6.7 weight % with sucrose concentration and (as the sugar soln before mixing with thalline, uses and contain the trehalose of 20 weight % and the sugar soln of sucrose respectively.) condition under the Powdered thalline of lyophilize (the circle numeral 2 among the figure) and the graphic representation of the preservation test-results of the Powdered thalline of lyophilize of usefulness (the circle numeral 1 among the figure) under 40 ℃ temperature relatively of the prepared bifidus longum bb that goes out.
Embodiment
Below the present invention is described in detail, but the variety of way that the present invention is not limited to narrate below.
As milk-acid bacteria of using among the present invention and lactobacillus bifidus, can example go out to belong to the mushroom that bifidus bacillus (Bifidobacterium) belongs to, specifically can enumerate for example bifidus longum bb (Bifidobacterium longum) strain, bifidobacteria infantis (Bifidobacterium infantis) strain, bifidobacterium breve (Bifidobacterium breve) strain, bifidumbacterium bifidum (Bifidobacterium bifidum) strain, bifidobacterium adolescentis (Bifidobacterium adolescentis) strain etc.Can example go out to belong to the mushroom that lactobacillus (Lactobacillus) belongs to, suis (Streptococcus) belongs in addition, specifically can enumerate for example Jia Shi lactobacillus (Lactobacillus gasseri) strain, lactobacillus bulgaricus (Lactobacillus bulgaricus) strain, thermophilus streptococcus (Streptococcus thermophilus) strain etc.But the present invention is not limited to these bacterial strains, and for these bacterial strains, can use separately or be used in combination more than 2 kinds.
As the preference in the above-mentioned bacterial strains, can enumerate bifidumbacterium bifidum (Bifidobacterium bifidum) strain, bifidus longum bb (Bifidobacterium longum) strain etc.
As the example of the preservation strain of these preferences, can enumerate bifidumbacterium bifidum (Bifidobacteriumbifidum) OLB6378 strain, bifidus longum bb (Bifidobacterium longum) OLB6001 strain etc.
(A) preservation of bifidumbacterium bifidum OLB6378 strain
Bifidumbacterium bifidum OLB6378 strain used among the present invention is carried out preservation with following condition.
(1) preservation organization names: independent administrative legal person's products assessment technique basal disc organization's patent microbial preservation center
(2) contact method: postcode 292-0818 Chiba county wood is total sickle foot 2-5-8 in Jinshi City more
Telephone number 0438-20-5580
(3) deposit number: NITE BP-31
(4) be used to the expression discerned: Bifidobacterium bifidum OLB6378
(5) former preservation date: put down into 16 years (2004) October 26
(6) shift the date based on the preservation of budapest treaty: on January 18th, 2006
(B) preservation of bifidus longum bb OLB6001 strain
Bifidus longum bb OLB6001 strain used among the present invention is carried out preservation with following condition.
(1) preservation organization names: the biological preservation of Independent Administrative Leged Industrial Technology Complex Inst's patent center
(2) contact method: postcode 305-8566 Ibaraki county builds east, ripple city 1-1-1 central authorities the 6th
Telephone number 029-861-6029
(3) deposit number: FERM P-13610
(4) be used to the expression discerned: Bifidobacterium longum No.7
(5) preservation date: put down into 5 years (1993) April 20
(6) shift based on the preservation of budapest treaty
Accept day: put down into 22 years (2010) March 2
Accept numbering: FERM ABP-11242
Bifidumbacterium bifidum OLB6378 strain and bifidus longum bb OLB6001 strain used among the present invention have following bacteriology character.
Bifidumbacterium bifidum OLB6378 strain is the Gram-positive obligate anaerobic bacillus from people's infant faeces.If this bacterium is seeded in the milk-acid bacteria MRS meat soup (BD) and under the anaerobic state that adopts AnaeroPackKENKI (gas chemistry society of Mitsubishi makes), cultivated 18 hours in 37 ℃, then observe the ne ar of Y-shaped.In addition, adopt interior Off ロ one ラ シ Application Port ジ ウ system 8 (the intestinal flora collections of thesis 8) of Auele Specific Primer (Intestinal of bifidumbacterium bifidum (Bifidobacteirum bifidum), the molecular Biological Detection of intestinal flora is identified; The light ridge is content with one's lot, Song Benlong is wide), specifically adopt the species-specific primer BiBIF-1:CCA CAT GAT CGC ATG TGA TT of 16S rDNA and BiBIF-2:CCG AAG GCT TGC TCC CAAA to observe the PCR product by PCR.
Bifidus longum bb OLB6001 strain is from the Gram-positive obligate anaerobic bacillus that becomes human faecal mass, and its bacterium is shaped as a bacillus or a catenate multiform shape, and no brood cell forms, no mobility.If be coated on this bacterial strain on BL nutrient agar (Rong Yan) flat board and utilize stainless steel wool method, form then that opaque circle is hemispheric to have a glossy bacterium colony 37 ℃ of cultivations 48 hours.This bacterial strain has fermentable for pectinose, wood sugar, ribose, glucose, fructose, semi-lactosi, sucrose, maltose, lactose, melibiose, raffinose, melizitose.
As the used protectant saccharic of the present invention is the mixture of trehalose and sucrose.From obtaining to be suitable for the aspect consideration of the Powdered thalline of lyophilize of prolonged preservation at high temperature, trehalose in the suspension before the lyophilize and sucrose concentration separately are more than the 4.5 weight %, are preferably more than the 8 weight %.Even consider from the aspect that the effect that excessively increases the prolonged preservation under the concentration high temperature is also identical, the upper limit of this concentration is preferably 15 weight %, 12 weight % more preferably.Weight ratio to trehalose and sucrose is not particularly limited, but considers from obtaining to be suitable at high temperature the aspect of the Powdered thalline of lyophilize of prolonged preservation, be preferably 5: 1～1: 5, more preferably 3: 1～1: 3, be preferably 1: 1～1: 1 especially.
Preferably, with these saccharics with the form of the aqueous solution that contains saccharic with cultivate bacterium and mix, make this bacterium resuspending.By the suspension that so obtains is carried out lyophilize, can obtain the Powdered thalline of lyophilize of the present invention.
In addition, in containing the aqueous solution of saccharic, except water and saccharic, can also contain for example milk protein, amino acid, xitix etc.
The manufacture method of the Powdered thalline of lyophilize of the present invention is not particularly limited, and for example is made of following step.
1) cultivates desired mushroom according to ordinary method.
2) will containing to some extent, the nutrient solution of the bacterium of cultivation is directly used in following 3) in, the nutrient solution that perhaps will contain to some extent the bacterium of cultivating concentrates or solid-liquid separation by centrifugation etc., obtain spissated nutrient solution (thalline liquid) or, use it for following 3 with the thalline that the isolated in form of solids component goes out) in.
3) resulting thalline liquid or thalline are mixed with the solution that contains normality saccharic (protective material), obtain suspension after, with this suspension lyophilize, obtain the Powdered thalline of lyophilize of the present invention.
As cryodesiccated method, can enumerate the method for use freeze drier etc., for example, can be under the low temperature (for example-30～-90 ℃) rapidly behind the precooling, (be preferably below the 1000Pa, more preferably the vacuum tightness below the 100Pa) carries out drying under room temperature (for example 0～20 ℃) and decompression, keeping under the condition of this vacuum tightness, the temperature of freeze drier is being risen to for example 30～70 ℃, further carrying out drying.
For the Powdered thalline of lyophilize of the present invention, owing to used trehalose and the sucrose that also can be used as food additives, thereby can directly absorb; And also it can be added in the various food compositions and use.In addition, even the Powdered thalline of lyophilize of the present invention also demonstrates specific bacterium survival rate after prolonged preservation, thereby also can be used as the raw materials such as kind bacterium of fermented milk etc.
Can enumerate various diet product (cold drink, fermented milk, sour milk, adjustment milk powder etc.) as food compositions.Food compositions can directly use, and perhaps can mix to wait with other food or food ingredient according to the ordinary method in the common food compositions and use.And the proterties of food compositions can be the state of normally used diet product, for example can be the random order of solid state (powder, particulate state etc.), pasty state, liquid state or suspension.
Other compositions in the food compositions are not particularly limited, can enumerate water, protein, saccharic, lipid, vitamins, mineral substance class, organic acid, organic bases, fruit juice, food flavouring class etc.These compositions can use a kind separately, perhaps also can be used in combination more than 2 kinds.In addition, as other compositions in the food compositions, also can use synthetics and/or contain the food of a large amount of syntheticss.
Embodiment
Below, the present invention will be described based on having used the embodiment that is difficult to the anerobe (lactobacillus bifidus) that preserves usually, but the present invention is not limited thereto.In addition, in this manual, for the expression of %, that represent under situation about not expressing is weight %.
[experimental example 1]
According to the step shown in following, make the Powdered thalline of lyophilize based on manufacture method of the present invention.
1) casein decompose substratum (with through zymolytic casein as proteic substratum) in the OLB6378 strain cultivation that neutralizes to bifidumbacterium bifidum (Bifidobacterium bifidum).
2) to the 320ml nutrient solution carry out centrifugation (4 ℃, carried out 20 minutes with 10000G) remove the 307.2ml supernatant liquor, obtain bacterial sediment thing part (12.8ml).
3) in bacterial sediment thing part (4ml), add the protective material liquid (2ml) that contains the specific concentrations saccharic, thalline is suspended, freezing at-80 ℃ to carry out lyophilize.
4) after lyophilize stops, in the Powdered thalline of 0.5g lyophilize, add normal saline solution immediately and carry out rehydration, utilize the viable count in the BL Agar Plating mensuration rehydration liquid.
5) further, the Powdered thalline of 0.5g lyophilize is added to Lami-Zip (registered trademark; The trade(brand)name of plastics bag) in,, utilize the BL Agar Plating to measure viable count similarly at 20 ℃ or the Powdered thalline of lyophilize of 30 ℃ of preservations after 53 days.
Protective material liquid used in this experimental example is shown in following table 1.
[table 1]
The above-mentioned preservation test-results based on bifidumbacterium bifidum in sample 1,2 of the present invention and the duplicate 1,2,3 is listed in table 2.In addition, because the survival rate in the duplicate 1,2 is low, thereby only show result under 7 days situation of 20 ℃ of preservations with reference to property ground.
[table 2]
*1: preserve fate: 7 days
As shown in table 2, in embodiment 1～4,, do not see the minimizing of viable count under the hot conditions of regulation even the Powdered thalline of lyophilize was preserved 53 days based on the inventive method yet.Especially as can be known, even under the high temperature that quite approaches the living environment temperature of embodiment 2,4, its survival rate also can obtain tremendous raising.
Hence one can see that, and manufacture method of the present invention is for fully being applicable to the technology in long solid state/powder food product of quality guaranteed period.
In addition, comparative example the 1, the 2nd about being the test of the system of only using trehalose of representative with non-patent literature 2, but only can not be reached purpose with trehalose as can be known fully.Particularly in non-patent literature 2, narrated the effect when under very low temperature, under humidity condition, preserving, but under the living environment temperature as this experiment, be to obtain desired effect as can be known.
In addition, in comparative example 3,4 at non-patent literature 2 in the trehalose/sucrose of same concentration share system and study, but viable count reduces by half under the temperature condition of living environment being assumed to as can be known.
Hence one can see that, utilizes the Powdered thalline of the resulting lyophilize of method of these comparative examples to be difficult to be applicable in the solid shape/powder food product of long quality-guarantee period, is not for being suitable for the technology to these food practicalities.
[experimental example 2]
For the Powdered thalline of the lyophilize that utilizes manufacture method of the present invention to obtain, further the effect that under hot conditions (20 ℃, 30 ℃, 40 ℃) are carried out under the situation of prolonged preservation (～6 months) experimentizes.
In addition,, charging capacity is increased to about 30 times for the Powdered thalline of employed lyophilize, in addition, according to making with sample 1 and 2 same manufacture method.Thereby the form with sample 1,2 illustrates below.
By resulting Fig. 1 and the Fig. 2 of the results are shown in of above-mentioned experiment.
By among the figure obviously as can be known, even with sample 1 (with reference to Fig. 1.The solution that will contain 20 weight % trehaloses mixes with the solution that contains 20 weight % sucrose and the trehalose that contains 6.6 weight % respectively that obtains and the protective material solution of sucrose) and sample 2 (with reference to Fig. 2.The solution that will contain 30 weight % trehaloses mixes with the solution that contains 30 weight % sucrose and the trehalose that contains 10 weight % respectively that obtains and the protective material solution of sucrose) all place under the storage temperature condition of 20 ℃ and 30 ℃; after 6 months, also can keep sufficient viable count, demonstrate very high viability.
For 40 ℃ preservation, although be the preservation of quite extreme living environment temperature condition, sample 1 still demonstrates 93.9% viability after preserving 1 month.Thereby as can be known, under this condition, as the concentration of protective material solution, more preferably used concentration in the sample 1.
[experimental example 3]
Use the sample shown in the following table 3 and preserved 3 months, in addition, experimentize similarly to Example 1 at 40 ℃.The results are shown in table 4.As shown in Table 4, be between 3: 1～1: 3 the time at the mass ratio of trehalose and sucrose, viability is 11～17%, does not see difference each other, has equal effect.
[table 3]
[table 4]
[experimental example 4]
According to the step shown in following, make the Powdered thalline of lyophilize based on manufacture method of the present invention.
1) casein decompose substratum (with through zymolytic casein as proteic substratum) in the OLB6001 strain cultivation that neutralizes to bifidus longum bb (Bifidobacterium longum).
2) to the 5400ml nutrient solution carry out centrifugation (4 ℃, carried out 20 minutes with 10000G) remove the 5200ml supernatant liquor, obtain bacterial sediment thing part (200ml).
3) in bacterial sediment thing part (200ml), add the protective material liquid 100ml that contains the normality saccharic, thalline is suspended, freezing at-80 ℃ to carry out lyophilize.
4) after lyophilize stops, in the Powdered thalline of 1g lyophilize, add normal saline solution immediately and carry out rehydration, utilize the viable count in the BL Agar Plating mensuration rehydration liquid.
5) further, the Powdered thalline of 2g lyophilize is added to Lami-Zip (registered trademark; The trade(brand)name of plastics bag) in, the Powdered thalline of lyophilize for after 40 ℃ of preservations during carrying out each of 8 days, 30 days, 82 days, 124 days utilizes the BL Agar Plating to measure viable count similarly.
Protective material liquid used in this experimental example is as follows.
(1) embodiment 8
Use contain respectively the trehalose of 20 weight % and the sugar soln of sucrose (below be called sample 6.)。The trehalose in the suspension before the lyophilize that this sugar soln and bacterial sediment thing are partly mixed and the content of sucrose are respectively 6.7 weight %.
(2) comparative example 5
Use contain 6 weight % skim-milks, 1.7 weight % lactose, 0.4 weight % amino acid (Methionin etc.), 4 other compositions of weight % (dextrin etc.) solution (below be called duplicate 4.)。
Result such as table 5 and shown in Figure 3.As shown in Table 5, in that the Powdered thalline of the lyophilize of comparative example 5 (the circle numeral 1 among Fig. 3) is preserved under about 4 months situation in 40 ℃, only demonstrated 0.002% viability; Relative therewith, in that the Powdered thalline of the lyophilize of embodiment 8 (the circle numeral 2 among Fig. 3) is preserved under about 4 months situation in 40 ℃, demonstrated 40.0% high survival probability.
[table 5]
Industrial applicibility
Utilization is based on the manufacture method of the Powdered thalline of lyophilize with long preservation period of the present invention, particularly guarantee that at needs such as solid matter/powder food products the situation of prolonged preservation is inferior, even after preservation, also can obtain the useful milk-acid bacteria and/or the effect of lactobacillus bifidus.In addition, it does not need special device and complicated procedures of forming, does not exist the cost that accompanies therewith to increase yet, and is favourable economically yet.Further, owing to the Powdered thalline of lyophilize of the present invention can directly use, thereby can after preservation, also directly absorb, can be used as the raw material in the food mfg, its utility value height effectively with powder type easily.
Claims (11)
1. the manufacture method of the Powdered thalline of lyophilize, this manufacture method is that resulting suspension carries out the manufacture method that lyophilize obtains the Powdered thalline of lyophilize of the Powdered thalline of lyophilize in the saccharic solution to milk-acid bacteria and/or lactobacillus bifidus are suspended in, wherein, described saccharic is trehalose and sucrose, and trehalose and sucrose concentration separately in the above-mentioned suspension before the lyophilize are more than the 4.5 weight %.
2. the manufacture method of the Powdered thalline of lyophilize as claimed in claim 1, wherein, trehalose and sucrose concentration separately in the above-mentioned suspension before the lyophilize are 4.5 weight %～15 weight %.
3. the manufacture method of the Powdered thalline of lyophilize as claimed in claim 1 or 2, wherein, trehalose and sucrose concentration separately in the above-mentioned suspension before the lyophilize are 8 weight %～12 weight %.
4. as the manufacture method of the Powdered thalline of each described lyophilize of claim 1～3, wherein, the weight ratio of trehalose and sucrose is 5: 1～1: 5.
5. as the manufacture method of the Powdered thalline of each described lyophilize of claim 1～4, wherein, described milk-acid bacteria and/or lactobacillus bifidus are for belonging to the mushroom that bifidus bacillus (Bifidobacterium) belongs to.
6. the manufacture method of the Powdered thalline of lyophilize as claimed in claim 5, wherein, described milk-acid bacteria and/or lactobacillus bifidus are bifidumbacterium bifidum (Bifidobacterium bifidum) strain.
7. the manufacture method of the Powdered thalline of lyophilize as claimed in claim 6, wherein, described milk-acid bacteria and/or lactobacillus bifidus are bifidumbacterium bifidum (Bifidobacterium bifidum) OLB6378 strain.
8. the manufacture method of the Powdered thalline of lyophilize as claimed in claim 5, wherein, described milk-acid bacteria and/or lactobacillus bifidus are bifidus longum bb (Bifidobacterium longum) strain.
9. the manufacture method of the Powdered thalline of lyophilize as claimed in claim 8, wherein, described milk-acid bacteria and/or lactobacillus bifidus are bifidus longum bb (Bifidobacterium longum) OLB6001 strain.
10. Powdered thalline of lyophilize, this thalline adopt the manufacture method of the Powdered thalline of each described lyophilize of claim 1～9 to make.
11. a food compositions, this food compositions contain the Powdered thalline of the described lyophilize of claim 10 of significant quantity.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
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| JP2009051118 | 2009-03-04 | ||
| JP2009-051118 | 2009-03-04 | ||
| PCT/JP2010/053421 WO2010101175A1 (en) | 2009-03-04 | 2010-03-03 | Freeze-dried microbial cell powder and method for producing same |
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| Publication Number | Publication Date |
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| CN102272287A true CN102272287A (en) | 2011-12-07 |
| CN102272287B CN102272287B (en) | 2014-12-10 |
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| JP (1) | JP5583114B2 (en) |
| CN (1) | CN102272287B (en) |
| HK (1) | HK1159688A1 (en) |
| SG (1) | SG172859A1 (en) |
| TW (1) | TWI501731B (en) |
| WO (1) | WO2010101175A1 (en) |
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| CN113005040A (en) * | 2021-01-22 | 2021-06-22 | 武汉微康益生菌研究院有限公司 | Bifidobacterium lactis freeze-drying protective agent and using method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EA020627B1 (en) * | 2008-06-26 | 2014-12-30 | Синва Фармасьютикал Ко., Лтд. | Process for producing cells of nano-level lactic acid bacterium and compositions based thereon |
| JP2012055288A (en) * | 2010-09-13 | 2012-03-22 | Kaneka Corp | Stabilized viable bacterial preparation, and method for producing the same |
| JP6032737B2 (en) * | 2012-09-27 | 2016-11-30 | 株式会社明治 | Method for detecting bifidobacteria |
| JP6230796B2 (en) * | 2013-03-08 | 2017-11-15 | 日清食品ホールディングス株式会社 | Freeze-dried bacteria sample and production method thereof |
| TW201636010A (en) * | 2014-12-26 | 2016-10-16 | Meiji Co Ltd | Organic-acid-production promoter, and agent for preventing and/or ameliorate inflammatory bowel disease |
| HUE065092T2 (en) * | 2016-07-15 | 2024-04-28 | Cj Cheiljedang Corp | Leuconostoc mesenteroides cjlm181 strain producing reduced amount of gas, and kimchi production method using same |
| CN108603164B (en) * | 2016-07-15 | 2020-04-10 | Cj第一制糖株式会社 | Leuconostoc mesenteroides CJLM119 strain with low gas production and method for producing kimchi using the same |
| JP6807677B2 (en) * | 2016-07-15 | 2021-01-06 | 株式会社明治 | Method of microbial culture concentration and microbial concentrate |
| EP3486312B1 (en) * | 2016-07-15 | 2023-11-22 | Cj Cheiljedang Corporation | Leuconostoc mesenteroides cjlm627 strain producing reduced amount of gas, and kimchi production method using same |
| CN111534434B (en) * | 2020-06-28 | 2022-07-05 | 江南大学 | Freeze-drying protective agent and application thereof in freeze-drying bifidobacterium adolescentis |
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| CN1922305A (en) * | 2004-02-24 | 2007-02-28 | 科·汉森有限公司 | Frozen lactic acid bacteria culture of individual pellets |
| JP2006273852A (en) * | 2005-03-03 | 2006-10-12 | Meiji Milk Prod Co Ltd | Preventive and/or therapeutic substance comprising bacillus bifidus as active component for allergy |
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| CN113005040A (en) * | 2021-01-22 | 2021-06-22 | 武汉微康益生菌研究院有限公司 | Bifidobacterium lactis freeze-drying protective agent and using method thereof |
| CN113005040B (en) * | 2021-01-22 | 2023-07-25 | 武汉微康益生菌研究院有限公司 | Bifidobacterium lactis freeze-drying protective agent and application method thereof |
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| Publication number | Publication date |
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| WO2010101175A1 (en) | 2010-09-10 |
| JP5583114B2 (en) | 2014-09-03 |
| CN102272287B (en) | 2014-12-10 |
| TWI501731B (en) | 2015-10-01 |
| SG172859A1 (en) | 2011-08-29 |
| JPWO2010101175A1 (en) | 2012-09-10 |
| HK1159688A1 (en) | 2012-08-03 |
| TW201043152A (en) | 2010-12-16 |
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