JP2019010073A - Thawing method of frozen lactic acid bacterium - Google Patents

Abstract

To improve survival property of lactic acid bacteria in a food product or beverage easily without restriction of each kind of a condition in a food product or beverage, without using a special additive, as a first purpose, and enhance vitality of a lactobacillus starter for achieving high production effect and shortening a fermentation time of a fermented milk, as a second purpose.SOLUTION: The invention relates to a thawing method of a frozen lactic acid bacterium in which the frozen lactic acid is thawed in a temperature condition in a range of 20°C-50°C.SELECTED DRAWING: None

Description

  The present invention relates to a method for thawing frozen lactic acid bacteria. Specifically, the present invention relates to a method for thawing frozen lactic acid bacteria under temperature conditions within a specific temperature range, and a method for producing a food or beverage composition containing lactic acid bacteria thawed using the thawing method.

  In recent years, strains having a probiotic function have attracted attention, and preventive medicine by improving and normalizing the gastrointestinal tract by ingesting these bacteria and the accompanying immunomodulating action has become popular. Among them, lactic acid bacteria are also called probiotic bacteria and have been ingested as various foods and drinks since ancient times. Recently, the development of functions has become more active than before, and the development of products incorporating new functionality is also progressing. Examples of such bacterial species include Lactobacillus gasseri, Lactobacillus bulgaricus, and Streptococcus thermophilus.

  In order to ingest lactic acid bacteria, lactic acid bacteria are often included in various foods and drinks, and improving the survival of lactic acid bacteria in foods and drinks means that lactic acid bacteria have the above probiotic functions. It has a great contribution to preventive medicine. However, it is known that fermented foods and drinks represented by fermented milk have a low pH environment and are not suitable for the survival of lactic acid bacteria. Therefore, it has been conventionally required to improve the survival of lactic acid bacteria in various foods and drinks, and various studies have been repeated so far.

Non-Patent Document 1 reports that the addition of cysteine, whey powder, WPC, casein hydrate, or tryptone improved the maintenance of the number of bacteria during storage in yogurt containing bifidobacteria and acidophilus bacteria. Has been.
Further, Non-Patent Document 2 reports that the number of probiotic bacteria (acidophilus bacteria) has been improved by adding ascorbic acid as an active oxygen scavenger.

  Moreover, it is a manufacturing method of the food / beverage products containing Lactobacillus gasseri in patent document 1, Comprising: The said manufacturing method including the process of making pH and sugar concentration of food / beverage products into a specific range, and mixing a lactobacillus gasseri and a specific lactic acid bacterium. Is disclosed.

  On the other hand, fermented milk such as yogurt is produced by adding lactic acid bacteria starters such as Lactobacillus bulgaricus and Streptococcus thermophilus to raw milk and proceeding fermentation under a predetermined temperature condition. Is done. In producing fermented milk, it is required to shorten the fermentation time without reducing the quality in order to cope with a rapid increase in the amount of substances and to reduce labor costs and electricity costs. In order to shorten the fermentation time, it is necessary to increase the “vitality (fermentation power)” of the lactic acid bacteria starter that maintains the ability to ferment raw material milk.

  As a method for improving the “viability” of this lactic acid bacteria starter and exhibiting high fermentation activity, for example, Patent Literature 2 discloses a culture medium for preparing a lactic acid bacteria starter. It is characterized by containing 3.5% or more of powder, and further containing insoluble calcium salt such as calcium carbonate and soluble magnesium salt as a lactic acid production promoting factor at 50 mM or more for pH adjustment.

International Publication No. 2013/085009 Japanese Patent Laid-Open No. 08-116872

R. I. Dave et al. (RI Dave and N.P. Shah), “Ingredient Supplementation Effects on Viability Bacteria in Yoghurt”, J. Am. Dairy Sci 81, 2804-2816 (1998) R. I. Dave et al. (Rajiv I. Dave and Negendra P. Shah), “Effectiveness of Ascorbic Acid as an Oxygen in Improving the Immobilizing Vibrate of Probably. Dairy Journal 7, 435-443 (1997)

  In the said nonpatent literature 1 and the nonpatent literature 2, it was going to improve the survival property of lactic acid bacteria by adding milk protein, an active oxygen removal agent, etc. However, these substances have a great influence on the flavor and physical properties of foods and drinks, and their use is limited. In addition, since an active oxygen scavenger that is an antioxidant exhibits its effect by auto-oxidation, a predetermined amount of the active oxygen scavenger cannot be expected to maintain its effect. In addition, when milk or a milk material is used, a specific process is required, and it takes time and effort to make adjustments, which leads to an increase in cost. Moreover, also in the said patent document 1, it was necessary to make pH and sugar concentration of food-drinks into a specific range, and it took time and effort for the adjustment.

  Therefore, a first object of the present invention is to easily improve the survival of lactic acid bacteria in food and drink without using special additives and without restricting various conditions in the food and drink. .

  On the other hand, a lactic acid bacteria starter used in the production of fermented milk can achieve high production efficiency of fermented milk if the time until completion of fermentation of the fermented milk is less than 3 hours. However, with conventional lactic acid bacteria, it has been difficult to complete the fermentation of raw milk in a fermentation time of less than 3 hours.

  Then, the 2nd objective of this invention aims at improving the vitality of a lactic acid bacteria starter, in order to shorten the fermentation time of fermented milk and to implement | achieve high production efficiency.

  The present inventors paid attention to the thawing conditions of frozen lactic acid bacteria in order to improve the survival of lactic acid bacteria in foods and beverages, or to improve the vitality of lactic acid bacteria starters and shorten the fermentation time of fermented milk. So far, when thawing frozen lactic acid bacteria, it has been common to thaw frozen lactic acid bacteria in running water at about 15 ° C. As a result of thawing the frozen lactic acid bacteria at a specific range of the thawing temperature when thawing the frozen lactic acid bacteria, surprisingly, the present inventors surprisingly found that the lactic acid bacteria produced when the thawed lactic acid bacteria were used in food and drink. It has been found that the persistence can be remarkably improved than before, the vitality of the lactic acid bacteria starter can be improved, and the fermentation time of the fermented milk can be shortened, and the present invention has been completed.

Therefore, according to the present invention, the following inventions are provided.
1. A method for thawing frozen lactic acid bacteria, wherein the frozen lactic acid bacteria are thawed under temperature conditions in the range of 20 ° C to 50 ° C.
2. 2. The method for thawing frozen lactic acid bacteria according to 1 above, wherein the lactic acid bacteria are Lactobacillus or Streptococcus.
3. The lactic acid bacterium is at least one selected from the group consisting of Lactobacillus gasseri, Lactobacillus bulgaricus, and Streptococcus thermophilus, wherein the lactic acid bacterium is at least one selected from the group consisting of Lactobacillus bulgaricus and Streptococcus thermophilus Of thawing frozen lactic acid bacteria.
4). The lactic acid bacteria, Lactobacillus gasseri OLL2716 (Lactobacillus gasseri OLL2716: FERM BP-6999), Lactobacillus bulgaricus OLL1255 (Lactobacillus delbrueckii subspecies bulgaricus OLL1255: NITE BP-76), and Streptococcus thermophilus OLS3294 (Streptococcus thermophilus OLS3294: NITE P -77) The method for thawing frozen lactic acid bacteria according to 1 above, which is at least one bacterium selected from the group consisting of:
5. A method for producing a food or beverage composition containing lactic acid bacteria, comprising a step of thawing frozen lactic acid bacteria,
The method for producing a food or beverage composition containing lactic acid bacteria, wherein the thawing step is a step of thawing frozen lactic acid bacteria under a temperature condition within a range of 20 ° C to 50 ° C.

According to the method for thawing frozen lactic acid bacteria of the present invention, when the thawed lactic acid bacteria are used in a food or drink, the survival of the lactic acid bacteria in the food or drink can be improved. Therefore, it is possible to provide a food or drink that can provide a high probiotic function even after long-term storage.
Furthermore, according to the method for thawing frozen lactic acid bacteria of the present invention, the vitality of the lactic acid bacteria starter can be improved. As a result, the fermentation time of fermented milk can be shortened, and high production efficiency can be realized.

  Hereinafter, the present invention will be described in detail.

[Method of thawing frozen lactic acid bacteria]
The present invention is a method for thawing frozen lactic acid bacteria, wherein the frozen lactic acid bacteria are thawed under temperature conditions in the range of 20 ° C to 50 ° C.
<Lactic acid bacteria>
The lactic acid bacteria in the present invention are those in which the amount of lactic acid produced is 50% or more with respect to assimilated lactose, and the type thereof is not particularly limited.
Examples of preferable lactic acid bacteria in the present invention include microorganisms belonging to the genus Lactobacillus, the genus Streptococcus, and the like.

As the genus Lactobacillus, for example, Lactobacillus gasseri and Lactobacillus bulgaricus are preferably used.
Moreover, as a Streptococcus genus (Streptococcus), for example, Streptococcus thermophilus (Streptococcus thermophilus) is preferably used.

  The Lactobacillus bulgaricus and Streptococcus thermophilus can be used as a lactic acid bacterium starter.

  In particular, as Lactobacillus gasseri (Lactobacillus gasseri), for example, Lactobacillus gasseri OLL2716 (Lactobacillus gasseri OLL2716: FERM BP-6999) strain is preferably used.

As Lactobacillus bulgaricus (Lactobacillus bulgaricus), for example, Lactobacillus bulgaricus subspecies subspecies subspecies OLL1255: NITE BP-76 is preferably used.
Further, Lactobacillus bulgaricus 2038 obtained by isolation from Bulgarian yogurt LB81 (manufactured by Meiji Co., Ltd.) can also be used.

As the Streptococcus thermophilus (Streptococcus thermophilus), for example, Streptococcus thermophilus OLS3294 (Streptococcus thermophilus OLS3294: NITE P-77) strain is preferably used.
Further, Thermophilus OLS3059 (Streptococcus thermophilus OLS3059) obtained by isolation from Bulgarian yogurt LB81 (manufactured by Meiji Corporation) can also be used.

  These strains may be used alone or in combination of two or more.

  Lactobacillus gasseri OLL2716 (Lactobacillus gasseri OLL2716: FERM BP-6999) strain, Lactobacillus bulgaricus OLL1255 (Lactobacillus delbrueckii subspecies bulgaricus OLL1255: NITE BP-76) strain, and Streptococcus thermophilus OLS3294 (Streptococcus thermophilus OLS3294: NITE P-77 ) Information on stock deposits is as follows.

Lactobacillus gasseri OLL2716 (FERM BP-6999) strain (1) Depositor name: Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry (Current depositor name: Patent Evaluation Organism, National Institute of Technology and Evaluation) Deposit Center)
(2) Contact information: 1-1-3 Higashi 1-chome, Tsukuba City, Ibaraki 305-8586, Japan Phone number 029-861-6029
(Current contact: 〒 292-0818, Kisarazu City, Chiba Prefecture, Kazusa Kamashiga 2-5-8 Room 120, telephone number 0438-20-5910)
(3) Accession number: FERM BP-6999
(4) Display for identification: Lactobacillus gasseri OLL2716
(5) Original deposit date: May 24, 1999 (6) Transfer date to deposit under the Budapest Treaty: January 14, 2000

Lactobacillus bulgaricus OLL1255 (Lactobacillus delbrueckii subspecies bulgaricus OLL1255: NITE BP-76) strain (1) Depositary institution name: National Institute of Technology and Evaluation Technology Patent Microorganism Depositary Center (2) 2-5-8 Kazusa City
(Currently: Room No. 2-5-8 Kazusa Kamashika, Kisarazu City, Chiba Prefecture)
(3) Accession number: NITE BP-76
(4) Display for identification: Lactobacillus delbrueckii subspecies bulgaricus OLL1255 (NITE BP-76)
(5) Date of original deposit: February 10, 2005 (6) Date of transfer to deposit under the Budapest Treaty: April 30, 2009

Streptococcus thermophilus OLS 3294 (Streptococcus thermophilus OLS 3294: NITE P-77) strain (1) Depositary name: National Institute of Technology and Evaluation, Patent Microorganism Depositary Center (2) Contact: Kazusa Kisarazu City, Chiba Prefecture 292-0818 2-5-8 sickle feet
(Currently: Room No. 2-5-8 Kazusa Kamashika, Kisarazu City, Chiba Prefecture)
(3) Accession number: NITE P-77
(4) Display for identification: Streptococcus thermophilus OLS3294
(5) Original deposit date: February 10, 2005

<Frozen lactic acid bacteria>
The frozen lactic acid bacterium in the present invention refers to a frozen one of the above lactic acid bacteria, and the freezing temperature is not particularly limited as long as it is in a frozen state, and may be, for example, about −80 ° C., − It may be about 40 ° C. From the viewpoint of the effect of the present invention, the freezing temperature is preferably lower than −15 ° C. to −30 ° C., which is an ice crystal growth temperature zone described later.

  The frozen lactic acid bacteria may be obtained by freezing a lactic acid bacteria culture obtained by culturing the lactic acid bacteria at the above temperature. Examples of the medium for culturing lactic acid bacteria include conventionally known culture media for culturing lactic acid bacteria, and the type of the medium is not particularly limited. That is, any medium can be used as long as it contains a nitrogen source, an inorganic substance and other nutrients in addition to the main carbon source. As the carbon source, for example, lactose, glucose, sucrose, fructose, starch hydrolyzate, and molasses can be used according to the assimilation ability of the bacteria used. As the nitrogen source, organic nitrogen-containing substances such as casein hydrolyzate, whey protein hydrolyzate, and soybean protein hydrolyzate can be used. In addition, as the growth promoter, for example, meat extract, fish extract, yeast extract and the like are used.

  The culture is desirably performed under anaerobic conditions, but may be performed under microaerobic conditions such as liquid stationary culture that is usually used. For anaerobic culture, known methods such as a method of culturing under a nitrogen gas layer can be applied, but other methods may be used. In general, the culture temperature is preferably 30 to 40 ° C., but may be other temperature conditions as long as the bacteria grow. The pH of the medium during the culture is preferably maintained at 6.0 to 7.0, but may be other pH conditions as long as the bacteria grow. It can also be cultured under batch culture conditions. The culture time is usually preferably 10 to 24 hours, but may be any other culture time as long as the bacteria can grow.

<Defrost temperature>
In the present invention, the temperature for thawing the frozen lactic acid bacteria is in the range of 20 ° C to 50 ° C. As long as the temperature for thawing the frozen lactic acid bacteria is in the range of 20 ° C. to 50 ° C., the thawing temperature may be constant, or the thawing temperature may be variable instead of being constant. The constant thawing temperature within the range of 20 ° C. to 50 ° C. means that the lactic acid bacterium is kept at a certain temperature (for example, 25 ° C.) within the above temperature range until the frozen lactic acid bacteria are thawed. However, an error of ± 0.5 ° C. may be included. For example, when setting a certain fixed temperature to 25 degreeC, the range of 24.5 degreeC-25.5 degreeC is said.
Conventionally, thawing of frozen lactic acid bacteria was generally performed by thawing with running water in order to quickly pass -15 ° C to -30 ° C (ice crystal growth temperature range), which is considered to cause cell damage. The thawing temperature was about 15 ° C.
In the present invention, it is possible to remarkably improve the survival of lactic acid bacteria by thawing frozen lactic acid bacteria under the above thawing temperature within the specific temperature range, and to improve the vitality of the lactic acid bacteria starter. Based on finding for the first time.
The temperature for thawing the frozen lactic acid bacteria is preferably 21 ° C or higher, more preferably 23 ° C or higher, and further preferably 25 ° C or higher. Moreover, it is preferably 40 ° C. or lower, more preferably 35 ° C. or lower, and further preferably 30 ° C. or lower.

  The thawing method is not particularly limited as long as the frozen lactic acid bacterium can be thawed at a temperature within the range of 20 ° C. to 50 ° C., for example, a constant temperature water bath (manufactured by Synics Co., Ltd., multithermal unit (CH-150i )) To thaw frozen lactic acid bacteria.

<Thawing time>
The time for thawing the frozen lactic acid bacteria is not particularly limited as long as the frozen lactic acid bacteria can be thawed, and may be appropriately adjusted according to the number of frozen lactic acid bacteria, the volume of the lactic acid bacteria culture, etc. As mentioned above, Preferably it is 0.8 hours or more, More preferably, it is 1 hour or more. For example, it is 5 hours or less, preferably 3 hours or less, more preferably 2 hours or less.

[Method for producing food and beverage composition containing lactic acid bacteria]
This invention also provides the method of manufacturing the food-drinks composition containing lactic acid bacteria using the thawing | decompressing lactic acid bacteria obtained through the thawing | decompression process which thaws frozen lactic acid bacteria.
That is, a method for producing a food or beverage composition containing lactic acid bacteria, comprising a step of thawing frozen lactic acid bacteria, wherein the thawing step is performed by thawing frozen lactic acid bacteria under a temperature condition within a range of 20 ° C to 50 ° C. The manufacturing method of the food-drinks composition containing the lactic acid bacteria which is the process to perform is provided.
The step of thawing the frozen lactic acid bacteria is a step of thawing the frozen lactic acid bacteria under temperature conditions within the range of 20 ° C to 50 ° C, and is as described above. If the thawed lactic acid bacterium is used, the food / beverage product composition of the present invention can be produced in the same manner as a food / beverage product containing a conventionally known lactic acid bacterium.

In the method for producing a food / beverage product composition containing lactic acid bacteria of the present invention, the number of viable lactic acid bacteria in the whole produced food / beverage product composition is, for example, 3 × 10 ⁷ cfu / g or more, preferably 5 ×. It is preferable to use the thawed lactic acid bacterium so that the concentration is 10 ⁷ cfu / g or more, more preferably 10 × 10 ⁷ cfu / g or more. The number of viable bacteria can be determined according to a conventional method.

  Examples of the form in which the thawed lactic acid bacteria are applied to foods and drinks include fermented milk and beverages containing live lactic acid bacteria, and can be used as other health foods, foods for specified health use, nutritional supplements, and the like. And, to these, food hygiene-acceptable formulations, for example, stabilizers, preservatives, colorants, fragrances, vitamins and the like are added to the phosphorylated polysaccharides as appropriate, mixed, and tableted by a conventional method. , Granular, granular, powder, capsule, liquid, jelly, cream, beverage and other foods.

  Although it does not specifically limit about another component, For example, water, protein, saccharide | sugar, a lipid, vitamins, minerals, organic acid, an organic base, fruit juice, flavors, etc. can be used as a main component.

  Examples of the protein include whole milk powder, skim milk powder, partially skimmed milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, κ-casein, β-lacto Globulin, α-lactalbumin, lactoferrin, soy protein, egg protein, meat protein and other animal and vegetable proteins, hydrolysates thereof (eg, butter, whey minerals, cream, whey, non-protein nitrogen, sialic acid, phospholipids) And various milk-derived components such as lactose).

  Examples of the saccharide include processed starch (in addition to dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and the like.

  Examples of lipids include animal oils such as lard, fish oil, etc., fractionated oils, hydrogenated oils, transesterified oils, and the like, such as palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, and the like. Examples include vegetable oils such as fractionated oil, hydrogenated oil, and transesterified oil.

  Examples of vitamins include vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline. And folic acid.

  Examples of the minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, and whey mineral.

  Examples of the organic acid include malic acid, citric acid, lactic acid, and tartaric acid.

  These components can be used in combination of two or more, and synthetic products and / or foods containing a large amount thereof may be used.

  In this invention, what added the lactic acid bacteria thawed | decompressed on the said predetermined temperature conditions as it is to various food-drinks, raw materials, etc. is good also as the food-drinks composition of this invention, and food-drinks which require fermentation (fermented milk etc.) In the case of), it is good also as a food-drinks composition obtained by mixing the thawed lactic acid bacteria with another raw material, and making it ferment. Manufacture of food / beverage products (fermented milk etc.) which require fermentation can be performed using a conventionally well-known method.

  Here, “fermented milk” may be any of “fermented milk”, “dairy lactic acid bacteria beverage”, and “lactic acid bacteria beverage” defined by the ordinances of yogurt, milk and the like. Specific examples of the yogurt include yogurts such as set type yogurt (solid fermented milk), soft yogurt (paste-like fermented milk), and drink yogurt (liquid fermented milk). Examples of the set type yogurt include plain yogurt. Generally, plain yogurt is manufactured by filling a raw material in a container and then fermenting it (post-fermentation). On the other hand, soft yogurt and drink yogurt are manufactured by filling fermented fermented milk into a container (pre-fermentation) after atomization or homogenization. In the present invention, it can be used in any of the above production methods.

  The method for producing fermented milk by thawing the frozen lactic acid bacteria according to the present invention and producing the fermented milk using the obtained lactic acid bacteria will be specifically described below.

The method for producing fermented milk includes the following steps, for example.
(I) Step of preparing raw milk (ii) Step of thawing frozen lactic acid bacteria under temperature conditions within the range of 20 ° C. to 50 ° C. (iii) Step of sowing and fermenting the thawed lactic acid bacteria in the prepared raw milk

(I) Step of preparing raw milk Raw milk is a liquid containing milk components such as raw milk, raw milk, full fat milk, skimmed milk, whey, and the type thereof is not particularly limited, and is conventionally A well-known thing can be used. Moreover, the method for preparing the raw milk is not particularly limited, and the raw milk is prepared by homogenization, sterilization, cooling, and the like, as in the conventional general fermented milk production process.

(Ii) Step of thawing frozen lactic acid bacteria under temperature conditions in the range of 20 ° C. to 50 ° C. The step of thawing frozen lactic acid bacteria is as described above.

(Iii) Steps of seeding and fermenting thawed lactic acid bacteria to the prepared raw milk The method for seeding and fermenting the thawed lactic acid bacteria to the prepared raw milk is not particularly limited, and conventional fermented milk production The same process can be performed. In a fermentation process, you may use a lactic acid bacteria starter separately as needed. For example, as shown in the Examples, a predetermined container (capacity: 100 g, made of plastic) can be filled and fermentation can be performed in a fermentation chamber at 43 ° C.

  The lactic acid bacteria thawed in (ii) above may be cultured by a conventionally known method before the step (iii) to increase (scale up) the number of bacteria to a necessary range.

  Since the method for producing fermented milk includes the step (ii) of thawing frozen lactic acid bacteria under a temperature condition in the range of 20 ° C. to 50 ° C., the survival of the lactic acid bacteria in the obtained fermented milk is improved.

Furthermore, since the method for producing the fermented milk includes the step (ii) of thawing the frozen lactic acid bacterium under a temperature condition in the range of 20 ° C. to 50 ° C., the lactic acid bacterium thawed by the method is measured by the following measurement method. The vitality estimation time of the lactic acid bacteria starter to be performed can be set to less than 3 hours.
[Measurement method of estimated time of vitality of lactic acid bacteria]
Raw milk: 500.0 g, skim milk powder: 49.8 g, fresh cream: 22.0 g, tap water: 398.2 g are mixed to prepare raw milk. The obtained raw milk is sterilized by heating at 95 ° C. for 5 minutes, and then cooled to 43 ° C. The above-prepared raw milk so that the thawed lactic acid bacteria are 40.5 × 10 ⁸ Lactobacillus bulgaricus and 25.5 × 10 ⁸ Streptococcus thermophilus After inoculation, the cup container (capacity: 100 g, made of plastic) is filled, and the time until the lactic acid acidity reaches 0.7% in a fermentation room at a room temperature of 43 ° C. is measured and used as the vitality estimation time.

  The vitality estimation time of the lactic acid bacteria is preferably within 170 minutes, more preferably less than 160 minutes.

[Food and beverage composition containing lactic acid bacteria]
The food / beverage product composition containing the lactic acid bacterium of the present invention, as described above, contains lactic acid bacteria thawed under specific temperature conditions, thereby improving the survival of the lactic acid bacteria than before, It is a food and beverage composition with high production efficiency obtained by using a starter with improved vitality.

  The form etc. of the food-drinks composition containing the lactic acid bacteria of this invention can apply similarly to what was mentioned above in the term of "the manufacturing method of the food-drinks composition containing lactic acid bacteria".

In the present invention, “survival of lactic acid bacteria” refers to how much viable bacteria are present after producing and storing a food and drink composition containing live lactic acid bacteria. Can be determined according to For example, it can obtain | require by measuring the colony on the culture medium after diluting the food / beverage products used for preservation | save suitably, smearing it on BL culture medium, and carrying out anaerobic culture | cultivation at 37 degreeC for 72 hours. A survival rate can be shown by the ratio of the viable count after preservation | save with respect to the viable count before the preservation | save of the culture solution and food-drinks used for preservation | save.
Specifically, as described later in Examples, for example, BL medium (Eiken Chemical Co., Ltd.) autoclaved at 121 ° C. for 15 minutes: 380 g and equine defibrinated blood (Japan Biotest Laboratories): A plate medium was prepared by mixing 20 g, and a thawed live lactic acid bacteria-containing culture solution was diluted with physiological saline and smeared on the surface. It can be measured as a lactic acid bacterium.
The survival rate of lactic acid bacteria is, for example, the ratio of the number of living lactic acid bacteria after 8 days of producing the food / beverage product composition to the number of living lactic acid bacteria after 1 day of producing the food / beverage product composition, as represented by the following formula: Can be evaluated.
Survival rate (%) = {Number of surviving lactic acid bacteria (cfu / g) 8 days after production of food / beverage composition containing lactic acid bacteria / Number of surviving lactic acid bacteria (cfu / g) 1 day after production of food / beverage composition containing lactic acid bacteria )} × 100

  The survival rate (%) of the lactic acid bacteria is preferably 30% or more, more preferably 40% or more, still more preferably 50% or more, still more preferably 60% or more, and particularly preferably 70% or more.

  EXAMPLES Hereinafter, although an Example and a comparative example are shown and this invention is demonstrated further more concretely, this invention is not limited to these examples.

Test Example 1 Survivability Improvement Test of Lactic Acid Bacteria In this test example, the effect of thawing temperature on the survival of the bacterium was examined for Lactobacillus gasseri OLL2716 (Lactobacillus gasseri OLL2716) strain.

[Example 1-1]
(Thawing of frozen lactic acid bacteria)
Lactobacillus gasseri OLL2716 frozen bacteria were thawed in a constant temperature water bath (manufactured by Synics Co., Ltd., Multi Thermal Unit (CH-150i)) at a water temperature of 25 ° C. The thawing time was 1 hour. In addition, after thawing for 1 hour, it was confirmed that the frozen bacteria of Lactobacillus gasseri OLL2716 was completely thawed.

(Preparation of yogurt)
Raw milk: 610 g, skim milk powder: 20 g, sugar: 36 g, tap water: 107 g were mixed to prepare raw milk (yogurt mix). The prepared raw milk was heated and sterilized at 95 ° C. for 5 minutes, and then cooled to 43 ° C. Thereafter, lactic acid bacteria starter composed of 10% skim milk powder (starter cultured with lactic acid bacteria isolated from Meiji Tokachi Yogurt Co., Ltd., Meiji Co., Ltd.) and 24 g (3% by weight of the total raw milk) were thawed as described above. After inoculating 0.24 g (0.03% by weight of the total amount of raw material milk) of “Lactobacillus gasseri OLL2716 frozen bacteria”, the raw material milk (including lactic acid bacteria starter) was filled into a paper cup container (capacity: 100 g). It was allowed to stand in a fermentation room at 0 ° C. and fermented. Then, when the lactic acid acidity reached 0.7%, the fermentation was terminated, and the yogurt was prepared by cooling to 5 ° C. in a refrigerator.

(Evaluation of survival of Lactobacillus gasseri OLL2716 strain in yogurt)
The yogurt produced by the above-mentioned method was stored at 5 ° C., and the number of bacteria of the Lactobacillus gasseri OLL2716 strain at the time of 1 day and 8 days after the production was counted. The number of bacteria was measured by the following method.

[Bacteria count]
BL medium (Eiken Chemical Co., Ltd.) sterilized by autoclaving at 121 ° C. for 15 minutes: 380 g was mixed with 20 g of equine defibrinated blood (Japan Biotest Laboratories Co., Ltd.) to prepare a plate medium. Then, 100 μL of a sample obtained by diluting the above-prepared yogurt 10 ⁵ times with physiological saline was smeared on the surface, cultured for 72 hours at 37 ° C., and the emerged rough colony was counted as Lactobacillus gasseri OLL2716 strain.
The results are shown in Table 1.

[Survival rate]
Based on the measured Lactobacillus gasseri OLL2716 strain, the survival rate represented by the following formula was calculated.
Survival rate (%) = {Number of surviving lactic acid bacteria (cfu / g) 8 days after production of food / beverage composition containing lactic acid bacteria / Number of surviving lactic acid bacteria (cfu / g) 1 day after production of food / beverage composition containing lactic acid bacteria )} × 100
The results are shown in Table 1 below.

[Example 1-2]
A yogurt was prepared in the same manner as in Example 1-1 except that the thawing time was 3 hours, and the count of bacteria and the survival rate were calculated. In addition, after thawing for 3 hours, it was confirmed that the frozen bacteria of Lactobacillus gasseri OLL2716 were completely thawed.
The results are shown in Table 1 below.

[Example 1-3]
A yogurt was prepared in the same manner as in Example 1-1 except that the frozen bacteria of Lactobacillus gasseri OLL2716 were thawed in a constant temperature water bath at a water temperature of 30 ° C., and the number of bacteria and the survival rate were calculated. In addition, after thawing for 1 hour, it was confirmed that the frozen bacteria of Lactobacillus gasseri OLL2716 was completely thawed.
The results are shown in Table 1 below.

[Example 1-4]
Except that the thawing time was 3 hours, yogurt was prepared in the same manner as in Example 1-3, and the number of bacteria was counted and the survival rate was calculated. In addition, after thawing for 3 hours, it was confirmed that the frozen bacteria of Lactobacillus gasseri OLL2716 were completely thawed.
The results are shown in Table 1 below.

[Example 1-5]
A yogurt was prepared in the same manner as in Example 1-3 except that the thawing time was 5 hours, and the number of bacteria was counted and the survival rate was calculated. In addition, after thawing for 5 hours, it was confirmed that the frozen bacteria of Lactobacillus gasseri OLL2716 was completely thawed.
The results are shown in Table 1 below.

[Example 1-6]
A yoghurt was prepared in the same manner as in Example 1-1 except that the frozen bacteria of Lactobacillus gasseri OLL2716 were thawed in a constant temperature water bath at a water temperature of 40 ° C., and the number of bacteria and the survival rate were calculated. In addition, after thawing for 1 hour, it was confirmed that the frozen bacteria of Lactobacillus gasseri OLL2716 was completely thawed.
The results are shown in Table 1 below.

[Comparative Example 1-1]
A yogurt was prepared in the same manner as in Example 1-1 except that the frozen bacteria of Lactobacillus gasseri OLL2716 were thawed in a constant temperature water bath at a water temperature of 15 ° C., and the number of bacteria and the survival rate were calculated. In addition, after thawing for 1 hour, it was confirmed that the frozen bacteria of Lactobacillus gasseri OLL2716 was completely thawed.
The results are shown in Table 1 below.

As can be seen from the above results, frozen lactic acid bacteria were thawed under temperature conditions within the range of 20 to 50 ° C. (Examples 1-1 to 1-6), and the frozen lactic acid bacteria were maintained at 15 ° C. (Comparative Example) Compared with 1-1), it was found that the survival of the Lactobacillus gasseri OLL2716 strain was significantly improved.
In addition, also when the preservation | save days after manufacture exceeded 8 days, Example 1-1 to 1-6 compared with Comparative Example 1-1 that the survival property with respect to 1 day after manufacture improved notably. confirmed.

Test Example 2 Activity Improvement Test of Lactic Acid Bacteria In this test example, Lactobacillus bulgaricus and Streptococcus thermophilus (Streptococcus thermophilus), which are lactic acid bacteria starters, are improved against the activity of the starter bacteria. The effect of thawing temperature was investigated.

[Example 2-1]
(Thawing of frozen lactic acid bacteria)
Frozen lactic acid bacteria (Lactobacillus bulgaricus OLL1255 (NITE BP-76) strain and Streptococcus thermophilus OLS3294 (NITE P-77) strain isolated from Meiji Bulgaria yogurt LB81 plain (Meiji Co., Ltd.)) at a water temperature of 20 ° C. Thawed in a constant temperature water bath. The thawing time was 1 hour. In addition, after thawing for 1 hour, it was confirmed that the frozen bacteria starter was completely thawed.

(Culture of thawed lactic acid bacteria)
In a stainless steel container, skim milk powder: 100 g and tap water: 870 g were mixed to prepare a medium, sterilized by heating at 95 ° C. for 5 minutes, and then cooled to 40 ° C. Thereafter, the medium was inoculated with 30 g (3% by weight) of the thawed lactic acid bacteria starter. The number of lactic acid bacteria contained in 30 g of this lactic acid bacteria starter is 13.5 × 10 ⁷ cfu / g for Lactobacillus bulgaricus OLL1255 (NITE BP-76) and Streptococcus thermophilus OLS 3294 (NITE P-77). The strain was 85.0 × 10 ⁷ cfu / g. This lactic acid bacteria starter was cultured by allowing it to stand at a temperature of 40 ° C. until the lactic acid acidity reached 0.70%. The medium container after the culture was immersed in cold water, the medium was cooled to 5 ° C., and a new scaled lactic acid bacterium was obtained.

(Preparation of yogurt)
First, raw milk: 500.0 g, skim milk powder: 49.8 g, fresh cream: 22.0 g, tap water: 398.2 g were mixed to prepare raw milk. The obtained raw milk was sterilized by heating at 95 ° C. for 5 minutes, and then cooled to 43 ° C. Subsequently, 40.5 × 10 ⁸ strains of Lactobacillus bulgaricus OLL1255 (NITE BP-76) and 25.5 × 10 ⁸ strains of Streptococcus thermophilus OLS3294 (NITE P-77) were prepared as described above. After inoculating the prepared raw milk so that it becomes individual, it is filled into a cup container (capacity: 100 g, made of plastic), and fermented in a fermentation room at a room temperature of 43 ° C. until the lactic acid acidity reaches 0.7%. A yogurt was prepared. At this time, the time (liveness estimation time) was measured from the inoculation of lactic acid bacteria to raw material milk until the lactic acid acidity reached 0.7%.
The results are shown in Table 2 below.

[Example 2-2]
A new lactic acid bacterium was produced in the same manner as in Example 2-1, except that the frozen lactic acid bacterium was thawed in a constant temperature water bath having a water temperature of 25 ° C., and the vitality estimation time was measured. In addition, after thawing for 1 hour, it was confirmed that the frozen lactic acid bacteria were completely thawed.
The results are shown in Table 2 below.

[Example 2-3]
A new lactic acid bacterium was produced in the same manner as in Example 2-1, except that the frozen lactic acid bacterium was thawed in a constant temperature water bath having a water temperature of 30 ° C., and the vitality estimation time was measured. In addition, after thawing for 1 hour, it was confirmed that the frozen lactic acid bacteria were completely thawed.
The results are shown in Table 2 below.

[Example 2-4]
A new lactic acid bacterium was produced in the same manner as in Example 2-1, except that the thawing time was 3 hours, and the vitality estimation time was measured. Further, after thawing for 3 hours, it was confirmed that the frozen lactic acid bacteria were completely thawed.
The results are shown in Table 2 below.

[Example 2-5]
A new lactic acid bacterium was produced in the same manner as in Example 2-2 except that the thawing time was 3 hours, and the vitality estimation time was measured. Further, after thawing for 3 hours, it was confirmed that the frozen lactic acid bacteria were completely thawed.
The results are shown in Table 2 below.

[Example 2-6]
A new lactic acid bacterium was produced in the same manner as in Example 2-3 except that the thawing time was 3 hours, and the vitality estimation time was measured. Further, after thawing for 3 hours, it was confirmed that the frozen lactic acid bacteria were completely thawed.
The results are shown in Table 2 below.

[Comparative Example 2-1]
A new lactic acid bacterium was produced in the same manner as in Example 2-1, except that the frozen lactic acid bacterium was thawed in a constant temperature water bath at a water temperature of 15 ° C., and the vitality estimation time was measured. In addition, after thawing for 1 hour, it was confirmed that the frozen lactic acid bacteria were completely thawed.
The results are shown in Table 2 below.

  As can be seen from the above results, when the frozen lactic acid bacteria were thawed at 15 ° C. by thawing the frozen lactic acid bacteria under temperature conditions within the range of 20-50 ° C. (Examples 2-1 to 2-6) (Comparative Example) Compared with 2-1), the vitality estimation time of the lactic acid bacteria starter was shortened, and could be less than 3 hours. That is, it was found that thawing the frozen lactic acid bacteria under a temperature condition in the range of 20 to 50 ° C. improves the vitality of the lactic acid bacteria starter and provides high production efficiency.

As Lactobacillus bulgaricus (Lactobacillus bulgaricus), for example, Lactobacillus bulgaricus subspecies subspecies subspecies OLL1255: NITE BP-76 is preferably used .

As the Streptococcus thermophilus (Streptococcus thermophilus), for example, Streptococcus thermophilus OLS3294 (Streptococcus thermophilus OLS3294: NITE P-77) strain is preferably used .

[Example 2-1]
(Thawing of frozen lactic acid bacteria)
Frozen lactic acid bacteria (La Kutobashirusu bulgaricus OLL1255 (NITE BP-76) strain, and Streptococcus thermophilus OLS3294 (NITE P-77) strain) were thawed in water temperature 20 ℃ constant temperature water bath. The thawing time was 1 hour. In addition, after thawing for 1 hour, it was confirmed that the frozen bacteria starter was completely thawed.

Claims (5)

  A method for thawing frozen lactic acid bacteria, wherein the frozen lactic acid bacteria are thawed under temperature conditions in the range of 20 ° C to 50 ° C.   The method for thawing frozen lactic acid bacteria according to claim 1, wherein the lactic acid bacteria are Lactobacillus or Streptococcus.   The lactic acid bacterium is at least one selected from the group consisting of Lactobacillus gasseri, Lactobacillus bulgaricus, and Streptococcus thermophilus. The method for thawing frozen lactic acid bacteria as described.   The lactic acid bacteria, Lactobacillus gasseri OLL2716 (Lactobacillus gasseri OLL2716: FERM BP-6999), Lactobacillus bulgaricus OLL1255 (Lactobacillus delbrueckii subspecies bulgaricus OLL1255: NITE BP-76), and Streptococcus thermophilus OLS3294 (Streptococcus thermophilus OLS3294: NITE P The method for thawing frozen lactic acid bacteria according to claim 1, which is at least one bacterium selected from the group consisting of -77). A method for producing a food or beverage composition containing lactic acid bacteria, comprising a step of thawing frozen lactic acid bacteria,
The method for producing a food or beverage composition containing lactic acid bacteria, wherein the thawing step is a step of thawing frozen lactic acid bacteria under a temperature condition within a range of 20 ° C to 50 ° C.