WO2012141244A1 - 微生物菌体乾燥粉末の製造方法 - Google Patents
微生物菌体乾燥粉末の製造方法 Download PDFInfo
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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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- 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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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
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- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
- C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
Definitions
- the present invention relates to a method for producing a microbial cell dry powder. More specifically, the enzyme activity of a microorganism can be stably maintained in a dry state for a long period of time regardless of whether the microorganism is live or dead.
- the present invention relates to a microbial cell dry powder that can be retained and a method for producing the same.
- microorganisms have useful enzyme activities and are widely used for the production of functional food materials such as carbohydrates, amino acids, and phospholipids.
- functional food materials such as carbohydrates, amino acids, and phospholipids.
- microorganisms that can be used for the production of carbohydrate materials, particularly oligosaccharides.
- carbohydrate materials particularly oligosaccharides.
- oligosaccharides For example, the ⁇ -galactosidase activity of yeast belonging to Sporobolomyces singularis is used to produce galactooligosaccharides.
- Patent Document 1 the production of mutant microorganisms with enhanced ⁇ -galactosidase activity of Sporoboromyces singularis has also been reported (Patent Document 2).
- the ⁇ -galactosidase possessed by Sporoboromyces singularis is technically free and purified industrially because the enzyme is firmly bound to the cell wall of the microorganism, just like the enzyme produced by some microorganisms. Both cost and cost. Therefore, when ⁇ -galactosidase possessed by Sporoboromyces singularis is industrially used, it is supplied as a diluted microbial cell fluid, but there is a problem due to this utilization form.
- an object of the present invention is to provide a method for producing a microbial cell dry powder that can stably maintain the enzyme activity possessed by a microorganism for a long time in a dry state.
- the present inventors have conducted intensive research to solve such problems, and in the coexistence state of dried microbial cells and carbohydrates, are the dried microorganisms live or dead? Regardless of whether or not the enzyme activity of the microorganisms can be maintained for a long time.
- microorganisms can be cultured without being restricted by demand, and the distribution cost can be greatly reduced, and the present invention has been completed.
- the present invention is a method for producing a dry powder of microbial cells with maintained enzyme titer, characterized in that a saccharide is added to a microbial cell solution having enzyme activity and then dried.
- the present invention is a microbial cell dry powder that can be stored for a long period of time and contains a microbial cell having enzyme activity and a saccharide.
- a microbial cell powder in which the enzyme activity of microorganisms hardly decreases even when stored for a long period of one year or longer is extremely economical from the viewpoint of both distribution and production. Furthermore, in the method of the present invention, even when dead bacteria are used as microorganisms, the enzyme titer can be maintained, so that deterioration of quality due to metabolites produced by microorganisms when live bacteria are used is avoided. Is something that can be done.
- the microbial cell dry powder obtained by the method of the present invention can be used without problems in practice in the oligosaccharide production reaction.
- the microorganism from which the microbial cell dry powder can be obtained by the method of the present invention may be any microorganism such as bacteria, yeast, mold, etc., as long as the enzyme is bound to the cell wall or produced in the cell. Further, the microorganism may be subjected to various treatments.
- bacteria include Streptococcus thermophilus, Lactobacillus bulgaricus, Streptococcus lactis, Lactobacillus salivalius, Lactobacillus reichmannii, Lactobacillus helvetics, Bacillus brevis, Bacillus stearothermophilus, Bifido Examples include Bifidobacterium, Bifidobacterium breve, Bifidobacterium longum and Bifidobacterium addresscentis.
- the enzyme is not particularly limited.
- a saccharide-degrading enzyme amylase, sucrose, ⁇ - and ⁇ -galactosidase, glucose isomerase, ⁇ - and ⁇ -glucosidase, ⁇ -fructofuranosidase, ⁇ - and ⁇ -mannosidase, xylanase and the like can be mentioned.
- yeast that produces ⁇ -galactosidase is preferable.
- yeasts that produce ⁇ -galactosidase include Sporoboromyces genus, Krivellomyces genus, Lipomyces genus, Candida genus, Cryptococcus genus, Sterigmatomyces genus, Brera genus, Bensintonia genus, Basosporo Myces, Ferromyces, Fibrobasidium, Shirobasidium, Tyresopsis, Itersoniria, Tyrecia, Saccharomyces, Schizosaccharomyces, Hansenula, Rhodotorula, Devalyomyces, Pichia, Toluropsis, etc.
- Sporoboromyces genus Sterigmatomyces genus, Kriveromyces genus, Cryptococcus genus, Rhodotorula genus, Shirobasidium genus, Lipomyces genus are preferable, Sporoboromyces genus Any of the microorganisms belonging to the genus Cass, Rhodotorula or Shirobasidium is more preferred, Sporoboromyces singularis, Sterigmatomyces evibiae, Cryptococcus lorenti, Rhodotorula lactosa, Rhodotorula minuta, Shirobasidium magnum, Lipomyces lipophor is particularly preferred, and Sporoboromyces singularis, Cryptococcus lorenti, Rhodotorula lactosa, Shirobasidium magnum, Rhodotorula minuta are more preferred.
- yeast that is preferably used is Sporoboromyces singularis, one of which is Sporoboromyces singularis JCM 5356, RIKEN BioResource Center (Tsukuba, Ibaraki 305-0074) It can be obtained from Ichitakanodai 3-1-1) for a fee.
- Another example is a yeast obtained as a mutant microorganism producing high ⁇ -galactosidase by the production method described in Patent Document 2.
- Sporoboromyces singularis ISK- is used as an example of yeast obtained by the steps (a) to (c) of the patent document using Sporoboromyces singularis JCM 5356 as the parent strain.
- # 4D4, # 5A5, and ### 2B6, which were dated April 10, 2002, the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1-1, Higashi 1-chome, Tsukuba City, Ibaraki 305-8566) 1 center 6) are deposited as FERM P-18818, FERM P-18819 and FERM P-18817, respectively.
- microorganisms are first cultured according to a conventional method, and then collected and washed using, for example, a DeLaval type continuous centrifuge or a membrane concentrator to prepare a microorganism cell solution.
- a saccharide as a stabilizer may be added to the microbial cell fluid according to a conventional method and dried. More specifically, when spray drying is used, a desired amount of saccharide is added to a microbial cell suspension in which microorganisms are suspended, and the target microbial cell is dried by spray drying in that state. A powder can be obtained.
- the amount of microbial cells in the microbial cell liquid is not particularly limited, but is preferably 1 to 10% by mass / volume% (hereinafter simply referred to as “%”), more preferably 2 to 6%.
- the carbohydrates used are not particularly limited, and monosaccharides, disaccharides, oligosaccharides of 3 or more sugars, and polysaccharides can be used.
- lactose As monosaccharides, glucose, galactose, fructose, mannose, and disaccharides as lactose, Examples of lactose isomers, maltose, sucrose, trehalose, trisaccharide or higher oligosaccharides include galactooligosaccharides, malto-oligosaccharides, fructooligosaccharides and other oligosaccharides, and polysaccharides include dextrin and starch.
- lactose one or more selected from the group consisting of lactose, maltose and dextrin is preferable from the viewpoint of the effect of stabilizing the enzyme titer, ease of drying and cost, and it is particularly preferable to use lactose and / or maltose. More preferably, lactose is used.
- the amount of carbohydrate added to the microbial cell liquid is not particularly limited, but from the viewpoint of the effect of stabilizing the enzyme titer, an amount of 0.1% or more with respect to the microbial cell liquid is preferable, and more preferable. Is an amount of 0.5% or more, more preferably 1% or more.
- the amount of saccharide added to the microbial cell fluid is too large, the enzyme titer per unit weight of the microbial cell dry powder will decrease, so the amount of saccharide is preferably 30% or less.
- the amount is preferably 15% or less, more preferably 10% or less, still more preferably 5% or less, and even more preferably 3% or less.
- the amount of carbohydrate added to the microbial cell fluid is preferably 0.1 to 30%, more preferably 0.5 to 15% of the microbial cell fluid, More preferably 0.5% to 10%, more preferably 1% to 10%, even more preferably 1% to 5%, more preferably 1% to 3%. It is.
- carbohydrates can serve as the substrate for the enzyme (for example, when lactose is added as a stabilizer to the microbial cell fluid that produces ⁇ -galactosidase), some or most of the period from the addition of carbohydrate to the completion of spraying
- saccharides may undergo a reaction, the effect of the saccharide is exerted regardless of the degree of reaction (degree of decomposition or degree of polymerization), so this is not a problem. For example, even if the reaction is performed at 5 ° C. to 40 ° C. for about 1 to 40 hours after the addition of carbohydrate, there is no problem in terms of stabilizing the enzyme titer.
- the microbial cell dry powder may contain a saccharide different from that in addition to the added saccharide.
- the microbial cell dry powder may contain a saccharide different from that in addition to the added saccharide.
- carbohydrates contained in the microbial cell dry powder include monosaccharides, disaccharides, oligosaccharides of 3 or more sugars, and polysaccharides. Examples of monosaccharides include glucose, galactose, fructose, mannose, and disaccharides.
- lactose lactose
- lactose isomers maltose
- sucrose trehalose
- trisaccharide or higher oligosaccharides include various oligosaccharides such as galactooligosaccharide and fructooligosaccharide, and polysaccharides include dextrin and starch.
- the saccharide contained in the microbial cell dry powder can include glucose, galactose, lactose, lactose isomers, galactooligosaccharides, Examples of the saccharide contained in the microbial cell dry powder when maltose is used include glucose, maltose and maltooligosaccharide.
- the saccharide contained in the microbial cell dry powder can include glucose, maltose, maltooligosaccharide and dextrin, and it is preferable to use at least one selected from the group consisting of lactose, maltose and dextrin as the saccharide added to the microbial cell fluid.
- body dry powder glucose, galactose, lactose, lactose isomers, galactooligosaccharide Maltose, maltooligosaccharides, it preferably contains one or more sugars selected from the group consisting of dextrin.
- lactose and / or maltose as the saccharide added to the microbial cell fluid, glucose, galactose, lactose, lactose isomer, galactooligosaccharide, maltose are contained in the microbial cell dry powder. More preferably, one or more carbohydrates selected from the group consisting of maltooligosaccharides are contained. Moreover, since it is more preferable to use lactose as the saccharide added to the microbial cell fluid, one or more saccharides selected from the group consisting of glucose, galactose, lactose, lactose isomers, and galactooligosaccharides are included. More preferably.
- the spray drying conditions may be such that the temperature at the inlet / outlet of the drying chamber is within a range where the enzyme is not inactivated significantly, and the number of atomizer rotations, stock solution feed amount, etc. vary slightly depending on the conditions. Although a dry powder of microbial cells is obtained, there is little influence on the final enzyme titer, so there is no need to pay attention to it.
- the inlet temperature of the drying chamber is 70 to 200 ° C., preferably 110 to 180 ° C.
- the outlet temperature is 50 to 120 ° C., preferably 70 to 90 ° C.
- the atomizer rotation speed can be exemplified as 10,000 to 30,000 rpm, and the stock solution feed rate can be exemplified as 0.2 to 200 kg / hour.
- These spray dryings can be performed by using a spray method such as a two-fluid nozzle in addition to the atomizer. The spray drying method is preferred because microorganisms are almost killed in this drying step, and a microbial cell dry powder with a small number of viable bacteria is obtained.
- a necessary amount of carbohydrate may be added to the microbial cell liquid by a conventional method, and then freeze-dried.
- the amount of carbohydrate added is also preferably 0.1 to 30%, more preferably 0.5 to 15%, more preferably 0.5% to 10% with respect to the microbial cell fluid. %, More preferably 1% to 10%, even more preferably 1% to 5%, more preferably 1% to 3%.
- the dried microbial cell powder obtained as described above can stably maintain the enzyme activity of the microorganism in a dry state for a long period of time.
- the microorganism may be live or dead, but is preferably dead in view of quality degradation due to metabolites produced by the microorganism.
- the microbial cell dry powder obtained as described above contains 0.01 to 30 times the amount of carbohydrate, preferably 0.05 to 15 times the amount of the dry microbial cell, and more preferably Is 0.05 to 10 times the amount, more preferably 0.1 to 10 times the amount, still more preferably 0.1 to 5 times the amount, still more preferably 0.1 to 3 times the amount. .
- the amount of water contained therein is not particularly defined, but is desirably 10% by mass or less.
- the enzyme titer after drying is 70% or more of the enzyme titer before drying, and when the drying method is spray-drying, the enzyme titer can be prevented from lowering only by the method described above. In the case of lyophilization, unlike spray drying, the enzyme titer does not decrease even by drying. Therefore, after lyophilizing only the microorganisms into a powder, a powdered saccharide may be added thereto.
- the microbial cell dry powder obtained in the present invention has a unique effect of maintaining the enzyme titer that the microorganisms immediately after drying have had for over 1 year even when stored at room temperature (25 ° C.). Is. That is, a high enzyme titer of 50% or more of the enzyme titer immediately after drying, or 80 to 90% or more can be maintained for a long time.
- Patent Documents 3 and 4 lactose or the like is used as an excipient in order to increase the survival of microorganisms.
- Patent Documents 3 and 4 There is no idea of enhancing and maintaining the system, and it is based on different technical ideas.
- the microorganism itself is killed and only the enzyme activity possessed by the microorganism is preserved.
- ⁇ -galactosidase titer measurement method (a) Preparation of test solution About 2.5 g of the test sample is concentrated, and about 150 to 350 mg of the test sample is a dry product Was accurately weighed into a 50 mL centrifuge tube, suspended in 50 mM sodium phosphate-citrate buffer (pH 4.0) (hereinafter referred to as “buffer”), and then centrifuged (20,000 G, 15 minutes). And washed to remove carbohydrates. After performing this washing operation three times, it was transferred to a 50 mL volumetric flask, made up to a constant volume with a buffer solution and sufficiently suspended to prepare a test solution.
- buffer sodium phosphate-citrate buffer
- the residual moisture content of the dried microbial cell powder (hereinafter referred to as “dried product”) in spray drying is 105 ° C. using an infrared moisture meter manufactured by Kett Science Laboratory. The measurement was performed for 15 minutes.
- the solid content of the freeze-dried product and the solid content of the dried stock solution and dry product used in calculating the ⁇ -galactosidase titer per solid content were calculated from the dry matter weight when treated at 105 ° C. for 16 hours. .
- Example 1 Preparation of yeast cell dry powder: Sporobolomyces singularis YIT 10047 (ISK-## 2B6, hereinafter referred to as “Ss”), glucose 5%, yeast extract 0.6%, monopotassium phosphate 0.1%, sulfuric acid
- the medium was aerobically cultured at 27 ° C. for 4 days in a medium (pH 5) containing 0.05% magnesium.
- Sterilized city water was added to the wet cells obtained by centrifuging this culture solution (10000 G, 30 minutes) and sufficiently suspended. This was centrifuged under the same conditions, and a suspension of wet cells in a small amount of city water was used as an Ss concentrate (solid content: 4.9%).
- Spray drying was performed under various operating conditions using a pilot device (Production Miner, GEA Process Engineering Co., Ltd.) equipped with a rotary atomizer.
- the operating conditions are shown in Table 1.
- Freeze-drying was performed using a freeze-dryer RLE-206 (Kyowa Vacuum Technology Co., Ltd.) at a shelf temperature of 25-30 ° C.
- the dried products obtained by spray drying and freeze-drying are each sealed in a plastic bag with a zipper, stored in a thermostatic chamber at 5 ° C and 25 ° C for about 1 year, and a part is extracted regularly.
- a storage test was performed by measuring the ⁇ -galactosidase titer.
- Table 1 shows the specific drying conditions of the dried product obtained by spray drying and the evaluation of the obtained dried product.
- SD-1 to SD-3 attempted to change the particle size of the dried product by increasing the droplet size by lowering the rotational speed of the rotary atomizer.
- SD-4 tried to increase production efficiency by raising the drying temperature by 5 ° C. and increasing the feed amount.
- Table 1 shows the spray drying conditions and the results of the obtained product.
- the residual titer ratio after drying of SD-1 to SD-3 (ratio of the titer per solid content of the dried product to the titer per solid content of the dry stock solution) is as high as about 95%. It was judged that it was a practical level. Even with SD-4 whose drying temperature was increased by 5 ° C., there was no significant difference in the residual titer.
- Dry product storage test The spray-dried product and freeze-dried product were subjected to a storage test for about 1 year at 5 ° C and 25 ° C. Spray-dried products are prepared from two specimens SD-2 and SD-4 with different drying temperatures, and freeze-dried products are prepared from Ss concentrate [no lactose: FD (-)] and dry stock solution [lactose 5%: FD (+)]. Two samples were used. Each specimen was sampled over time, and the residual titer (the ratio of the titer per solid content of the dried product after storage to the titer per solid content of the dried product immediately after drying) was calculated. The results for the spray-dried product are shown in Table 2 and FIG. 1, and the results for the freeze-dried product are shown in Table 3 and FIG.
- the presence or absence of a stabilizer also affects the residual titer rate in storage of freeze-dried products, and both FD with lactose added at 5 ° C and 25 ° C.
- the residual titer of (+) was higher than that of FD ( ⁇ ) without addition of lactose, and the residual titer after 1 year storage was about 85 to 90%.
- the titer residual ratio which is an index of the titer yield in the entire drying process, increases with the concentration of lactose, and the effect is recognized at a concentration of 0.1% or more. A clear effect was observed at a concentration of 0.5% or more.
- Example 3 (Preparation of dry powder of yeast cells) For dextrin (NSD # 300, # 500; both Sanei saccharification Co., Ltd.), maltose, and lactose, the enzyme titer maintenance effect (stabilization effect) during spray drying was compared. 25 mL of a 4, 20, 40% stabilizer solution was added to 75 mL of an Ss concentrate (solid content: 5.0%), and 100 mL each of a dry stock solution containing 1, 5, 10% stabilizer was prepared.
- the residual titer was higher when the stabilizer coexisted than when no stabilizer was added (34.4%, Table 4).
- disaccharides such as lactose and maltose had a higher titer residual rate than dextrin, and among disaccharides, lactose had a higher titer residual rate and was excellent as a stabilizer.
- Example 4 Examination of sugar composition (Preparation of dry powder of yeast cells) Three solutions were prepared by adding 2.6 L of a 20% lactose solution to 7.8 L of an Ss concentrate (solid content: 5.3%), and mixing them well. 10 ° C-1 hour, 5 ° C-40 hours, 40 ° C The reaction was carried out under the respective conditions of ° C-18 hours to prepare dry stock solutions having different sugar compositions. These were dried using a pilot device (Production Miner, GEA Process Engineering Co., Ltd.) under the conditions of inlet temperature: 120 ° C., outlet temperature: about 80 ° C., atomizer rotation speed: 12500 rpm, and stock solution treatment amount: 4 kg / hour. did.
- a pilot device Production Miner, GEA Process Engineering Co., Ltd.
- the cells were aerobically cultured at 26 ° C. in a medium (pH 5) containing 0.3% yeast extract, 0.1% monopotassium phosphate and 0.05% magnesium sulfate.
- Sterilized city water was added to the wet cells obtained by centrifuging this culture solution (10000 G, 30 minutes) and sufficiently suspended. This was centrifuged under the same conditions, and a wet cell suspension in a small amount of city water was used as a cell concentrate (solid content of about 4%).
- a 20% lactose solution was added to 75 mL of the bacterial cell concentrate and stirred sufficiently to prepare a dry stock solution. This operation was performed at 20 ° C. or lower.
- yeast powder was prepared by spray drying for each of the dry stock solutions prepared using water instead of the lactose solution. The solid content (calculated value) in the dry stock solution was adjusted to be about 3% for bacterial cells and about 5% for lactose.
- Spray drying was performed using a two-fluid nozzle type spray drying apparatus (SD-1000) manufactured by Tokyo Science Instruments Co., Ltd., at an inlet temperature of 120 ° C. and an outlet temperature of 70 to 90 ° C.
- SD-1000 two-fluid nozzle type spray drying apparatus manufactured by Tokyo Science Instruments Co., Ltd.
- Rhodotorula lactosa The remaining titers after spray drying in the three strains other than Rhodotorula lactosa (R. lactosa) were all higher when lactose was added at 5% than when lactose was not added. Rhodotorula lactosa was extremely high at 99.8% when 5% of lactose was added. From these results, it was shown that the ⁇ -gal titer lowering suppression effect after spray drying exhibited by the addition of lactose is not specific to Ss and can be widely applied to yeast.
- the yeast cell dry powder obtained by the method of the present invention can stably maintain the enzyme activity of this yeast in a dry state for a long period of time. That is, even if it preserve
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Abstract
Description
(a)検液の調製
被験試料が濃縮液の場合は、その約2.5gを、また、被験試料が乾燥品の場合は、その150~350mg程度を50mL容の遠沈管に正確に秤量し、50mMリン酸ナトリウム-クエン酸緩衝液(pH4.0)(以下、「緩衝液」という)に懸濁した後に、遠心分離(20000G、15分間)して洗浄し、糖質を除いた。この洗浄操作を3回行なった後に、50mL容メスフラスコに移し、緩衝液で定容して十分に懸濁させて検液とした。
o-ニトロフェニル-β-D-ガラクトピラノシド(o-nitrophenyl-β-D-galacto-pyranoside;ONPG)0.3766gを100mL容メスフラスコに秤量し、緩衝液に溶解・定容して12.5mMの溶液を調製した。試験管に、このONPG溶液を0.8mL入れ、30℃の恒温水槽中で5分間保持した。これに検液を0.2mL添加してよく混合し、30℃で10分間反応させた後に0.25M炭酸ナトリウム溶液を4mL加えて反応を停止した(試験系)。別に、試験管にONPG溶液0.8mLと0.25M炭酸ナトリウム溶液4mLを入れ、さらに検液を0.2mL加えてよく混合した(盲検系)。試験系および盲検系のそれぞれを、遠心分離(2000G、10分間、15~20℃)にかけ、得られた上清について、波長420nmで吸光度を測定し、次式により単位数を算出した。なお、上記の反応条件で、1分間に1μmolのo-ニトロフェノール(o-nitrophenol;ONP)を遊離するのに要する酵素量を1Uとした。
噴霧乾燥における微生物菌体乾燥粉末(以下、「乾燥品」という)の残留水分は、(株)ケット科学研究所製の赤外線水分計を用いて105℃、15分間の条件で測定した。また、凍結乾燥品の固形分含量および固形分あたりのβ-ガラクトシダーゼ力価を算出する際に用いる乾燥原液と乾燥品の固形分含量は、105℃で16時間処理したときの乾物重量から算出した。
噴霧乾燥品の粒度分布は、シンパテック社製のレーザー回折式粒度分布測定装置(HELOS&RODOSシステム)を用いて乾式で測定した。
ラクトース2.5%、酵母エキス0.5%、リン酸一カリウム0.1%、硫酸マグネシウム0.05%、寒天1.5%となるようにこれらを水に溶解し、2N塩酸でpH5.0に調整した後に、オートクレーブ滅菌(121℃、10分間)し、平板プレート(φ90mm)を作成した。これに、生理食塩水で溶解・希釈したサンプルを100μL塗抹し、25℃で約一週間培養した後に、生じたコロニーを計測し、スポロボロマイセス・シンギュラリス生菌数とした。
酵母菌体乾燥粉末の調製:
スポロボロマイセス・シンギュラリス(Sporobolomyces singularis)YIT 10047(ISK-##2B6、以下、「Ss」と表記)を、グルコース5%、酵母エキス0.6%、リン酸一カリウム0.1%、硫酸マグネシウム0.05%を含む培地(pH5)で、27℃、4日間好気的に培養した。この培養液を遠心分離(10000G、30分間)して得られた湿菌体に、滅菌した市水を加えて十分に懸濁した。これを同条件で遠心分離し、湿菌体を少量の市水に懸濁したものをSs濃縮液(固形分4.9%)とした。Ss濃縮液20Lに、25%乳糖溶液を5L加えて十分に撹拌し、乾燥原液を得た。この操作は20℃以下で行なった。この乾燥原液を試料として用い、酵母粉末を凍結乾燥法および噴霧乾燥法により調製した。乾燥原液中の固形分含量(計算値)は、Ssが約3.9%、乳糖が約5%となる。
噴霧乾燥で得られた乾燥品について、その具体的な乾燥条件と得られた乾燥品の評価を表1に示した。SD-1~3は、ロータリーアトマイザーの回転数を下げて液滴を大きくすることで、乾燥品粒度の変更を試みた。また、SD-4は、乾燥温度を5℃上げてフィード量を増やすことで、生産効率を上げることを試みた。噴霧乾燥の条件と、得られた製品の結果を表1に示す。
噴霧乾燥品と凍結乾燥品について、5℃、25℃で約1年間の保存試験を行なった。噴霧乾燥品は乾燥温度の異なるSD-2とSD-4の2検体、凍結乾燥品はSs濃縮液[乳糖なし:FD(-)]と乾燥原液[乳糖5%:FD(+)]から調製した2検体とした。それぞれの検体について、経日的にサンプリングし、力価残存率(乾燥直後の乾燥品の固形分あたり力価に対する、保存後の乾燥品の固形分あたり力価の割合)を算出した。噴霧乾燥品についての結果を表2、図1に、凍結乾燥品についての結果を表3、図2に示す。
乳糖添加量の検討
酵母菌体乾燥粉末の調製
乾燥原液、乾燥品の力価を測定し、乾燥原液の固形分あたり力価に対する乾燥品の固形分あたり力価の割合(力価残存率)を算出した。得られた結果を表4、表5、図3に示す。
他の安定化剤の検討:
(酵母菌体乾燥粉末の調製)
デキストリン(NSD#300、#500;ともにサンエイ糖化(株))、マルトース、および乳糖について、噴霧乾燥時の酵素力価維持効果(安定化効果)を比較した。Ss濃縮液(固形分5.0%)75mLに、4、20、40%の安定化剤溶液25mLを加え、安定化剤を1、5、10%含む乾燥原液を100mLずつ調製した。これらを、二流体ノズル式の実験用噴霧乾燥装置(SD-1000、東京理科器械(株))を用いて、入口温度:120℃、出口温度:約80℃、原液処理量:4mL/分の条件で処理し、サイクロン部に付着した分も含めて回収したものを乾燥品とした。
乾燥原液と乾燥品の力価を測定し、乾燥原液の固形分あたり力価に対する乾燥品の固形分あたり力価の割合(力価残存率)を算出した。その結果を表6に示す。
糖組成の検討:
(酵母菌体乾燥粉末の調製)
Ss濃縮液(固形分5.3%)7.8Lに、20%乳糖溶液を2.6L加えて十分に混合したものを3つ準備し、10℃-1時間、5℃-40時間、40℃-18時間のそれぞれの条件で保持して反応させ、糖組成の異なる乾燥原液を調製した。これらを、パイロット装置(プロダクションマイナ、GEAプロセスエンジニアリング(株))を用いて、入口温度:120℃、出口温度:約80℃、アトマイザー回転数:12500rpm、原液処理量:4kg/時間の条件で乾燥した。
以下のHPLC条件で、反応後の乾燥原液中の糖組成を分析した。その結果を表7に示す。
カラム:ShodexSUGAR KS-802(昭和電工(株)
溶媒:純水
流速:0.5mL/分
温度:80℃
検出器:示差屈折計
乾燥原液と乾燥品の力価を測定し、乾燥原液の固形分あたり力価に対する乾燥品の固形分あたり力価の割合(力価残存率)を算出した。その結果を表7に示す。
乾燥温度の検討:
(酵母菌体乾燥粉末の調製)
Ss濃縮液(固形分5.0%)に5N水酸化ナトリウム溶液を加えてpH4.5に調整し、45℃で9時間保持することにより、Ssを死菌化した。この液の8Lに、25%乳糖溶液を2L加えて十分に混合したものを乾燥原液として、乾燥室の入口温度を120℃、150℃、180℃とし、これに対して出口温度が80℃になるよう原液供給量を調整して噴霧乾燥した。
乾燥原液と乾燥品の力価を測定し、乾燥原液の固形分あたり力価に対する乾燥品の固形分あたり力価の割合(力価残存率)を算出した。その結果を表8に示す。
Ss以外の酵母についての検討:
(酵母菌体乾燥粉末の調製)
クリプトコッカス・ローレンティ(C. laurentii IFO18803)、ロドトルラ・ラクトーザ(R. lactosa JCM1546)、シロバシディウム・マグナム(S. magnum JCM6876)およびロドトルラ・ミヌタ(R. minuta JCM8101)を、それぞれ単独で、乳糖5%、酵母エキス0.3%、リン酸一カリウム0.1%、硫酸マグネシウム0.05%を含む培地(pH5)中、26℃で好気的に培養した。この培養液を遠心分離(10000G、30分間)して得られた湿菌体に、滅菌した市水を加えて十分に懸濁した。これを同条件で遠心分離し、湿菌体を少量の市水に懸濁したものを菌体濃縮液(固形分約4%)とした。
それぞれの酵母の乾燥原液と乾燥品の力価を測定し、乾燥原液の固形分あたり力価に対する乾燥品の固形分あたり力価の割合(力価残存率)を算出した。また、乳糖を添加していない菌体濃縮液についても同様に力価を測定し、力価残存率を算出した。その結果を表9に示す。
オリゴ糖生成試験:
(噴霧乾燥品の懸濁液の調製)
上記実施例5で得られた乾燥品(入口温度120℃)を45U相当量秤量し、これを10mlのイオン交換水に加え、懸濁させて懸濁液を得た。
(オリゴ糖生成反応)
60%乳糖溶液800mLに、前記で調製した乾燥品の懸濁液の全量を添加して混合し、65℃、pH6で22時間反応させた。このときの糖組成を調べた結果を表10に示す。
Claims (13)
- 酵素活性を有する微生物菌体液に糖質を添加し、次いでこれを乾燥することを特徴とする酵素力価が維持された微生物菌体乾燥粉末の製造方法。
- 乾燥が、噴霧乾燥または凍結乾燥である請求項1記載の酵素力価が維持された微生物菌体乾燥粉末の製造方法。
- 糖質を、微生物菌体液に対して、0.1ないし30質量/体積%となるよう添加する請求項1または2記載の酵素力価が維持された微生物菌体乾燥粉末の製造方法。
- 乾燥後の酵素力価が、乾燥前の酵素力価の70%以上である請求項1ないし3の何れかの項記載の酵素力価が維持された微生物菌体乾燥粉末の製造方法。
- 常温で1年間保存後の酵素力価が、乾燥直後の酵素力価の80%以上である請求項1ないし4の何れかの項記載の酵素力価が維持された微生物菌体乾燥粉末の製造方法。
- 酵素活性が、β-ガラクトシダーゼ活性である請求項1ないし5の何れかの項記載の酵素力価が維持された微生物菌体乾燥粉末の製造方法。
- 微生物が、スポロボロマイセス属、クリプトコッカス属、ロドトルラ属またはシロバシディウム属に属する微生物の何れかである請求項1ないし6の何れかの項記載の酵素力価が維持された微生物菌体乾燥粉末の製造方法。
- 糖質が、乳糖、マルトースおよびデキストリンからなる群から選ばれる1種以上である請求項1ないし7の何れかの項記載の酵素力価が維持された微生物菌体乾燥粉末の製造方法。
- 酵素活性を有する微生物菌体と、糖質とを含有する長期保存可能な微生物菌体乾燥粉末。
- 常温で1年間保存後の酵素力価が、乾燥直後の酵素力価の80%以上保存される請求項9記載の微生物菌体乾燥粉末。
- 糖質が、グルコース、ガラクトース、乳糖、乳糖異性体、ガラクトオリゴ糖、マルトース、マルトオリゴ糖およびデキストリンからなる群から選ばれる1種以上である請求項9または10記載の微生物菌体乾燥粉末。
- 酵素活性を有する微生物菌体液に糖質を添加し、次いでこれを乾燥することにより得られる請求項9ないし11の何れかの項記載の微生物菌体乾燥粉末。
- 乾燥が、噴霧乾燥または凍結乾燥で行われる請求項9ないし12の何れかの項記載の微生物菌体乾燥粉末。
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EP2711416A4 (en) * | 2011-05-20 | 2014-12-31 | Yakult Honsha Kk | METHOD FOR DESTRUCTION OF MICROORGANISM |
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MX2019004558A (es) * | 2016-10-31 | 2019-08-05 | Kimberly Clark Co | Metodo para archivar un microbioma. |
US11306336B2 (en) * | 2017-01-13 | 2022-04-19 | Tata Chemicals Limited | Process for production of Galacto-oligosaccharides |
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WO2023077007A1 (en) * | 2021-10-27 | 2023-05-04 | Board Of Regents, The University Of Texas System | Dry powder compositions comprising eukaryotic cells and method of their manufacture and use |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0558714B2 (ja) | 1986-03-07 | 1993-08-27 | Yakult Honsha Kk | |
JPH07203950A (ja) * | 1994-01-26 | 1995-08-08 | Kanegafuchi Chem Ind Co Ltd | 被覆されたファフィア・ロドチーマ酵母及びその造粒物 |
JPH0919283A (ja) * | 1995-07-06 | 1997-01-21 | Hisatoki Komaki | 乳酸菌及び酵母の菌体成分複合体の製造方法 |
JPH1057031A (ja) | 1996-07-09 | 1998-03-03 | Soc Prod Nestle Sa | 食用微生物組成物の噴霧乾燥法 |
JP2002017337A (ja) | 2000-05-02 | 2002-01-22 | Biofuerumin Seiyaku Kk | 噴霧乾燥による菌体乾燥物 |
JP2006223268A (ja) * | 2005-02-21 | 2006-08-31 | Yakult Honsha Co Ltd | ガラクトシル2糖類の製造法 |
JP2008043206A (ja) * | 2006-08-10 | 2008-02-28 | Yakult Honsha Co Ltd | 経口摂取用固体組成物及びその製造方法 |
JP4071037B2 (ja) | 2002-05-09 | 2008-04-02 | 株式会社ヤクルト本社 | β−ガラクトシダーゼ高産生変異微生物の作出方法および当該方法により作出される変異微生物ならびにその利用 |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US2773002A (en) * | 1955-03-31 | 1956-12-04 | Nat Dairy Res Lab Inc | Purification of lactase enzyme and spray-drying with sucrose |
JP2711095B2 (ja) * | 1986-09-27 | 1998-02-10 | ユニチカ株式会社 | ビフイドバクテリウム菌の増殖促進剤の製造法 |
US4857469A (en) * | 1987-04-09 | 1989-08-15 | Toyo Jozo Co., Ltd. | Process for preparing optically active mercapto compound |
JP3130972B2 (ja) * | 1991-08-30 | 2001-01-31 | 京セラ株式会社 | セラミック基板およびその製造方法 |
JPH07203850A (ja) * | 1994-01-14 | 1995-08-08 | Kawasaki Kiko Co Ltd | 製茶揉機の作動点検機構 |
BRPI0621953B1 (pt) * | 2006-10-16 | 2016-09-13 | Ct De Investigacíon Em Alimentacíon Y Dessarolo A C | composição sólida, seca, eficaz no controle biológico de colletotrichum gloeosporioides; método para produção de uma composição; método para o controle biológico da enfermidade causada por colletotrichum gloeosporioides e método para reduzir a perda de peso durante a armazenagem de manga |
JP5901625B2 (ja) * | 2011-05-20 | 2016-04-13 | 株式会社ヤクルト本社 | 微生物の死菌化方法 |
-
2012
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Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0558714B2 (ja) | 1986-03-07 | 1993-08-27 | Yakult Honsha Kk | |
JPH07203950A (ja) * | 1994-01-26 | 1995-08-08 | Kanegafuchi Chem Ind Co Ltd | 被覆されたファフィア・ロドチーマ酵母及びその造粒物 |
JPH0919283A (ja) * | 1995-07-06 | 1997-01-21 | Hisatoki Komaki | 乳酸菌及び酵母の菌体成分複合体の製造方法 |
JPH1057031A (ja) | 1996-07-09 | 1998-03-03 | Soc Prod Nestle Sa | 食用微生物組成物の噴霧乾燥法 |
JP2002017337A (ja) | 2000-05-02 | 2002-01-22 | Biofuerumin Seiyaku Kk | 噴霧乾燥による菌体乾燥物 |
JP4071037B2 (ja) | 2002-05-09 | 2008-04-02 | 株式会社ヤクルト本社 | β−ガラクトシダーゼ高産生変異微生物の作出方法および当該方法により作出される変異微生物ならびにその利用 |
JP2006223268A (ja) * | 2005-02-21 | 2006-08-31 | Yakult Honsha Co Ltd | ガラクトシル2糖類の製造法 |
JP2008043206A (ja) * | 2006-08-10 | 2008-02-28 | Yakult Honsha Co Ltd | 経口摂取用固体組成物及びその製造方法 |
Non-Patent Citations (4)
Title |
---|
BOZA, Y. ET AL.: "Effect of spray-drying on the quality of encapsulated cells of Beijerinckia sp", PROCESS BIOCHEM., vol. 39, no. 10, 2004, pages 1275 - 1284, XP055127081 * |
BOZA, Y. ET AL.: "Exopolysaccharide production by encapsulated Beijerinckia cultures", PROCESS BIOCHEM., vol. 39, 2004, pages 1201 - 1209, XP055127085 * |
LODATO, P. ET AL.: "Viability and thermal stability of a strain of Saccharomyces cerevisiae freeze-dried in different sugar and polymer matrices", APPL. MICROBIOL. BIOTECHNOL., vol. 52, 1999, pages 215 - 220, XP055127078 * |
NOMURA, Y. ET AL.: "Preparation and preservation of freeze-dried cells of acetic acid bacteria with aldehyde oxidase acitivity", BIOSCI. BIOTECHNOL. BIOCHEM., vol. 62, no. 6, 1998, pages 1134 - 1137, XP055127079 * |
Cited By (4)
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EP2711416A4 (en) * | 2011-05-20 | 2014-12-31 | Yakult Honsha Kk | METHOD FOR DESTRUCTION OF MICROORGANISM |
US10760066B2 (en) | 2011-05-20 | 2020-09-01 | Kabushiki Kaisha Yakult Honsha | Method for killing microorganism |
JP2016044173A (ja) * | 2014-08-22 | 2016-04-04 | 国立大学法人山形大学 | 微生物触媒及びその使用 |
JP2016042827A (ja) * | 2014-08-22 | 2016-04-04 | 国立大学法人山形大学 | ポリ塩化ビフェニル類分解用微生物触媒及びその組み合わせ |
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EP2698428A1 (en) | 2014-02-19 |
EP2698428A4 (en) | 2015-01-14 |
AU2012243770B2 (en) | 2016-06-23 |
JPWO2012141244A1 (ja) | 2014-07-28 |
EP2698428B1 (en) | 2018-10-24 |
KR101920899B1 (ko) | 2019-02-13 |
CN103403141B (zh) | 2016-03-23 |
CN103403141A (zh) | 2013-11-20 |
JP5997692B2 (ja) | 2016-09-28 |
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US10465160B2 (en) | 2019-11-05 |
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