SG172859A1 - Freeze-dried microbial cell powder and method for producing same - Google Patents
Freeze-dried microbial cell powder and method for producing same Download PDFInfo
- Publication number
- SG172859A1 SG172859A1 SG2011049061A SG2011049061A SG172859A1 SG 172859 A1 SG172859 A1 SG 172859A1 SG 2011049061 A SG2011049061 A SG 2011049061A SG 2011049061 A SG2011049061 A SG 2011049061A SG 172859 A1 SG172859 A1 SG 172859A1
- Authority
- SG
- Singapore
- Prior art keywords
- freeze
- bacterial cell
- cell powder
- dried bacterial
- dried
- Prior art date
Links
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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
- 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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Virology (AREA)
- General Engineering & Computer Science (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Mycology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Description
FREEZE-DRIED BACTERIAL CELL POWDER AND
METHOD OF PRODUCING THE SAME
The present invention relates to a freeze-dried bacterial cell powder and to a method of producing this freeze-dried bacterial cell powder. More particularly, the present invention relates to a freeze-dried bacterial cell powder that is capable of long-term storage even under prescribed high- temperature conditions and to a method of producing this freeze-dried bacterial cell powder.
Microbial strains that have a probiotic function have been the subject of increasing interest over the last few year, and preventive medicine, which is based on digestive tract improvement and normalization brought about by the consumption of these microorganisms, and which is also based on the accompanying immunoregulatory action, has been flourishing.
Among such microorganisms, the lactic acid bacteria and bifidobacteria, which are also called probiotic microorganisms, have been consumed in various foods since antiquity. The development of their functionalities has quite recently become even more energetic than in the past, and their development in the form of products that incorporate new functionalities is also underway.
A large number of processed food groups that include these microorganisms have been developed for the above- mentioned purposes such as improvement and normalization of digestive tract, but efforts are also underway to make consumption even more convenient and more effective. Yogurt is a typical example of one of these food groups, but these microorganisms are also being put into powdery form in order to further facilitate consumption. This bacterial cell powder can also be used as a novel starting material that makes possible the simple and easy production of applied and processed foods, and this bacterial cell powder is therefore quite useful.
However, when these microorganisms are converted into powdery form, their advantageous effect cannot be realized unless at least a necessary amount of the actual microorganism remains alive at the time of consumption or food production using the microorganisms. Accordingly, there is desire for effective methods of preserving microorganisms and, methods of manufacturing the bacterial cell powder easily by using the microorganisms. For example, there is desire for methods of improving the survivability when the microorganisms are freeze-dried.
Various investigations have been made to date in pursuit of these goals.
For example, Japanese Patent Application Laid-open No.
H5-186337 (JP H5-186337 A) is directed to improving the intestinal solubility while avoiding dissolution in gastric fluids by adopting a preservation technique of coating and granulation of useful bacteria such as lactic acid bacteria, bifidobacteria and so forth that may be killed by acidity or high temperatures, . More specifically, a granulate is made of a physiologically active substance using a three-layers coating wherein a saccharide is used for one layer.
In order to improve the survivability of bacteria belonging to the genus Bifidobacterium, Japanese Patent
Application Laid-open No. H11-137172 (JP H11-137172 A) provides a method in which a survivability enhancer, which contains a saccharide selected from glycerol, xylitol, adonitol, arabitol, and mannitol, is added to the culture medium. The goal of this method is to maintain the survivability and cell count (i.e. the number of living cells) during storage under aerobic conditions and/or under low pH conditions. In particular, the main motivation of this art is to improve the survival rate in the culture medium system.
Also, Japanese Patent Application Laid-open No. 2001- 64189 (JP 2001-64189 A) provides an enterosoluble lactic acid bacterial composition, that exhibits an excellent long-term stability, an excellent acid resistance, and an excellent enterosolubility, that is obtained by mixing a polyglycerol/ fatty acid ester into a lactic acid bacterial cell powder. The main focus of this art is storage and robustness under specific conditions.
On the other hand, various art has also been developed with regard to improving microbial survivability in cryostorage (i.e. freeze storage) and freezing dry storage.
For example, with the goal of introducing a production method that exhibits a high survival rate and little cell damage or mortality in freezing or freezing dry, a freezing dry method is disclosed in Japanese Patent Application Laid- open No. H7-313140 (JP H7-313140 A) in which a polysaccharide partial hydrolyzate originating from konjac powder is added to a liquid cell dispersion of lactic acid bacteria. The polysaccharide partial hydrolyzate has a specific molecular weight which is obtained by enzymatic degradation, and a complex process 1s required for its production. In addition, the main motivation of this art is to improve the survival rate in freezing and thawing, and suggestions of or references to the post-storage survival rate of a freeze-dried bacterial cell powder produced by this art are entirely absent.
Japanese Patent Application Laid-open No. 2001-327280 (JP 2001-327280 A) provides a preservation solution that gives an excellent bacterial cell viability rate and activity, even after the frozen bacterial cells have been thawed. This bacterial cell cryopreservation solution contains trehalose and/or polyethylene glycol as effective components. As a consequence, this preservation solution is characteristically able to inhibit damage during freezing and makes possible use immediately after thawing. This art relates to a water-
containing system and in particular solves the problems in the process of thawing after freezing.
Also, in Japanese Patent Application Laid-open No. 2005- 52100 (Jp 2005-52100 A), Lactobacillus fermentum is made into a spray-dried powder by being coated with a starch- and sugar- containing protective coating. This art is an art essentially directed to improving the bacterial cell survivability during spray drying.
Furthermore, the storage stability in the dry state is improved in the art of International Publication No. 2006/106806 (WO 2006/106806 A) by combining a dried microorganism such as a dried lactic acid bacterium with an acidic amino acid salt of L-arginine. The main motivation of this art is to improve the storage stability of microorganisms during the formulation of microorganisms. The result, which is given for the two-week storage at 40°C of a bifidobacterial formulation produced by this art, is that the survival rate is low at about 25%. This result exhibits that this art is not an art that can provide tolerance to long-term storage extending to several months.
Also, "Samuel B. Leslie et al., Trehalose and Sucrose
Protect Both Membranes and Proteins in Intact Bacteria during
Drying, Applied and Environmental Microbiology, October, 1995, pp. 3592-3597 (USA)" reports the results of research in which it was ascertained, based on an analysis of the mechanism in freezing dry, that the damage during the freezing dry of a microorganism is largely due to changes in the physical state of the lipids making up the cell membrane, and to structural changes in proteins, and in which, in response to this finding, freezing dry was performed on, for example, E. coli, in the presence of trehalose or sucrose. However, suggestions of or references to the use of a mixture of trehalose and sucrose are entirely absent in this report. Moreover, absolutely no investigations were performed that were directed to the long- term storage of a specific freeze-dried material or to storage at prescribed high-temperature conditions.
Furthermore, "Gaber Zayed et al., Influence of trehalose and moisture content on survival of Lactobacillus salivarius subjected to freeze-drying and storage, Process Biochemistry, 2004, Volume 39, pp. 1081-1086" reports on investigations performed, with the same objectives as above, on the freezing dry and storage of Lactobacillus salivarius. This study investigated the use of 4% of trehalose, 4% of sucrose, 18% of skim milk powder, 4% of trehalose + 18% of skim milk powder, 4% of sucrose + 18% of skim milk powder, 4% of trehalose + 4% of sucrose, and 4% of trehalose + 4% of sucrose + 18% of skim milk powder, as freezing-dry protective agents, and investigated the survival rate when the freeze-dried bacterial cell powder was stored at -85°C. It should be noted that in the cell suspension at the time of freezing dry, the concentration of saccharide is approximately 3.2% and the concentration of skim milk powder is 14.4%. According to the results disclosed in this document, the 4% of sucrose was reported to be unable to improve the -85°C storability of the freeze-dried bacterial cell powder sufficiently, while the 4% of sucrose + 18% of skim milk powder was reported to be able to improve the storability at —-85°C of the freeze-dried bacterial cell powder.
Moreover, 4% of trehalose by itself was reported to improve the -85°C storability of the freeze-dried bacterial cell powder.
In addition, results are also reported for the storage for 7 weeks at room temperature (the value of temperature not given) of a bacterial cell powder obtained by freeze-drying the bacterial cells with a mixed solution of 4% of trehalose + 4% of sucrose + 18% of skim milk powder.
It is reported in the results that a high preservability is maintained when storage was carried out in an atmosphere with a specific humidity (i.e. humidity: 2.8 to 5.6%, moisture activity: 0.028 to 0.056). However, a high preservability could not be maintained in an atmosphere of 0% or 8.8% of humidity (the survival rate was, respectively, approximately 40% and 10%). The conditions for room-temperature preservability are thus limited to a very narrow humidity range at very low humidity, and these conditions are impractical.
Thus, the art of this document shows the results of preservation tests under environmental conditions that are remote from the usual living environment.
Moreover, in this document, suggestions of or references to storage under high-
temperature conditions are entirely absent, and the application of this art to food products in general is still guite problematic.
As described in the preceding, various investigations have been carried out to date on the storage or preservation of useful microorganisms and on effective formats.
Particularly with regard to freeze-dried bacterial cell powders or dried cell powders, various approaches have been taken in terms of the convenience of the product format.
However, there is still much room for investigation with regard to the retention of microbial function and the survival rate after long-term storage. For example, there are approaches that have effects only for a specific microorganism, whereas there are approaches that have effects for various microorganisms in very limited conditions for storage such as very low temperatures and low humidities. Thus, an industrially effective means has not be developed yet. In particular, in order to use a bacterial cell powder as a bacterial cell powder that can be applied to foods that are capable of long-term storage at a room air temperature (i.e. ambient temperature), it is necessary that the bacterial cell powder has a high survivability under high-temperature conditions (30 to 40°C) in the range presumed for the temperature in the normal living environment and under conditions that are not limited to special storage conditions.
Bacterial cell powders that do not have such a high survivability do not have the quality to be secured, and cannot be practically used as starting materials of foods.
Accordingly, an object of the present invention is to provide a freeze-dried bacterial cell powder which is suitable for long-term storage and which is obtained by a simple and effective freeze-dry method, and a method of manufacturing the freeze-dried bacterial cell powder. In particular, the present invention provides a freeze-dried bacterial cell powder which can be stored at high survival rates of useful microorganisms such as a lactic acid bacteria, a bifidobacteria and the like, even in the event of long-term storage after freezing dry and even in the event of long-term storage in the living temperature zone (particularly during the summer) which seems to have an undesired influence on the storability because of the relatively high temperature compared to a very low temperature. The present invention also provides a method of producing the freeze-dried bacterial cell powder described above. Furthermore, the present invention provides a food composition which contains the obtained freeze-dried bacterial cell powder.
The present inventors carried out intensive investigations in view of the above-described problems of the prior art, and discovered that the survivability, especially the survivability in high-temperature storage, of a cell powder of useful microorganisms such as a lactic acid bacteria, a bifidobacteria and the like is increased in a great degree by freeze-drying a suspension of bacterial cells of a lactic acid bacterium and/or a bifidobacterium suspended in a saccharides' solution. Thus, the present invention has been accomplished.
The present invention provides a freeze-dried bacterial cell powder which is suitable for long storage and which is obtained by freeze-dry process which is simple and effective.
That is, an aspect of the present invention is a method of producing a freeze-dried bacterial cell powder wherein a suspension of bacterial cells of a lactic acid bacterium and/or a bifidobacterium suspended in a saccharides' solution is freeze-dried to obtain a freeze-dried bacterial cell powder, and wherein the saccharides are trehalose and sucrose, and the concentration of each of trehalose and sucrose in the suspension prior to freeze-drying is not less than 4.5% by weight (hereinafter abbreviated to "wt %").
In the present invention, the concentration of each of trehalose and sucrose in the suspension prior to freeze-drying is preferably 4.5 to 15 wt %, more preferably 8 to 12 wt %.
In the present invention, the weight ratio of sucrose/trehalose is preferably 1/5 to 5/1.
In the present invention, preferable examples of the lactic acid bacterium and/or bifidobacterium include a microorganism belonging to the genus Bifidobacterium.
Preferable examples of the genus Bifidobacterium includes the strain Bifidobacterium bifidum and the strain
Bifidobacterium longum.
Preferable examples of the strain Bifidobacterium bifidum includes Bifidobacterium bifidum OLB6378.
Preferable examples of the strain Bifidobacterium longum includes Bifidobacterium longum OLB6001.
Also, another aspect of the present invention is a freeze-dried bacterial cell powder produced by the method according to any of the preceding for producing a freeze-dried bacterial cell powder.
Yet another aspect of the present invention is a food composition comprising the aforementioned freeze-dried bacterial cell powder in an effective amount.
The method of the present invention for producing a long- term storable freeze-dried bacterial cell powder exhibits its effects in particular when long-term storage must be secured in solid or powdered foods. That is, the bacterial cell powder produced by the production method according to the present invention has advantageous effects of a lactic acid bacteria and/or a bifidobacteria being useful even after storage, because the survival rate during high-temperature storage is remarkably improved.
Also, the production method of the present invention is simple and easy. Accordingly, a special apparatus and a complex procedure are not required, and the accompanied cost increase does not occur.
Also, the bacterial cell powder produced by the production method of the present invention can be used directly as is, and as a consequence can also be administered as a powder in an easy way even after storage or can be effectively utilized as a starting material for food production.
Figure 1 is a graph that shows the results of storage testing at the temperature of 20°C, 30°C or 40°C of a
Bifidobacterium bifidum cell powder produced using a slurry having a trehalose concentration of 6.6 wt % and a sucrose concentration of 6.6 wt % prior to freeze-drying, which is a slurry obtained using a saccharides' solution having a trehalose concentration of 20 wt % and a sucrose concentration of 20 wt % prior to be mixed with the microorganism.
Figure 2 is a graph that shows the results of storage testing at the temperature of 20°C, 30°C or 40°C of a
Bifidobacterium bifidum cell powder produced using a slurry having a trehalose concentration of 10 wt % and a sucrose concentration of 10 wt % prior to freeze-drying, which is a slurry obtained using a saccharides' solution having a trehalose concentration of 30 wt % and a sucrose concentration of 30 wt % prior to be mixed with the microorganism.
Figure 3 is a graph that shows the results of storage testing at the temperature of 40°C of a freeze-dried
Bifidobacterium longum cell powder (represented as circled number 2) produced using a slurry having a trehalose concentration of 6.7 wt % and a sucrose concentration of 6.7 wt % prior to freeze-drying, which is a slurry obtained using a saccharides' solution having a trehalose concentration of 20 wt % and a sucrose concentration of 20 wt % prior to be mixed with the microorganism, and a freeze-dried bacterial cell powder for reference (represented as circled number 1).
The present invention is described in detail in the following, but the present invention is not limited to the individual embodiments described below.
The lactic acid bacteria and/or bifidobacteria used in the present invention can be exemplified by microorganisms belonging to the genus Bifidobacterium and can be specifically exemplified by the strains Bifidobacterium longum,
Bifidobacterium infantis, Bifidobacterium breve,
Bifidobacterium bifidum, Bifidobacterium adolescentis, and so forth. Other examples are microorganisms belonging to the genus Lactobacillus and the genus Streptococcus. Specific examples of other examples are the strain Lactobacillus gasseri, the strain Lactobacillus bulgaricus, and the strain
Streptococcus thermophilus. However, the present invention is not limited to these species. Moreover, a single one of these microbial strains can be used, or two or more of these microbial strains can be used in combination.
Particularly preferred strains among the preceding are the strain Bifidobacterium bifidum, the strain Bifidobacterium longum, and so forth.
Examples of deposited strains include the strains
Bifidobacterium bifidum OLB6378, Bifidobacterium longum OLB6001, and so forth. (A) Deposit of the strain Bifidobacterium bifidum OLB6378
The deposit of the Bifidobacterium bifidum OLB6378 strain used by the present invention has been made as follows. (1) Name of Authority Depository: Patent Microorganisms
Depositary of the National Institute of Technology and
Evaluation established as an Independent Administrative Legal
Entity (2) Contact address: 2-5-8 Kazusakamatari, Kisarazu-shi,
Chiba-ken, postal code 292-0818 telephone: 0438-20-5580 (3) Deposit number: NITE BP-31 (4) Presentment for identification: Bifidobacterium bifidum
OLB6378 (5) Original deposit date: 26 October 2004 (6) Date of transfer to a deposit based on the Budapest
Treaty: 18 January 2006
(B) Deposit of the strain Bifidobacterium longum OLB6001
The deposit of the Bifidobacterium longum QLB6001 strain used by the present invention has been made as follows. (1) Name of Authority Depository: International Patent
Organism Depositary of the National Institute of Advanced
Industrial Science and Technology established as an
Independent Administrative Legal Entity (2) Contact address: Chuo dai-6, 1-1-1 Higashi, Tsukuba-shi,
Ibaragi-ken, postal code 305-8566 telephone: 029-861-6029 (3) Deposit number: FERM P-13610 (4) Presentment for identification: Bifidobacterium longum
No.7 (5) Original deposit date: 20 April 1993 (6) Transfer to a deposit based on the Budapest Treaty
Receipt date: 2 March 2010
Receipt Number: FERM ABP-11242
The Bifidobacterium bifidum OLB6378 strain and the
Bifidobacterium longum OLB6001 used in the present invention have the following bacteriological properties.
The strain Bifidobacterium bifidum OLB6378 is Gram- positive obligate anaerobic bacillus originating from human infant feces. A Y-shaped cell morphology is seen when this organism is inoculated to Lactobacilli MRS Broth (BD) and anaerobically cultured for 18 hours at 37°C using an
AnaeroPack KENKI (manufactured by Mitsubishi Gas Chemical
Company, Inc.). A PCR product was observed by PCR using a specific primer for Bifodobacteirum bifidum (Tomotari MITSUOKA and Takahiro MATSUMOTO, Molecular Biological Detection and
Identification of Intestinal Flora, Proceedings of the 8th
Symposium on Intestinal Flora) and specifically BiBIF-1: CCA
CAT GAT CGC ATG TGA TT and BiBIF-2: CCG AAG GCT TGC TCC CAA A, which are species-specific primers for 16S rDNA.
The strain Bifidobacterium longum OLB6001 is Gram-positive obligate anaerobic microorganism originating from adult human feces which is a rod or a branched pleomorphic microorganism, and does not have motility and the ability of forming spore. A colony having obscure and hemispherical glossy is formed when this organism is inoculated to BL agar plate (EIKEN) and cultured for 48 hours at 37°C using steel wool method. The microorganism has a fermentability of arabinose, xylose, ribose, glucose, fructose, galactose, sucrose, maltose, melibiose, raffinose and melicitose.
The saccharide used as a protective agent in the present invention is a mixture of trehalose and sucrose. The concentration of each of trehalose and sucrose in the slurry prior to freeze-drying is not less than 4.5 wt %, and preferably not less than 8 wt % in view of obtaining a freeze- dried bacterial cell powder suitable for long-term storage in high temperature. Its upper limit is preferably 15 wt %, and more preferably 12 wt %. The weight ratio of sucrose/trehalose is not particularly limited, but preferably 1/5 to 5/1, more preferably 1/3 to 3/1, most preferably 1/1 in view of obtaining a freeze-dried bacterial cell powder suitable for long-term storage in high-temperature.
It is preferable that these saccharides are mixed with the cultured bacterial cells as a saccharide-containing agueous solution to resuspend the cultured bacterial cells.
The freeze-dried bacterial cell powder of the present invention can be obtained by freeze-drying the suspension obtained in this manner.
The saccharide-containing aqueous solution may include milk protein, amino acids, ascorbic acid or the like in addition to water and saccharides.
The method of producing a freeze-dried bacterial cell powder of the present invention is not particularly limited, but can comprise, for example, the following sequence. 1) The desired microorganism is cultured according to the usual method. 2) The culture liquid including cultured bacterial cells is used as it is in the following 3), or is concentrated or divided into solid and liquid components by for example, centrifugal separation to obtain a concentrated culture liquid (i.e. cell liquid) or bacterial cells separated as the solid component, and then used in the following 3).
3) By mixing the obtained bacterial cell liquid or bacterial cells with a solution that contains the saccharides (i.e. the protective agent) having prescribed concentrations of these saccharides, a suspension is obtained. After that, this suspension is freeze-dried to obtain the freeze-dried bacterial cell powder of the present invention.
Freeze-drying can be done by for example, using a freeze- dry machine. For example, prior freezing is done rapidly at a low temperature (for example, -30 to -90°C). After that, drying is done at a room air temperature (for example, 0 to 20°C) and under a reduced pressure (degree of vacuum of preferably not more than 1,000Pa, more preferably not more than 100Pa). After that, the temperature of the freeze-dry machine is raised to for example, a temperature of from 30 to 70°C while maintaining the reduced pressure. After that, drying is kept for a certain period of time.
The freeze-dried bacterial cell powder of the present invention can also be consumed as 1s, because trehalose and sucrose used in the present invention can be also used as food additives. The freeze-dried bacterial cell powder of the present invention can also be used by adding to various food compositions. In addition, the freeze-dried bacterial cell powder of the present invention can also be used as a starting material such as a starting culture for fermented milk products and so forth, because the freeze-dried bacterial cell powder of the present invention exhibits a certain cell survival rate even after long-term storage.
The food composition can be exemplified by various beverages and foods (e.g., soft drinks, fermented milk products, yogurt, modified milk powder, and so forth). This food composition may be used as is, or may be used according to the usual methods in the usual food product compositions, e.g., by mixing with another food or with a food component.
The form of the food composition may be the generally used formats of beverage or food, such as a solid (i.e. powder, granular, and so forth), paste, liquid, or suspension.
The other components in the food composition are also not particularly limited. For example, one or more selected from water, proteins, saccharides, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, flavors and so forth can be used as components in the food composition. These components can be used as a single component, or two or more of these components can be used in combination. It is possible to use synthetic products and/or foods that contain a large amount of synthetic products as the other components in the food composition.
The present invention is described below using Examples, but the present invention is not limited to these Examples. In this Description, “%” indicates “percent by weight (wt %)”
unless specifically indicated otherwise.
The present invention is described in additional detail based on the Examples provided below. [Experimental Example 1]
Using the procedures given below, a freeze-dried bacterial cell powder was produced based on the production method according to the present invention. 1) Neutral cultivation of the strain Bifidobacterium bifidum
OLB6378 is carried out on a digested casein medium (i.e. a medium in which enzymatically digested casein is used as the protein). 2) 320 mL of the culture broth is centrifugally separated for minutes at 10,000 G and 4°C; 307.2 mL of supernatant is removed; and a cell pellet fraction (12.8 mL) is obtained. 3) 2 mL of the protective agent which includes saccharides having prescribed concentrations is added to the bacterial cell pellet fraction (4 mL), and a bacterial cell suspension (abbreviated as "cell suspension" in Table 1) is prepared and freeze-dried by freezing at -80°C. 4) Immediately after the completion of freeze-drying, 0.5 g of the freeze-dried bacterial cell powder was covered over with water by the addition of physiological saline aqueous solution, and the number of living cells (i.e. living cell count) in the liquid was measured on flat BL agar culture medium.
5) Furthermore, 0.5 g of the freeze-dried bacterial cell powder was placed in a lami-zip (trademark; a product name of a plastic bag), and stored for 53 days at 20°C or 30°C. After that, the number of living cells in the freeze-dried bacterial cell powder was similarly measured using flat BL agar culture medium.
The protective agent solutions used in this Experimental
Example were as shown in Table 1 below.
Table 1 sample trehalose sucrose trehalose sucrose number concentration in | concentration in concentration concentration the protective the protective in the cell in the cell agent solution agent solution suspension suspension (%) (%) (%) (%)
Sew [po [po | es | e.s
Comparative 10 3.3
Sample 1
Comparative 26 8.7
Sample 2
Comparative 10 10 3.3 3.3
Sample 3
The results are given in Table 2 for the Bifidobacterium bifidum storage tests described above using Samples 1 and 2 and Comparative Samples 1, 2 and 3. Due to the low survival rates with Comparative Samples 1 and 2, only the results for storage for 7 days at 20°C are given for Comparative Samples 1 and 2 in Table 2.
Table 2 number sample storage living cell living cell survival number temperature count count after rate immediately storage (%) after drying (cfu/qg) (cfu/q) 5.41107 | Tao" | i150
L110" | 1.20" | 1095
Comparative | Comparative 20°C 1 ox101t 3 8x10 *1 30.4
Example 1 Sample 1 : :
Comparative | Comparative 20°C 1 1x10! 5 3x10 *1 48.2
Example 2 Sample 2 : :
Comparative | Comparative 20°C 1 0x10! 7 2x101° 70.0
Example 3 Sample 3 : :
Comparative | Comparative 30°C 1 0x10! 5 2x101° 49.9
Example 4 Sample 3 : : *1: Number of days of storage: 7 days
As is clear from Table 2, a reduction in the living cell count (i.e. the number of the living cells) was not seen in each of Examples 1 to 4 done by the method of the present invention, even when the freeze-dried bacterial cell powder was stored under a prescribed high-temperature condition for 53 days. In particular, it was shown that the survival rate could undergo a dramatic increase even at a high temperature close to the temperature of the living environment, as in
Examples 2 and 4. This demonstrated that the production method of the present invention was an art that was well qualified for application to solid or powdered foods with a relatively long-term quality guarantee.
On the other hand, Comparative Examples 1 and 2, which were tests using trehalose only modeled on the document "Gaber
Zayed et al." described above, demonstrated that the target could not be achieved sufficiently by trehalose only. In particular, while the document "Gaber Zayed et al." describes an effect for storage under ultra-low temperature and humidity conditions, the results of Comparative Examples 1 and 2 show that the desired effects could not be obtained at the living environment temperatures as in these experiments.
In addition, investigations were carried out in
Comparative Examples 3 and 4 in which trehalose and sucrose are used at the same concentrations as in the document "Gaber
Zayed et al.". The results of Comparative Examples 3 and 4 show that the living cell count decreased by about half under the temperature conditions assumed for the living environment.
Consequently, it was found that the freeze-dried bacterial cell powders provided by the method of these
Comparative Examples were difficult to be applied to solid or powdered foods with a long-term quality guarantee, and that the methods of these Comparative Examples were not arts that were well qualified for application to such foods. [Experimental Example 2]
The freeze-dried bacterial cell powder provided by the production method of the present invention was subjected to experiments on the effects under long-term storage (up to six months) under high-temperature conditions (20°C, 30°C or 40°C).
The cell powder used was produced by the same production method as for Samples 1 and 2, with the exception that the amounts of the initial inputs were increased to about 30 times as that in Samples 1 and 2. Accordingly, the following discussion retains the designation of Sample 1 and Sample 2.
The results obtained in this experiment are given in
Figures 1 and 2.
As is clear from the Figures, Sample 1 (see Figure 1; a protective agent solution including trehalose and sucrose at the concentration of 6.6 wt % each, obtained by mixing a solution including trehalose at the concentration of 20 wt % with a solution including sucrose at the concentration of 20 wt %) and Sample 2 (Figure 2; a protective agent solution including trehalose and sucrose at the concentration of 10 wt % each, obtained by mixing a solution including trehalose at the concentration of 30 wt % with a solution including sucrose at the concentration of 30 wt %) were both able to maintain a sufficient number of living cells even after six months at a storage temperature of 20°C or 30°C, and a very high survivability was shown by Samples 1 and 2.
For storage at 40°C, which is a fairly extreme living environment temperature condition, a high survivability of 93.9% is seen with Sample 1 after storage for 1 month. Thus, the concentration used for Sample 1 was shown to be more preferred concentration of a protective agent for this condition (i.e. storage at 40°C).
[Experimental Example 3]
Experiments were done in the same way as in Example 1 except that Samples shown in Table 3 were used and the storage temperature and period were 40°C and 3 months. The results are shown in Table 4. From Table 4, it can be seen that the results in survivability of Examples 5 to 7 are not substantially different to one another because the survival rates are from 11 % to 17 % when the weight ratio of sucrose/trehalose ranges from 1/3 to 3/1, and Examples 5 to 7 show almost equal effects to one another.
Table 3 sample trehalose sucrose trehalose sucrose number concentration in | concentration in concentration concentration the protective the protective in the cell in the cell agent solution agent solution suspension suspension (%) (%) (%) (%)
Gls | p00 ee |e.
Table 4 number sample storage living cell living cell survival number temperature count count after rate immediately storage (%) after drying (cfu/qg) (cfu/qg) 7.1107 | a0” 5.510" | 6.1407 [Experimental Example 4]
Using the procedures given below, a freeze-dried bacterial cell powder was produced based on the production method according to the present invention.
1) Neutral cultivation of the strain Bifidobacterium longum
OLB6001 is carried out on a digested casein medium (i.e. a medium in which enzymatically digested casein is used as the protein). 2) 5400 mL of the culture broth is centrifugally separated for minutes at 10,000 G and 4°C; 520 mL supernatant is removed; and a bacterial cell pellet fraction (200 mL) is obtained. 3) 100 mL of the protective agent which includes saccharides having the prescribed concentrations is added to the bacterial cell pellet fraction (200 mL), and a bacterial cell suspension is prepared and freeze-dried by freezing at -80°C. 4) Immediately after the completion of freeze-drying, 1 g of the freeze-dried bacterial cell powder was covered over with water by the addition of physiological saline aqueous solution, and the number of living cells (i.e. living cell count) in the liquid was measured on flat BL agar culture medium. 5) Furthermore, 2 g of the freeze-dried bacterial cell powder was placed in a lami-zip (trademark; a product name of a plastic bag), and stored for 8 days, 30 days, 82 days or 124 days at 40°C. After that, the number of living cells (i.e. living cell count) in the freeze-dried bacterial cell powder was similarly measured using flat BL agar culture medium.
The protective agent solutions used in this experimental example were as shown below.
(1) Example 8
A saccaride solution (referred to "Sample 6" hereinafter) including trehalose and sucrose at the concentration of 20 wt % each is used. The concentration of each of trehalose and sucrose in the suspension prior to freeze-drying obtained by mixing this saccaride solution with the cell pellet fraction was 6.7 wt %. (2) Comparative Example 5
A solution (hereinafter referred to as Comparative Sample 4) including 6 wt % of powdered skim milk, 1.7 wt % of lactose, 0.4 wt % of amino acids (lysine and so forth), 4 wt % of other components (dextrin and so forth) was used.
The results are shown in Table 5 and Figure 3. Table 5 shows that the freeze-dried bacterial cell powder of Example 8 (shown as circled number 2 in Figure 3) has a high survival rate of 40.0% for storage of about 4 months at 40°C, whereas the freeze-dried bacterial cell powder of Comparative Example (shown as circled number 1 in Figure 3) has a survival rate of 0.002% for storage of about 4 months at 40°C
Table 5
CE re Tre Tre ee ea compara | COMPATET | 40°C | 6.5x10% | 23x10 | 1.9x10% | 2.0x107 | 1.1x10°
Example Sample 4
The method according to the present invention for producing a long-term storable freeze-dried bacterial cell powder makes it possible to obtain the effects of useful lactic acid bacteria and/or bifidobacteria even after storage, particularly with respect to solid foods and powdered foods and particularly in those instances where long-term storage must be secured.
In addition, special equipment and a complicated procedure are not required, which also eliminates the extra expenses associated therewith and thus also makes this method economically advantageous.
Moreover, since the bacterial cell powder can be used as 1s, 1t can also be conveniently consumed as a powder directly after storage, or it can be effectively utilized as a starting material for food production.
As a consequence, the bacterial cell powder of the present invention is highly useful.
Claims (11)
1. A method of producing a freeze-dried bacterial cell powder wherein a suspension of bacterial cells suspended in a saccharides' solution, which includes a lactic acid bacterium and/or a bifidobacterium, 1s freeze-dried to obtain a freeze- dried bacterial cell powder, and wherein the saccharides are trehalose and sucrose, and the concentration of each of trehalose and sucrose in the suspension prior to freeze-drying is not less than 4.5% by weight.
2. The method of producing a freeze-dried bacterial cell powder according to claim 1, wherein the concentration of each of trehalose and sucrose in the suspension prior to freeze- drying is 4.5% to 15% by weight.
3. The method of producing a freeze-dried bacterial cell powder according to claim 1 or 2, wherein the concentration of each of trehalose and sucrose in the suspension prior to freeze-drying is 8% to 12% by weight.
4. The method of producing a freeze-dried bacterial cell powder according to any of claims 1 to 3, wherein the weight ratio of sucrose/trehalose is 1/5 to 5/1.
5. The method of producing a freeze-dried bacterial cell powder according to any of claims 1 to 4, wherein the lactic acid bacterium and/or bifidobacterium is a microorganism belonging to the genus Bifidobacterium.
6. The method of producing a freeze-dried bacterial cell powder according to claim 5, wherein the lactic acid bacterium and/or bifidobacterium is the strain Bifidobacterium bifidum.
7. The method of producing a freeze-dried bacterial cell powder according to claim 6, wherein the lactic acid bacterium and/or bifidobacterium is the strain Bifidobacterium bifidum OLB6378.
8. The method of producing a freeze-dried bacterial cell powder according to claim 5, wherein the lactic acid bacterium and/or bifidobacterium is the strain Bifidobacterium longum.
9. The method of producing a freeze-dried bacterial cell powder according to claim 8, wherein the lactic acid bacterium and/or bifidobacterium is the strain Bifidobacterium longum OLB60O01.
10. A freeze-dried bacterial cell powder produced by the method of producing a freeze-dried bacterial cell powder according to any of claims 1 to 9.
11. A food composition comprising the freeze-dried bacterial cell powder according to claim 10 in an effective amount.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2009051118 | 2009-03-04 | ||
| PCT/JP2010/053421 WO2010101175A1 (en) | 2009-03-04 | 2010-03-03 | Freeze-dried microbial cell powder and method for producing same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| SG172859A1 true SG172859A1 (en) | 2011-08-29 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| SG2011049061A SG172859A1 (en) | 2009-03-04 | 2010-03-03 | Freeze-dried microbial cell powder and method for producing same |
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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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| US20110189343A1 (en) * | 2008-06-26 | 2011-08-04 | Hideo Hasegawa | Nano-sized lactic acid bacteria |
| 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 |
| JP6807677B2 (en) * | 2016-07-15 | 2021-01-06 | 株式会社明治 | Method of microbial culture concentration and microbial concentrate |
| CN110177866B (en) * | 2016-07-15 | 2023-06-20 | Cj第一制糖株式会社 | Leuconostoc mesenteroides CJLM627 strain with low gas production and method for preparing pickle by using strain |
| RU2731642C2 (en) * | 2016-07-15 | 2020-09-07 | СиДжей ЧЕИЛДЗЕДАНГ КОРПОРЕЙШН | Cjlm119 leuconostoc mesenteroides strain, which produces reduced amount of gas, and method for preparing kimchi using said strain |
| 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 |
| CN111534434B (en) * | 2020-06-28 | 2022-07-05 | 江南大学 | Freeze-drying protective agent and application thereof in freeze-drying bifidobacterium adolescentis |
| 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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| JPH0638704A (en) * | 1991-06-19 | 1994-02-15 | Morinaga Milk Ind Co Ltd | Fermented seasoning and its production |
| JPH0551321A (en) * | 1991-08-23 | 1993-03-02 | Yotsuba Nyugyo Kk | Immune activating agent |
| JPH08187071A (en) * | 1995-01-11 | 1996-07-23 | Morinaga Milk Ind Co Ltd | Method for preserving food |
| EP1493806A1 (en) * | 2003-07-02 | 2005-01-05 | Chr. Hansen A/S | Use of compounds involved in biosynthesis of nucleic acids as cryoprotective agents |
| DK1720974T4 (en) * | 2004-02-24 | 2022-01-31 | Chr Hansen As | Frozen lactic acid bacterial culture of individual pellets |
| JP5273695B2 (en) * | 2005-03-03 | 2013-08-28 | 株式会社明治 | Allergy prevention and / or treatment agent containing bifidobacteria as an active ingredient |
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2010
- 2010-03-03 CN CN201080004172.6A patent/CN102272287B/en active Active
- 2010-03-03 SG SG2011049061A patent/SG172859A1/en unknown
- 2010-03-03 WO PCT/JP2010/053421 patent/WO2010101175A1/en active Application Filing
- 2010-03-03 JP JP2011502775A patent/JP5583114B2/en active Active
- 2010-03-04 TW TW099106326A patent/TWI501731B/en active
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2012
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Also Published As
| Publication number | Publication date |
|---|---|
| WO2010101175A1 (en) | 2010-09-10 |
| TW201043152A (en) | 2010-12-16 |
| CN102272287B (en) | 2014-12-10 |
| JP5583114B2 (en) | 2014-09-03 |
| CN102272287A (en) | 2011-12-07 |
| HK1159688A1 (en) | 2012-08-03 |
| JPWO2010101175A1 (en) | 2012-09-10 |
| TWI501731B (en) | 2015-10-01 |
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