Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
  • A23C9/00—Milk preparations; Milk powder or milk powder preparations
  • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
  • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
  • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
  • A23L29/269—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of microbial origin, e.g. xanthan or dextran
• • 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; 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

Definitions

• the present invention relates to a method for producing exopolysaccharides and their use.
• Exopolysaccharides (EPS) produced by lactic acid bacteria are known to have various functionalities, such as immunomodulatory effects, prebiotic effects, anticancer effects, and cholesterol-lowering effects (Non-Patent Document 1).
• polysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 (OLL1073R-1) have been shown to activate NK cells (Patent Document 1) and have anti-influenza effects (Patent Document 2). Therefore, efforts have been made to increase the amount of polysaccharides produced by lactic acid bacteria, including OLL1073R-1. For example, there is a medium for Lactobacillus delbrueckii subsp.
• bulgaricus which is an exopolysaccharide-producing lactic acid bacterium, characterized in that formic acid and/or a formate are added to skim milk and/or reduced skim milk, and/or the skim milk and/or skim milk powder are heat-treated to a total concentration of formic acid and/or a formate of 0.4 to 10 mM (Patent Document 3);
• a pH buffer which is an alkali metal salt and a mixture of lactic acid bacteria of Streptococcus thermophilus species and lactic acid bacteria of Lactobacillus delbrueckii subsp.
• bulgaricus species which produce an extracorporeal functional product of lactic acid bacteria are blended, and the milk raw material is fermented while the growth of the lactic acid bacteria of Streptococcus thermophilus species is inhibited by the pH buffer and the growth of the lactic acid bacteria of Lactobacillus delbrueckii subsp. bulgaricus species is promoted.
• a pH buffering agent which is an alkali metal salt of phosphoric acid, in an amount ranging from 0.3% by weight to 0.55% by weight, and a mixture of lactic acid bacteria of the Streptococcus thermophilus species and lactic acid bacteria of the Lactobacillus delbrueckii subsp. bulgaricus species which produce an extracorporeal functional product of lactic acid bacteria, with the pH buffering agent.
• a method for increasing the production of an exofunctional product of lactic acid bacteria is developed in which the production of the exofunctional product of lactic acid bacteria is increased by fermenting a raw milk material while suppressing the growth of the lactic acid bacteria of the Mophilus species and promoting the growth of the Lactobacillus delbrueckii subsp. bulgaricus species, and the amount of EPS is 1.05 to 4.2 times the amount of EPS contained in fermented milk obtained by fermenting the raw milk material without decomposing the lactose contained in the raw milk (Patent Document 5).
• a method for promoting the amount of exopolysaccharide production in a microorganism which comprises subjecting an exopolysaccharide-producing microorganism of the genus Lactococcus to a pulsed electric field treatment, the conditions of which include an integrated value of the pulse width and the total number of pulses per liter of culture liquid
• a method for producing exopolysaccharides which comprises inoculating a lactic acid bacterium capable of producing exopolysaccharides into a medium obtained by saccharifying rice bran obtained by polishing brown rice to a weight polishing ratio of 80% or more with rice koji or a saccharifying enzyme, and fermenting the medium to produce exopolysaccharides
• Patent Document 7 a method for controlling the amount of neutral polysaccharides produced by lactic acid bacteria, which is one of the exopolysaccharides (EPS), which comprises culturing the lactic acid bacteria in
• EPS is a biopolymer secreted by microorganisms, including lactic acid bacteria, to cope with harsh environmental conditions. It is one of the main components involved in the formation of an extracellular biofilm matrix that protects microorganisms from adverse conditions, and the production of EPS by lactic acid bacteria is known to be related to environmental stress (Non-Patent Document 2). It has been reported that exposing the stress-sensitive Bifidobacterium bifidum to a sublethal temperature of 42°C for 100 to 300 seconds significantly increases the resistance of the cells to freeze-drying compared to cells cultured in a conventional bioreactor (Non-Patent Document 3).
• the lag phase is a temporary period in which logarithmic growth is not observed when bacteria are introduced into a new medium, and has been thought to be a preparatory stage for bacteria to take up nutrients and adapt to the new environment.
• Recent studies have shown that the lag phase is a dynamic, organized, adaptive, and evolvable process that protects bacteria from threats and promotes their growth ability, and is widely related to research on bacterial evolution, host-pathogen interactions, antibiotic resistance, environmental biology, molecular microbiology, and food safety (Non-Patent Document 4).
• the effect of temperature on the growth rate of Lactobacillus rhamnosus GG has been reported, and it has been reported that the lag phase of L. rhamnosus GG in milk increased with decreasing temperature at 6 to 41°C (Non-Patent Document 5).
• JP 2005-194259 A Patent No. 5177728
• International Publication WO2011/065300 Patent No. 5971949
• JP 2014-27925 A Patent No. 6209371
• International Publication WO2014-084340 Patent No. 6392668
• JP 2019-62782 A Patent No. 7109895, JP 2022-103317
• JP 2017-2169276 A Patent No. 6621065
• JP 2020-156341 A JP 2022-45812 A JP 2010-104376 A Patent No. 4759643
• Exopolysaccharides Produced by Lactic Acid Bacteria From Biosynthesis to Health-Promoting Properties.
• Exopolysaccharide production by lactic acid bacteria the manipulation of environmental stresses for industrial applications.
• Stochastic exposure to sub-lethal high temperature enhancesexopolysaccharides (EPS) excretion and improves Bifidobact erium bifidum cell survival to freeze-drying.
• the present invention provides the following: [1] treating an exopolysaccharide-producing bacterium under conditions that lengthen the lag period by 10% or more; A method for producing exopolysaccharides, comprising the step of culturing the treated exopolysaccharide-producing bacterium in a medium to produce exopolysaccharides. [2] The method according to 1, wherein the step of producing exopolysaccharide is carried out for 4 to 72 hours. [3] The method according to 1 or 2, wherein the step of producing exopolysaccharide includes a 10% longer induction period.
• exopolysaccharide-producing bacterium is any one selected from the group consisting of bacteria belonging to Lactobacillus delbrueckii, bacteria belonging to Lactococcus lactis, bacteria belonging to Streptococcus thermophilus, and bacteria belonging to Bifidobacterium breve.
• Lactobacillus delbrueckii is a bacterium belonging to Lactobacillus delbrueckii ssp. bulgaricus.
• a composition for utilizing an exopolysaccharide as a functional component comprising an exopolysaccharide-producing bacterium selected from the group consisting of a bacterium belonging to the genus Lactobacillus, a bacterium belonging to the genus Lactococcus, a bacterium belonging to the genus Streptococcus, and a bacterium belonging to the genus Bifidobacterium, which has been treated under conditions that lengthen the induction period by 10% or more.
• composition according to 8, wherein the exopolysaccharide-producing bacterium is any one of a bacterium belonging to Lactobacillus delbrueckii, a bacterium belonging to Lactococcus lactis, a bacterium belonging to Streptococcus thermophilus, and a bacterium belonging to Bifidobacterium breve.
• a composition comprising any one of a bacterium belonging to the genus Lactobacillus delbrueckii having an exopolysaccharide productivity of 30 mg/kg or more, a bacterium belonging to the genus Lactococcus lactis having an exopolysaccharide productivity of 2.2 mg/kg or more, a bacterium belonging to the genus Streptococcus thermophilus having an exopolysaccharide productivity of 69 mg/kg or more, and a bacterium belonging to the genus Bifidobacterium breve having an exopolysaccharide productivity of 4.2 mg/kg or more.
• a culture of Lactobacillus delbrueckii comprising 300 mg/kg or more of exopolysaccharide produced by Lactobacillus delbrueckii, and a composition comprising the same.
• a culture of a bacterium belonging to the genus Lactococcus lactis the culture containing 2.2 mg/kg or more of exopolysaccharides produced by Lactococcus lactis, and a composition containing the same.
• a culture of Streptococcus thermophilus the culture containing 69 mg/kg or more of exopolysaccharide produced by Streptococcus thermophilus, and a composition containing the same.
• a culture of Bifidobacterium breve comprising 4.2 mg/kg or more of exopolysaccharide produced by Bifidobacterium breve, and a composition comprising the same.
• a method for producing fermented milk comprising a step of fermenting raw milk with one or more lactic acid-producing bacteria, including an exopolysaccharide-producing bacterium that has been treated so as to lengthen the induction period by 10% or more, to obtain fermented milk containing exopolysaccharides.
• the method according to 16, wherein the step of producing exopolysaccharides is carried out for 4 to 72 hours.
• the method according to 16 or 17, wherein the step of producing exopolysaccharides comprises a 10% longer induction period.
• the exopolysaccharide-producing bacteria treated to lengthen the lag period by 10% or more include bacteria belonging to L. delbrueckii ssp. bulgaricus, and the exopolysaccharide content of the fermented milk after the fermentation step is 30 mg/kg or more;
• the exopolysaccharide-producing bacteria treated to lengthen the induction period by 10% or more include bacteria belonging to the genus Lactococcus lactis, and the exopolysaccharide content of the fermented milk after the fermentation process is 2.2 mg/kg or more; 19.
• exopolysaccharide-producing bacteria treated to lengthen the lag period by 10% or more comprise bacteria belonging to Streptococcus thermophilus, and the exopolysaccharide content in the fermented milk after the fermentation step is 69 mg/kg or more; or the exopolysaccharide-producing bacteria treated to lengthen the lag period by 10% or more comprise bacteria belonging to Bifidobacterium breve, and the exopolysaccharide content in the fermented milk after the fermentation step is 4.2 mg/kg or more.
• a method for improving the exopolysaccharide-producing ability of an exopolysaccharide-producing bacterium which comprises treating the bacterium under conditions that lengthen the induction period.
• the production method according to any one of items 1 to 7 and 16 to 21, or the method according to item 22, wherein the treatment under conditions that prolong the induction period includes any treatment selected from the group consisting of heat treatment, osmotic pressure treatment, and pH treatment.
• the present invention also provides the following: [1] treating an exopolysaccharide-producing bacterium under conditions that lengthen the lag period by 10% or more; A method for producing exopolysaccharides, comprising the step of culturing the treated exopolysaccharide-producing bacterium in a medium to produce exopolysaccharides. [2] The method according to 1, further comprising at least one of a step of concentrating the exopolysaccharide and a step of purifying the exopolysaccharide.
• exopolysaccharide-producing bacterium is any one selected from the group consisting of bacteria belonging to the genus Lactobacillus, bacteria belonging to the genus Lactococcus, bacteria belonging to the genus Streptococcus, and bacteria belonging to the genus Bifidobacterium.
• exopolysaccharide-producing bacterium is any one selected from the group consisting of bacteria belonging to Lactobacillus delbrueckii, bacteria belonging to Lactococcus lactis, bacteria belonging to Streptococcus thermophilus, and bacteria belonging to Bifidobacterium breve.
• Lactobacillus delbrueckii is a bacterium belonging to Lactobacillus delbrueckii ssp. bulgaricus.
• a composition for utilizing an exopolysaccharide as a functional component comprising an exopolysaccharide-producing bacterium selected from the group consisting of a bacterium belonging to the genus Lactobacillus, a bacterium belonging to the genus Lactococcus, a bacterium belonging to the genus Streptococcus, and a bacterium belonging to the genus Bifidobacterium, which has been treated under conditions that lengthen the induction period by 10% or more.
• composition according to 6, wherein the exopolysaccharide-producing bacterium is any one of a bacterium belonging to Lactobacillus delbrueckii, a bacterium belonging to Lactococcus lactis, a bacterium belonging to Streptococcus thermophilus, and a bacterium belonging to Bifidobacterium breve.
• a composition comprising any one of a bacterium belonging to the genus Lactobacillus delbrueckii having an exopolysaccharide productivity of 30 mg/kg or more, a bacterium belonging to the genus Lactococcus lactis having an exopolysaccharide productivity of 2.2 mg/kg or more, a bacterium belonging to the genus Streptococcus thermophilus having an exopolysaccharide productivity of 69 mg/kg or more, and a bacterium belonging to the genus Bifidobacterium breve having an exopolysaccharide productivity of 4.2 mg/kg or more.
• a culture of Lactobacillus delbrueckii containing 300 mg/kg or more of exopolysaccharides produced by Lactobacillus delbrueckii and a composition containing the same.
• a culture of a bacterium belonging to the genus Lactococcus lactis the culture containing 2.2 mg/kg or more of exopolysaccharides produced by the bacterium belonging to the genus Lactococcus lactis, and a composition containing the same.
• a culture of Streptococcus thermophilus the culture containing 69 mg/kg or more of exopolysaccharides, and a composition containing the same.
• a culture of Streptococcus thermophilus the culture containing 69 mg/kg or more of exopolysaccharides produced by Streptococcus thermophilus, and a composition containing the same.
• a culture of Bifidobacterium breve, the culture containing 4.2 mg/kg or more of exopolysaccharide, and a composition containing the same Preferably, a culture of Bifidobacterium breve, the culture containing 4.2 mg/kg or more of exopolysaccharide produced by Bifidobacterium breve, and a composition containing the same.
• a method for producing fermented milk comprising a step of fermenting raw milk with one or more lactic acid-producing bacteria, including an exopolysaccharide-producing bacterium that has been treated so as to lengthen the induction period by 10% or more, to obtain fermented milk containing exopolysaccharides.
• the exopolysaccharide-producing bacteria treated to lengthen the lag period by 10% or more include bacteria belonging to L. delbrueckii ssp. bulgaricus, and the exopolysaccharide content of the fermented milk after the fermentation step is 30 mg/kg or more;
• the exopolysaccharide-producing bacteria treated to lengthen the induction period by 10% or more include bacteria belonging to the genus Lactococcus lactis, and the exopolysaccharide content of the fermented milk after the fermentation process is 2.2 mg/kg or more; 15.
• exopolysaccharide-producing bacteria treated to lengthen the lag period by 10% or more comprise bacteria belonging to Streptococcus thermophilus, and the exopolysaccharide content in the fermented milk after the fermentation step is 69 mg/kg or more; or the exopolysaccharide-producing bacteria treated to lengthen the lag period by 10% or more comprise bacteria belonging to Bifidobacterium breve, and the exopolysaccharide content in the fermented milk after the fermentation step is 4.2 mg/kg or more.
• the lactic acid-producing bacteria further include bacteria belonging to Streptococcus thermophilus.
• lactic acid-producing bacteria further include bacteria belonging to L. bulgaricus.
• a method for improving the exopolysaccharide-producing ability of an exopolysaccharide-producing bacterium comprising treating the bacterium under conditions that lengthen the induction period.
• the treatment under conditions that prolong the induction period includes any treatment selected from the group consisting of heat treatment, osmotic pressure treatment, and pH treatment.
• exopolysaccharides from exopolysaccharide-producing bacteria.
• the exopolysaccharide-producing ability of an exopolysaccharide-producing bacterium can be increased by a simple treatment. By increasing the amount of exopolysaccharide, the desired function in food can be improved.
• the present invention will be described in detail below based on an embodiment of the present invention (referred to as the present embodiment).
• the following embodiment is an example for explaining the present invention, and is not intended to limit the present invention to this embodiment alone.
• the type and number are arbitrary. Unless otherwise specified, percentages regarding content and concentration are based on mass.
• the present embodiment relates to a method for producing an exopolysaccharide, the method comprising the steps of treating an exopolysaccharide-producing bacterium under conditions that lengthen the induction period; and culturing the treated bacterium in a medium to produce an exopolysaccharide. Regarding.
• the exopolysaccharide-producing bacteria is not particularly limited as long as it is a bacterium capable of producing exopolysaccharide (EPS), but is preferably a lactic acid bacterium.
• EPS exopolysaccharide
• lactic acid bacteria are used as an example of exopolysaccharide-producing bacteria, but the explanation also applies to exopolysaccharide-producing bacteria other than lactic acid bacteria.
• the exopolysaccharide-producing bacteria used in this embodiment are not particularly limited as long as they are used in food production and have the ability to produce exopolysaccharide (EPS).
• They may be bacilli (such as bacteria belonging to the genus Lactobacillus) or cocci (such as bacteria belonging to the genus Lactococcus, Leuconostoc, Bediococcus, or Streptococcus). They may also be bacteria belonging to the genus Bifidobacterium. In this embodiment, only one type of exopolysaccharide-producing bacteria may be used, or two or more types may be used.
• the exopolysaccharide-producing bacteria are selected from bacteria belonging to the genus Lactobacillus, bacteria belonging to the genus Lactococcus, bacteria belonging to the genus Streptococcus, bacteria belonging to the genus Thermophilus, and bacteria belonging to the genus Bifidobacterium.
• Lactobacillus bacteria include Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus acidophilus, and Lactobacillus plantarum.
• bacteria belonging to the genus Lactobacillus refers to the bacterium described in the paper "A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of La” in INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, Volume 70, Issue 4, published on April 15, 2020.
• the exopolysaccharide-producing bacteria may be bacteria belonging to the genus Lactobacillus, excluding the genus Lacticaseibacillus, particularly Lacticaseibacillusrhamnosus, and more specifically Lactobacillus rhamnosus GG described in Non-Patent Document 5 (which is classified as Lacticaseibacillusrhamnosus after reorganization based on the above paper).
• bacteria belonging to the genus Lactobacillus are preferred, and bacteria belonging to Lactobacillus delbrueckii subsp. bulgaricus are even more preferred.
• the exopolysaccharide-producing bacterium is Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 (accession number: FERM BP-10741) (sometimes referred to as OLL1073R-1).
• OLL1073R-1 has been internationally deposited with the National Institute of Technology and Evaluation (IPOD, NITE) (2-5-8 Kazusa Kamatari, Kisarazu, Chiba, Japan) in accordance with the Budapest Treaty (depositor: Meiji Co., Ltd., date of deposit: November 29, 2006, accession number: FERM BP-10741).
• Lactobacillus delbrueckii or Lactobacillus delbrueckii subsp. bulgaricus include the following. Lactobacillus delbrueckiisubsp. bulgaricus OLL205013 (NITE BP-02411), Lactobacillus delbrueckiisubsp. bulgaricus OLL1171 (NITE BP-01569), Lactobacillus delbrueckiiOLL204989 (NITE BP-02874)
• Another example of a preferred exopolysaccharide-producing bacterium is any one selected from the group consisting of bacteria belonging to Lactococcus lactis, bacteria belonging to Streptococcus thermophilus, and bacteria belonging to Bifidobacterium breve.
• Lactococcus lactis examples include: Lactococcus lactis spp. lactis OLS3789 (NITE BP-1387)
• Streptococcus thermophilus examples include: Streptococcus thermophilus OLS3618 (NITE BP-01815), Streptococcus thermophilus OLS 3290 (FERM BP-19638)
• Examples of preferred strains belonging to Bifidobacterium breve include the following: B. breve JCM1192 T JCM1192 T is the type strain and is available from the Japan Collection of Microorganisms (JCM), RIKEN BioResource Research Center.
• the method for producing EPS in this embodiment includes a step of pretreating the exopolysaccharide-producing bacteria under conditions that lengthen the lag phase before culturing the bacteria in a specific medium.
• the present inventors have found that, in culturing an exopolysaccharide-producing bacteria, pretreating the bacteria before culturing, specifically exposing the bacteria to conditions that lengthen the lag phase in the culture, increases the concentration of EPS produced in the culture solution compared to the untreated case, and that there is a positive correlation between the length of the lag phase and the concentration of EPS in the culture solution (see Figures 1 to 6). Note that "untreated” can also be referred to as "untreated.”
• EPS is a biopolymer secreted by microorganisms, including lactic acid bacteria, to cope with harsh environmental conditions. It is one of the main components involved in the formation of an extracellular biofilm matrix that protects microorganisms from adverse conditions, and the production of EPS by lactic acid bacteria is known to be related to environmental stress (Non-Patent Document 2, cited above). It has been reported that exposing the stress-sensitive Bifidobacterium bifidum to a sublethal temperature of 42°C for 100 to 300 seconds significantly increases the resistance of the cells to freeze-drying compared to cells cultured in a conventional bioreactor (Non-Patent Document 3, cited above).
• the lag phase generally refers to the temporary period during which no logarithmic growth is observed when bacteria are introduced into a new medium. Recent studies have shown that the lag phase is a dynamic, organized, adaptive, and evolvable process that protects bacteria from threats and promotes their growth potential, and is widely relevant to the study of bacterial evolution, host-pathogen interactions, antibiotic resistance, environmental biology, molecular microbiology, and food safety (Non-Patent Document 4, cited above).
• whether a certain pretreatment condition can be said to be a condition that lengthens the induction period can be said to be a condition that lengthens the induction period if, when a bacterium treated under that condition is cultured, the time required for the pH of the culture medium to drop to a certain degree (induction period) is longer in the early stages of cultivation (some time from the start of cultivation) than when the bacterium is not treated. More specifically, the time required for the pH of the culture medium to drop to a certain degree can be the time required for the pH of the culture medium at the start of cultivation to drop by 0.2.
• the pH of the culture medium at the start of cultivation can be the pH of a fresh medium.
• pH can vary depending on temperature, but when a pH value is indicated in relation to the present invention, it is the value measured at the temperature during cultivation of the exopolysaccharide-producing bacterium, unless otherwise specified. This temperature is often 40°C, and in the case of Lactococcus lactis, it is 30°C.
• the culture for determining the time required for the treated bacteria to change the pH by 0.2 from the initial pH level and the culture for determining the time required for the untreated bacteria to change the pH by 0.2 from the initial pH level are performed using the same medium and under the same conditions.
• the medium and culture conditions may be those standardly used for untreated bacteria or those used for the production of EPS.
• Examples of media that can be used when determining the retardation rate are MRS medium for bacteria belonging to L. delbrueckii, MRS medium for bacteria belonging to L. lactis, M17 medium for bacteria belonging to S. thermophilus, and GAM medium for bacteria belonging to B. breve.
• a skimmed milk medium may be used if appropriate.
• the above can be achieved by treating the exopolysaccharide-producing bacteria under conditions that lengthen the lag period of the exopolysaccharide-producing bacteria.
• the conditions that lengthen the lag period of the exopolysaccharide-producing bacteria include high temperature treatment, low temperature treatment, high osmotic pressure treatment, low osmotic pressure treatment, low pH treatment, high pH treatment, high pressure treatment, drying treatment, freezing treatment, freeze-thawing treatment, nutrient deficiency treatment (starvation of nutrients such as nitrogen, sugar, and carbon dioxide), nutrient oversupply, CO2 treatment, oxidative load, co-culture treatment, chemical substance treatment, antibiotic treatment, cell wall decomposition enzyme treatment, antimicrobial peptide treatment, ultrasonic treatment, and ultraviolet irradiation treatment. Only one or more of the conditions may be used to lengthen the lag period of the exopolysaccharide-producing bacteria.
• the induction period is lengthened by any of the following treatments, which are known to have a relatively high desired effect and are easy to implement: heat (high temperature) treatment, osmotic pressure (high osmotic pressure) treatment, and pH (high pH) treatment.
• heat high temperature
• osmotic pressure high osmotic pressure
• pH high pH
• the condition that lengthens the lag period by 10% or more is heat (high temperature) treatment, regardless of which exopolysaccharide-producing bacterium is used and regardless of which medium is used in the culture after the treatment.
• the temperature is preferably 45°C or higher, more preferably 50°C or higher, and even more preferably 58°C or higher.
• the upper limit of the temperature is preferably 86°C or lower, more preferably 84°C or lower, and even more preferably 82°C or lower.
• the temperature is preferably 40°C or higher, more preferably 45°C or higher, and even more preferably 50°C or higher.
• the upper limit of the temperature is preferably 76°C or lower, more preferably 74°C or lower, even more preferably 72°C or lower, and especially preferably 70°C or lower.
• the temperature is preferably 50 to 86°C, and more preferably 58 to 82°C. If the bacteria belong to Lactococcus lactis, the temperature is preferably 45 to 70°C, and more preferably 50 to 70°C.
• the treatment time can be appropriately determined depending on the temperature.
• the treatment time is preferably 0.5 minutes or more, preferably 1 minute or more, preferably 2 minutes or more, preferably 3 minutes or more, more preferably 4 minutes or more, and even more preferably 8 minutes or more.
• the upper limit is preferably 20 minutes or less, more preferably 18 minutes or less, preferably 16 minutes or less, and even more preferably 14 minutes or less.
• the treatment time is preferably 0.5 minutes or more, preferably 1 minute or more, more preferably 2 minutes or more, and even more preferably 2.5 minutes or more.
• the upper limit is preferably 6 minutes or less, more preferably 5 minutes or less, and even more preferably 4 minutes or less.
• the processing time is preferably 0.25 seconds or more, and can be, for example, 0.5 minutes or more, 0.75 minutes or more.
• the upper limit is preferably 6 minutes or less, and can be, for example, 5 minutes or less, 4 minutes or less, or 3 minutes or less.
• the treatment time is preferably 0.5-20 minutes, more preferably 2.5-18 minutes, and even more preferably 3-10 minutes.
• the treatment time is preferably 0.5-6 minutes, more preferably 0.5-16 minutes, and even more preferably 1-3 minutes.
• the treatment time is preferably 0.5-2 minutes, and more preferably 0.5-1 minute.
• the condition for lengthening the lag period by 10% or more is high osmotic pressure treatment (high salt concentration treatment) regardless of which exopolysaccharide-producing bacterium is used and which medium is used in the culture after the treatment.
• the salt concentration is preferably 0.8% or more, more preferably 3% or more, and even more preferably 5% or more.
• the upper limit is preferably 20% or less, more preferably 16% or less, and even more preferably 12% or less.
• the treatment time can be appropriately determined depending on the salt concentration.
• the treatment time is preferably 1 hour or more, more preferably 2 hours or more, and even more preferably 3 hours or more.
• the upper limit is preferably 9 hours or less, more preferably 7 hours or less, and even more preferably 3 hours or less.
• the condition for lengthening the lag period by 10% or more is high pH treatment, regardless of which exopolysaccharide-producing bacterium is used and which medium is used in the culture after the treatment.
• the high pH treatment can be performed by setting the pH at the start of the culture high.
• the pH at the start of the culture is preferably greater than 6.3, more preferably 6.5 or more, and even more preferably 6.8 or more.
• the upper limit is preferably 7.4 or less, more preferably 7.2 or less, and even more preferably 7.0 or less.
• Examples of treatments other than heat, osmotic pressure, and pH that are believed to have a relatively high desired effect include treatments using any one selected from the group consisting of ultrasound, ultraviolet light, high pressure, cell wall-decomposing enzymes, antimicrobial peptides, low temperature, and freezing and thawing. It is believed that the desired effect cannot be obtained by exposing the exopolysaccharide-producing bacteria to a low temperature, for example, 21° C. or lower, for a short period of time, for example, a few minutes, but the desired effect can be expected from treatment of exposing the bacteria to a low temperature for several hours, for example, 4 hours or more.
• the step of pretreating the exopolysaccharide-producing bacteria does not include refrigerated storage, freezing, and freeze-drying, or is a treatment other than these.
• the induction period can also be lengthened by adding a relatively small amount of bacteria to the medium at the start of cultivation and by culturing at a relatively low temperature, but adding a small amount to the medium and culturing at a relatively low temperature do not constitute a step of pretreating the exopolysaccharide-producing bacteria.
• the production method of this embodiment includes a step of culturing in a medium an exopolysaccharide-producing bacterium that has been treated under conditions that lengthen the lag period by 10% or more to produce exopolysaccharide.
• the conditions for this step can be the same as those for culturing an untreated bacterium to produce EPS.
• the medium for producing EPS may be a medium containing a milk ingredient or a synthetic medium.
• a medium containing a milk ingredient can be used.
• the method for producing fermented milk will be described later.
• the synthetic medium can be selected depending on the exopolysaccharide-producing bacteria used.
• the culture may be performed under anaerobic or aerobic conditions.
• the dissolved oxygen concentration of the medium is not particularly limited as long as a lag period extended by 10% or more is ensured. Although it depends on the bacteria used, it is considered that a low dissolved oxygen concentration promotes the growth of lactic acid bacteria and shortens the lag period (Patent Document 9). Therefore, from the viewpoint of ensuring a long lag period, it is better that the dissolved oxygen concentration of the medium is not too low.
• the dissolved oxygen concentration of the medium at the start of culture is 1 ppm or more, and can be 2 ppm or more, 3 ppm or more, 4 ppm or more, 5 ppm or more, more than 5 ppm, 5.1 ppm or more, 5.2 ppm or more, 5.3 ppm or more, 5.4 ppm or more, or 5.5 ppm or more.
• the dissolved oxygen concentration of the medium at the start of culture can be 10 ppm or less, regardless of other culture conditions, and can be 9 ppm or less, 8 ppm or less, 7 ppm or less, or 6 ppm or less.
• the method for producing exopolysaccharides of this embodiment does not include a treatment or step for reducing dissolved oxygen in the medium.
• the culture temperature is preferably 37 to 43°C
• the culture time is preferably 4 to 72 hours from the viewpoint of the proliferation of the exopolysaccharide-producing bacteria and the EPS produced, and may be, for example, 4 to 48 hours, 4 to 36 hours, 4 to 24 hours, 6 to 72 hours, 6 to 48 hours, 6 to 36 hours, 6 to 24 hours, 8 to 72 hours, 8 to 48 hours, 8 to 36 hours, 8 to 24 hours, 12 to 72 hours, 12 to 48 hours, 12 to 36 hours, 12 to 24 hours, 14 to 72 hours, 14 to 48 hours, 14 to 36 hours, 14 to 24 hours, 16 to 36 hours, or 16 to 24 hours. If the culture time is 4 hours or more, sufficient EPS can be obtained.
• the pH of the culture medium is preferably 3.5 to 7.5 throughout the culture period, more preferably 4.5 to 7.0, and even more preferably 5.5 to 6.5.
• the pH of the culture medium of an exopolysaccharide-producing bacterium usually decreases over the culture time, so neutralizing the culture medium during culture to maintain the pH may increase the amount of EPS produced.
• the step of producing exopolysaccharides in the manufacturing method of this embodiment may include an induction period that is 10% or more longer.
• An induction period that is 10% or more longer means an induction period that is 10% or more longer than when the same bacteria without pretreatment are cultured under the same conditions.
• the induction period here may be the period during which no logarithmic growth is observed temporarily, as seen in bacteria introduced into a new medium, or the time required for the pH at the start of culture to decrease to a predetermined level, for example, 0.2.
• the process of producing exopolysaccharides preferably includes an lag period that is 10% or more longer, more preferably includes an lag period that is 30% or more longer, and even more preferably includes an lag period that is 90% or more longer.
• the induction period can be lengthened by relatively reducing the amount of lactic acid bacteria added to the medium at the start of culture (Patent Document 9) and by culturing at a relatively low temperature (Non-Patent Document 5), but the step of producing exopolysaccharides in the production method of this embodiment, in one aspect, does not include an induction period lengthened by reducing the amount of bacteria added to the medium at the start of culture, regardless of the bacteria used or other culture conditions (or includes an induction period lengthened by means other than reducing the amount of bacteria added to the medium at the start of culture), and does not include an induction period lengthened by culturing at a relatively low temperature (or includes an induction period lengthened by means other than culturing at a relatively low temperature).
• the step of producing exopolysaccharides in the production method of this embodiment does not include an aspect in which raw milk with reduced lactose is used in advance, regardless of the bacteria used or other culture conditions (or exopolysaccharides are increased by means other than using raw milk with reduced lactose in advance).
• the manufacturing method of this embodiment may include steps other than those described above. Examples of such steps include a step of filtering the culture solution obtained by culturing the bacteria, a step of centrifuging, a step of membrane separation, a step of sterilization, a step of concentrating, a step of purifying the produced EPS, a step of drying, etc.
• the purification step may include: 1. Remove the cells from the culture by centrifugation. 2. Add trichloroacetic acid to a final concentration of about 5-10% by weight to precipitate proteins, then centrifuge. 3. High molecular weight polysaccharides and proteins are recovered as precipitates by ethanol precipitation. 4. Remove proteins and nucleic acids. a) Degrade nucleic acids using DNase and RNase. b) Proteinase is used to break down proteins. c) The protein is heat denatured, followed by centrifugation and dialysis. 5. After the acidic polysaccharides are adsorbed by an anion exchange resin, they are eluted and recovered.
• the neutral polysaccharides can be isolated by the method described in JP-A-2000-247895 and, if necessary, purified and used.
• EPS can also be isolated by the following procedure. 1. Add trichloroacetic acid to the medium to a final concentration of 10% by weight to denature proteins. 2. Remove denatured proteins and cells from the culture by centrifugation. 3. High molecular weight polysaccharides are precipitated and collected by ethanol precipitation. 4. Acidic polysaccharides are adsorbed using an anion exchange resin, and neutral polysaccharides are recovered from the remaining eluate. 5. Degrade nucleic acids by treatment with DNase or RNase. 6. Degrade proteins by proteinase treatment. 7. Heat at 90°C for 10 minutes to inactivate the enzyme. 8. Purify the neutral polysaccharide by ethanol precipitation and dialysis.
• EPSs can be produced by the exopolysaccharide-producing bacteria in the production method of this embodiment.
• the EPSs produced by the exopolysaccharide-producing bacteria are structurally classified into homopolysaccharides and heteropolysaccharides (e.g., those composed of galactose and glucose) and may be modified by phosphorylation, sulfation, or the like, but any of them can be produced in the production method of this embodiment.
• the EPS comprises at least one of neutral polysaccharides and acidic polysaccharides in which phosphate groups have been added to neutral polysaccharides.
• Such EPS are known to be produced by Lactobacillus delbrueckii subsp. bulgaricus, Lactococcus lactis spp. lactis, and Lactococcus lactis subsp. cremoris.
• the EPS may include an acidic exopolysaccharide having a repeating structure in which repeating units represented by the following formula (I) are linked together:
• n represents an integer of 0 or 1, independently for each repeating unit.
• each repeating unit represented by formula (I) constituting the acidic EPS may or may not have one glycerol 3-phosphate group, but the acidic polysaccharide as a whole has at least one glycerol 3-phosphate group added.
• the acidic polysaccharide may have a repeating structure represented by the following formula (II), in which the repeating units represented by formula (I) are linked together.
• n is an integer of 0 or 1, and is independent for each repeating unit.
• m is an integer and may be 1 to 300, or may be 1 to 200.
• ⁇ -D-Galp represents a pyranose-type ⁇ -D-galactose residue
• ⁇ -D-Galp represents a pyranose-type ⁇ -D-galactose residue
• ⁇ -D-Galf represents a furanose-type ⁇ -D-galactose residue
• Gro3P represents a glycerol 3-phosphate group.
• (1-2), (1-3), (1-5), and (1-6) represent the 1-2 bond (i.e., the 1-position carbon-2-position carbon bond), 1-3 bond, 1-5 bond, and 1-6 bond, respectively, between residues.
• a particularly preferred embodiment of the method for producing exopolysaccharides of this embodiment is described below, in which a specific exopolysaccharide-producing bacterium is used and cultured for a specific period of time.
• a method for producing exopolysaccharides comprising the step of culturing the treated exopolysaccharide-producing bacteria in a medium for 4 to 48 hours to produce exopolysaccharides.
• the culture time may be 4 to 36 hours, 4 to 24 hours, 6 to 48 hours, 6 to 36 hours, 6 to 24 hours, 8 to 48 hours, 8 to 36 hours, 8 to 24 hours, 12 to 48 hours, 12 to 36 hours, 12 to 24 hours, 14 to 48 hours, 14 to 36 hours, 14 to 24 hours, 16 to 36 hours, or 16 to 24 hours.
• the present embodiment relates to a composition comprising an exopolysaccharide-producing bacterium treated under conditions that lengthen the lag period by 10% or more, and an exopolysaccharide-producing bacterium with an EPS productivity increased by about 5% compared to conventional bacteria.
• bacteria with an EPS productivity increased by about 5% compared to conventional bacteria can be obtained.
• the exopolysaccharide-producing bacteria of this embodiment can be specified as follows based on their exopolysaccharide-producing ability (hereinafter also referred to as EPS-producing ability) when a skim milk powder medium is used.
• EPS-producing ability exopolysaccharide-producing ability
• a bacterium belonging to the genus Lactobacillus preferably a bacterium belonging to Lactobacillus delbrueckii, more preferably a bacterium belonging to Lactobacillus delbrueckii ssp. bulgaricus, and even more preferably Lactobacillus delbrueckii ssp.
• bulgaricus OLL1073R-1 (FERM BP-10741), having an EPS production ability of more than 125 mg/kg, preferably 150 mg/kg or more, more preferably 175 mg/kg or more, and even more preferably 300 mg/kg or more; a bacterium belonging to the genus Lactococcus, preferably a bacterium belonging to Lactococcus lactis spp. lactis, more preferably Lactococcus lactis spp.
• lactis OLS3789 (NITE BP-1387), having an EPS-producing ability of 2.2 mg/kg or more, preferably 5 mg/kg or more, more preferably 10 mg/kg or more, and even more preferably 15 mg/kg or more; a bacterium belonging to the genus Streptococcus, preferably a bacterium belonging to Streptococcus thermophilus, more preferably Streptococcus thermophilus OLS 3290 (FERM BP-19638), having an EPS production ability of 69 mg/kg or more, preferably 72 mg/kg or more, more preferably 75 mg/kg or more, and even more preferably 80 mg/kg or more; A bacterium belonging to the genus Bifidobacterium, preferably a bacterium belonging to Bifidobacterium breve, more preferably Bifidobacterium breve JCM1192T, having an EPS production ability of 4.2 mg/kg or more, preferably 7 mg/kg or more, more preferably
• the concentration or amount of EPS refers to the concentration or amount measured by the phenol-sulfuric acid method (Ziadi et al., BioMed Research International, (2016) Vol. 2018, doi: 10.1155/2018/1896240.), unless otherwise specified. Detailed examples of the conditions for the phenol-sulfuric acid method are described in the Examples section of this specification.
• the concentration or amount of EPS in the target substance may be determined by converting a value measured by a method other than the phenol-sulfuric acid method into a value measured by the phenol-sulfuric acid method, as necessary.
• exopolysaccharide-producing bacteria can be identified as follows based on their ability to produce EPS when a skim milk powder medium is used.
• Lactobacillus delbrueckii subsp. bulgaricus OLL205013 (NITE BP-02411), which has an EPS production ability of 2.1 mg/kg or more, preferably 5 mg/kg or more, more preferably 7 mg/kg or more, and even more preferably 7.5 mg/kg or more; Lactobacillus delbrueckii subsp.
• bulgaricus OLL1171 (NITE BP-01569), which has an EPS production ability of 16.4 mg/kg or more, preferably 20 mg/kg or more, more preferably 24 mg/kg or more, and even more preferably 28 mg/kg or more; Lactobacillus delbrueckii OLL204989 (NITE BP-02874), which has an EPS productivity of 7.7 mg/kg or more, preferably 20 mg/kg or more, more preferably 30 mg/kg or more, and even more preferably 40 mg/kg or more.
• exopolysaccharide-producing bacteria can be identified as follows based on their ability to produce EPS when a skim milk powder medium is used.
• Streptococcus thermophilus OLS3618 (NITE BP-01815), which has an EPS productivity of 4.4 mg/kg or more, preferably 6 mg/kg or more, more preferably 7.5 mg/kg or more, and more preferably 9 mg/kg or more.
• the exopolysaccharide-producing bacteria of this embodiment can be specified as follows based on their EPS production ability when a synthetic medium is used: a bacterium belonging to the genus Lactobacillus, preferably a bacterium belonging to Lactobacillus delbrueckii, more preferably a bacterium belonging to Lactobacillus delbrueckii ssp. bulgaricus, and even more preferably Lactobacillus delbrueckii ssp.
• bulgaricus OLL1073R-1 having an EPS production ability of 301 mg/kg or more, preferably 305 mg/kg or more, more preferably 310 mg/kg or more, and even more preferably 314 mg/kg or more;
• a bacterium belonging to the genus Lactococcus preferably a bacterium belonging to Lactococcus lactis spp. lactis, more preferably Lactococcus lactis spp. lactis OLS3789 (NITE BP-1387), having an EPS productivity of 32 mg/kg or more, preferably 45 mg/kg or more, more preferably 70 mg/kg or more, and even more preferably 80 mg/kg or more.
• the synthetic medium can be selected depending on the exopolysaccharide-producing bacterium used.
• exopolysaccharide-producing bacteria can be identified as follows based on their ability to produce EPS when a synthetic medium is used.
• Lactobacillus delbrueckii subsp. bulgaricus OLL205013 (NITE BP-02411), which has an EPS productivity of 255 mg/kg or more, preferably 260 mg/kg or more, more preferably 270 mg/kg or more, and further preferably 280 mg/kg or more.
• bulgaricus OLL1171 (NITE BP-01569), which has an EPS productivity of 209 mg/kg or more, preferably 240 mg/kg or more, more preferably 275 mg/kg or more, and even more preferably 305 mg/kg or more.
• Lactobacillus delbrueckii OLL204989 (NITE BP-02874) with an EPS productivity of 218 mg/kg or more, preferably 230 mg/kg or more, more preferably 240 mg/kg or more, and even more preferably 250 mg/kg or more.
• the exopolysaccharide-producing bacteria of this embodiment can be specified as follows based on the length of the lag period when a skim milk powder medium is used.
• a bacterium belonging to the genus Lactobacillus preferably a bacterium belonging to Lactobacillus delbrueckii, more preferably a bacterium belonging to Lactobacillus delbrueckii ssp. bulgaricus, and even more preferably Lactobacillus delbrueckii subsp.
• bulgaricus OLL1171 (NITE BP-01569), having an induction period of 152 minutes or more, preferably 400 minutes or more, more preferably 700 minutes or more, and even more preferably 1000 minutes or more;
• a bacterium belonging to the genus Lactococcus preferably a bacterium belonging to Lactococcus lactis spp. lactis, more preferably Lactococcus lactis spp.
• lactis OLS3789 (NITE BP-1387), having an induction period of 236 minutes or more, preferably 240 minutes or more, more preferably 244 minutes or more, and even more preferably 248 minutes or more;
• a bacterium belonging to the genus Streptococcus preferably a bacterium belonging to Streptococcus thermophilus, more preferably Streptococcus thermophilus OLS3618 (NITE BP-01815), having a length of induction period of 199 minutes or more, preferably 206 minutes or more, more preferably 212 minutes or more, and even more preferably 219 minutes or more;
• a bacterium belonging to the genus Bifidobacterium preferably a bacterium belonging to Bifidobacterium breve, more preferably Bifidobacterium breve JCM1192T, having an induction period of 425 minutes or more, preferably 431 minutes or more, more preferably 436 minutes or more, and even more preferably 442
• the length refers to the time required to change the pH of the target exopolysaccharide-producing bacteria by 0.2 at the initial stage of culture when the bacteria are cultured in a medium containing a milk raw material, unless otherwise specified.
• a medium using a milk raw material it is preferable to use a skim milk medium, and it is more preferable to use a 10% reconstituted skim milk medium (pH 6.5) in any case of using bacteria.
• the 10% reconstituted skim milk medium refers to a medium containing 10% (W/W) skim milk components (reconstituted skim milk) that has been sterilized by an appropriate means, for example, by reaching a temperature of 95°C.
• the composition of the skim milk powder is, for example, 1% fat by mass, 34% protein by mass, 54% lactose by mass, 8% ash by mass, and 96% non-fat milk solids by mass.
• the 10% reconstituted skim milk medium contains, for example, 5.4% lactose by mass and 9.6% non-fat milk solids by mass.
• the conditions and medium for activation culture can be prepared, specifically, according to the method described in the Examples or the description in the section entitled "Exopolysaccharide production ability (EPS production ability)".
• exopolysaccharide-producing bacteria can be identified based on the length of induction period using a skim milk powder medium as follows. Lactobacillus delbrueckiis sp. bulgaricus OLL1073R-1 (FERM BP-10741), the length of the induction period being 126 minutes or more, preferably 380 minutes or more, more preferably 630 minutes or more, and even more preferably 900 minutes or more; Lactobacillus delbrueckii subsp.
• bulgaricus OLL205013 (NITE BP-02411), in which the length of the induction period is 89 minutes or more, preferably 102 minutes or more, more preferably 115 minutes or more, and even more preferably 128 minutes or more; The length of the induction period is 99 minutes or more, preferably 100 minutes or more, more preferably 102 minutes or more, and even more preferably 103 minutes or more. Lactobacillus delbrueckii OLL204989 (NITE BP-02874)
• exopolysaccharide-producing bacteria can be identified as follows based on the length of the lag phase when a skim milk powder medium is used. Streptococcus thermophilus OLS3290 (FERM BP-19638), in which the length of the induction period is 99 minutes or more, preferably 100 minutes or more, more preferably 102 minutes or more, and even more preferably 103 minutes or more.
• an upper limit is set for the induction period from the viewpoint of fermented milk production, etc., it is 1000 minutes or less, preferably 700 minutes or less, and more preferably 300 minutes or less.
• the exopolysaccharide-producing bacteria of this embodiment can be specified as follows based on the length of the lag phase when a synthetic medium is used: A bacterium belonging to the genus Lactobacillus, preferably a bacterium belonging to Lactobacillus delbrueckii, more preferably a bacterium belonging to Lactobacillus delbrueckii ssp. bulgaricus, and even more preferably Lactobacillus delbrueckii subsp.
• bulgaricus OLL1073R-1 (FERM BP-10741), having an induction period of 136 minutes or more, preferably 355 minutes or more, more preferably 574 minutes or more, and even more preferably 793 minutes or more;
• a bacterium belonging to the genus Lactococcus preferably a bacterium belonging to Lactococcus lactis spp. lactis, more preferably Lactococcus lactis spp. lactis OLS3789 (NITE BP-1387), having an induction period of 198 minutes or more, preferably 191 minutes or more, more preferably 194 minutes or more, and even more preferably 196 minutes or more.
• the synthetic medium can be selected depending on the exopolysaccharide-producing bacterium used.
• an upper limit is set for the induction period from the viewpoint of fermented milk production, etc., it is 1000 minutes or less, preferably 700 minutes or less, and more preferably 300 minutes or less.
• exopolysaccharide-producing bacteria can be identified as follows based on the length of the lag phase when a synthetic medium is used.
• Lactobacillus delbrueckii subsp. bulgaricus OLL205013 (NITE BP-02411), in which the length of the induction period is 105 minutes or more, preferably 129 minutes or more, more preferably 152 minutes or more, and even more preferably 176 minutes or more.
• Lactobacillus delbrueckii subsp. bulgaricus OLL1171 (NITE BP-01569), in which the length of the induction period is 115 minutes or more, preferably 167 minutes or more, more preferably 219 minutes or more, and even more preferably 271 minutes or more.
• the length of the induction period is 89 minutes or more, preferably 91 minutes or more, more preferably 92 minutes or more, and even more preferably 93 minutes or more.
• Lactobacillus delbrueckii OLL204989 NITE BP-02874.
• an upper limit is set for the induction period from the viewpoint of fermented milk production, etc., it is 500 minutes or less, preferably 300 minutes or less.
• the EPS production ability of the exopolysaccharide-producing bacteria when a skim milk powder medium is used can be defined as the EPS content (mg/kg) of the culture when a bacterial solution of 1.0 ⁇ 10 4 to 10 ⁇ 10 10 cfu/g is added to the skim milk powder medium to give a concentration of 1% by mass, and the culture is cultured at an optimal temperature for 24 hours (main culture).
• the optimal temperature is around 30°C for Lactococcus lactis spp. lactis and 37 to 43°C for other lactic acid bacteria.
• the skim milk powder medium refers to a 10% skim milk powder medium unless otherwise specified.
• the amount of bacteria added to the milk powder medium or MRS medium is preferably an amount that results in a viable cell count of 2 x 10 to 5 x 10 cfu/g, more preferably an amount that results in a viable cell count of 4 x 10 to 4 x 10 cfu/g, and even more preferably an amount that results in a viable cell count of 1 x 10 to 2 x 10 cfu/g.
• the skim milk medium may be used with the addition of a fermentation promoter as necessary.
• a fermentation promoter for bacteria belonging to the genus Lactobacillus, such as bacteria belonging to L. delbrueckii, inosinic acid is added at an effective concentration; for bacteria belonging to the genus Streptococcus, such as bacteria belonging to S. thermophilus, casein peptides are added at an effective concentration; for bacteria belonging to the genus Bifidobacterium, such as bacteria belonging to B. breve, inosinic acid is added at an effective concentration; and for bacteria belonging to the genus Lactococcus, such as bacteria belonging to L. lactis, yeast extract is added at an effective concentration.
• the skim milk medium does not contain formic acid, malic acid, fumaric acid, or salts thereof.
• the milk-derived raw material used is one in which lactose has not been reduced by lactase (one that does not contain lactase in the raw material).
• the activation culture is preferably performed in a medium suitable for the target exopolysaccharide-producing bacteria at 37°C under anaerobic conditions for 16 to 18 hours (preferably 16 hours).
• the amount of bacteria used in the activation culture is preferably an amount that results in a viable cell count of 1 x 10 4 to 2 x 10 8 cfu/g in the medium suitable for the target bacteria, and more preferably an amount that results in a viable cell count of 1 x 10 4 to 1 x 10 6 cfu/g.
• Examples of media for activation culture include skim milk medium or MRS medium for bacteria belonging to L. delbrueckii and L. lactis, MRS medium for bacteria belonging to L. lactis, M17 medium for bacteria belonging to S. thermophilus, and GAM medium for bacteria belonging to B. breve.
• the EPS production ability can also be defined when a synthetic medium is used.
• the EPS production ability can be defined as the EPS content (mg/kg) of the culture when 1.0 ⁇ 10 4 to 10 ⁇ 10 10 cfu/g of lactic acid bacteria liquid is added to a medium suitable for the target bacteria so as to be 1% by mass, and the culture is cultured at the optimal temperature for 24 hours.
• synthetic media include MRS medium for bacteria belonging to L. delbrueckii, MRS medium for bacteria belonging to L. lactis, M17 medium for bacteria belonging to S. thermophilus, and GAM medium for bacteria belonging to B. breve.
• the synthetic medium does not contain any other components than the standard components.
• composition of the present embodiment can be used to utilize EPS as a functional component (sometimes called an active component).
• EPS produced by lactic acid bacteria is known to have various functionalities, such as immunomodulatory activity, prebiotic activity, anticancer activity, and cholesterol level lowering activity (Non-Patent Document 1, supra). Therefore, in one aspect, a composition containing EPS can be used to utilize EPS for immunomodulation, as a prebiotic, for cancer treatment (risk reduction, prevention, treatment, etc.), and for cholesterol level control.
• the EPS produced by OLL1073R-1 has been shown to have an NK cell activation effect (Patent Document 1, supra) and an anti-influenza effect (Patent Document 2, supra). Therefore, a composition containing OLL1073R-1 can be used to utilize the EPS produced by OLL1073R-1 for immunomodulation, as a prebiotic, for cancer treatment (risk reduction, prevention, treatment, etc.), and for cholesterol level control.
• the composition of this embodiment may include a culture.
• the culture may include lactic acid bacteria and EPS.
• Examples of cultures provided by this embodiment are as follows: A culture of a bacterium belonging to Lactobacillus, preferably a bacterium belonging to Lactobacillus delbrueckii, more preferably a bacterium belonging to Lactobacillus delbrueckii ssp. bulgaricus, even more preferably Lactobacillus delbrueckii ssp.
• bulgaricus OLL1073R-1 which contains 300 mg/kg or more, preferably 304 mg/kg or more, more preferably 308 mg/kg or more, even more preferably 312 mg/kg or more of EPS, preferably EPS produced by the bacterium;
• lactis OLS3789 which contains 2.2 mg/kg or more, preferably 5 mg/kg or more, more preferably 10 mg/kg or more, even more preferably 15 mg/kg or more of EPS, preferably an EPS produced by the bacterium;
• a culture of a bacterium belonging to the genus Bifidobacterium preferably a bacterium belonging to Bifidobacterium breve, more preferably Bifidobacterium breve JCM1192T, which contains 4.2 mg/kg or more, preferably 7 mg/kg or more
• the amount of EPS referred to here is the amount measured by the phenol-sulfuric acid method (due to the nature of the measurement, polysaccharides other than EPS produced by the bacterial cells, such as polysaccharides derived from medium components, may be included).
• the composition may be in the form of a fermented food.
• fermented foods include fermented milk, cheese, fermented products (such as fermented yogurt made from plant-based ingredients) using plant-based raw materials (soy milk, almond milk, coconut milk, etc.), pickles, kimchi, soy sauce, miso, and other fermented foods, and preferably fermented milk, cheese, and fermented products using plant-based raw materials.
• the yogurt may be, for example, plain yogurt, hard yogurt, drink yogurt, soft yogurt, or frozen yogurt.
• the fermented products may contain various substances such as food raw materials and food additives used in the food industry, and nutritional components and additives for culture.
• the present embodiment relates to a method for producing fermented milk containing EPS, using an exopolysaccharide-producing bacterium that has been treated to lengthen the induction period by 10% or more, as described above, or an exopolysaccharide-producing bacterium that has been treated to increase the concentration of EPS produced in the culture medium.
• the production method of this embodiment includes a step of fermenting raw milk with one or more lactic acid producing bacteria including exopolysaccharide producing bacteria treated to lengthen the induction period by 10% or more to obtain fermented milk containing EPS.
• the exopolysaccharide producing bacteria treated to lengthen the induction period by 10% or more used in this embodiment are the same as the "exopolysaccharide producing bacteria treated under conditions to lengthen the induction period by 10% or more" in the section "exopolysaccharide producing bacteria treated under conditions to lengthen the induction period by 10% or more and compositions containing the same".
• the production method of this embodiment includes a step of fermenting raw milk with one or more lactic acid producing bacteria including exopolysaccharide producing bacteria treated to increase the concentration of EPS produced in the culture medium to obtain fermented milk containing EPS.
• the lactic acid bacteria treated to lengthen the induction period by 10% or more in this embodiment are the same as the treatment described in the "step of pretreating exopolysaccharide producing bacteria" in the section "method of producing exopolysaccharide”.
• the one or more lactic acid-producing bacteria may include, in addition to the treated exopolysaccharide-producing bacteria, bacteria belonging to the genus Streptococcus, preferably bacteria belonging to Streptococcus thermophilus.
• the exopolysaccharide content of the fermented milk after the fermentation step, or the exopolysaccharide content of the obtained fermented milk is as follows.
• one or more lactic acid bacteria including a bacterium belonging to Lactobacillus, preferably a bacterium belonging to Lactobacillus delbrueckii, more preferably a bacterium belonging to Lactobacillus delbrueckii ssp. bulgaricus, and even more preferably Lactobacillus delbrueckii ssp.
• the exopolysaccharide content of the obtained fermented milk is 300 mg/kg or more, preferably 304 mg/kg or more, more preferably 308 mg/kg or more, and even more preferably 312 mg/kg or more;
• one or more lactic acid bacteria including bacteria belonging to the genus Lactococcus preferably bacteria belonging to Lactococcus lactis spp. lactis, more preferably Lactococcus lactis spp.
• the exopolysaccharide content of the obtained fermented milk is 2.2 mg/kg or more, preferably 5 mg/kg or more, more preferably 10 mg/kg or more, and even more preferably 15 mg/kg or more;
• the exopolysaccharide content of the obtained fermented milk is 69 mg/kg or more, preferably 72 mg/kg or more, more preferably 75 mg/kg or more, and even more preferably 80 mg/kg or more;
• one or more lactic acid bacteria including a bacterium belonging to the genus Bifidobacterium preferably a bacterium belonging to Bifidobacterium breve, more preferably Bifidobacterium breve JCM1192T, are used.
• the production method of this embodiment includes a step of obtaining EPS from an exopolysaccharide-producing bacterium that has been treated so as to lengthen the induction period by 10% or more, and a step of adding the obtained EPS to fermented milk.
• the method includes a step of obtaining EPS from an exopolysaccharide-producing bacterium that has been treated so as to increase the concentration of EPS produced in the culture medium, and a step of adding the obtained EPS to fermented milk.
• the raw milk used to obtain fermented milk includes dairy products.
• dairy products that can be used include animal milk from cows, goats, etc., pasteurized milk, skim milk, whole milk powder, skim milk powder, whole fat concentrated milk, skim concentrated milk, cream, butter, buttermilk, and whey.
• Milk proteins include milk protein concentrate (MPC), whey protein concentrate (WPC), whey protein isolate (WPI), ⁇ -lactalbumin ( ⁇ -La), ⁇ -lactoglobulin ( ⁇ -Lg), heat-denatured whey protein, and enzyme-treated whey protein.
• the raw milk may contain other ingredients.
• other ingredients include sugar, sweeteners, sugars, flavorings, water, etc.
• gelling agents, thickeners, and stabilizers such as gelatin, agar, pectin, carboxymethylcellulose (CMC), xanthan gum, carrageenan, tamarind seed gum, guar gum, gum arabic, locust bean gum, gellan gum, soybean polysaccharides, and modified starch may be included as necessary.
• raw milk with unreduced lactose levels is used.
• EPS production can be increased, but this embodiment does not include the embodiment in which raw milk in which lactose has been reduced in advance with lactase is used.
• the method for producing fermented milk in this embodiment may include a homogenization step, a sterilization step, a step of filling the fermented milk into a container, etc.
• the present embodiment relates to a method for improving the exopolysaccharide production ability of lactic acid bacteria, which comprises treating the lactic acid bacteria under conditions that lengthen the lag period, and a method for improving the exopolysaccharide production ability of lactic acid bacteria, which comprises subjecting the lactic acid bacteria to any treatment selected from the group consisting of heat (high heat) treatment, osmotic pressure (high osmotic pressure) treatment, and pH (high pH) treatment.
• heat high heat
• osmotic pressure high osmotic pressure
• pH high pH
• the lactic acid bacteria applicable to this embodiment are the same as those described in the section on the method for producing exopolysaccharides.
• This embodiment is based on the inventors' findings that in culturing lactic acid bacteria, the concentration of EPS produced in the culture medium increases when the exopolysaccharide-producing bacteria are exposed to conditions that lengthen the lag phase by 10% or more prior to culturing, and that there is a positive correlation between the length of the lag phase and the concentration of EPS in the culture medium (see Figures 1 to 6).
• Example 1 Increase in polysaccharide production by L. delbrueckii by heat treatment (skim milk medium)] The effect of heat treatment of four L. delbrueckii strains, including OLL1073R-1, on polysaccharide production during fermentation of skim milk powder was examined.
• Heat treatment conditions 250 ⁇ L of each bacterial solution (bacteria cultured in a specified medium containing protein components (skim milk powder, lactose monohydrate, yeast extract, fish extract, SMO (Sunsoft Q17S), water). Bacterial count approximately 5.8 x 109 cfu/g) was placed in a 1.5 mL tube and heated at 60°C for 10 minutes in a heat block. The heat-treated bacterial solutions were frozen and stored as necessary.
• a specified medium containing protein components skim milk powder, lactose monohydrate, yeast extract, fish extract, SMO (Sunsoft Q17S), water.
• Bacterial count approximately 5.8 x 109 cfu/g
• Fermentation medium 10% reduced skim milk medium (10% aqueous solution of skim milk powder (fat 1%, protein 34%, lactose 54%, ash 8%, non-fat solids 96%) (Meiji Co., Ltd.) to which inosinic acid was added to a final concentration of 1 mM. Lactose content: 5.4%, non-fat solids: 9.6%) in the medium, sterilized at 95°C, pH 6.5
• the 10% reconstituted skim milk medium means a medium containing 10% (W/W) skim milk components (reconstituted skim milk).
• Fermentation conditions 1% of the bacterial liquid (frozen bacteria) was inoculated into the fermentation medium, fermented at 40°C, and fermentation was stopped after 24 hours by placing on ice.
• Polysaccharide concentration measurement method Phenol-sulfuric acid method
• Pretreatment After 24 hours of fermentation, 10 g of the fermented product was added with 1 mL of 100 w/v% trichloroacetic acid solution, stirred, and then centrifuged at 13400 g, 4 ° C, for 10 minutes. The supernatant was collected, 5 mL of 10% w/v trichloroacetic acid solution was added, stirred, and centrifuged at 13400 g, 4 ° C, for 10 minutes. The supernatant was collected, 100% ethanol was added in an amount twice that of the supernatant, mixed, and left to stand overnight at 10 ° C.
• ⁇ pH measurement method The pH was measured continuously by attaching a pH sensor (pH Sensor SE 555, Knick) to a pH monitoring device (Horiba, Ltd.) and inserting the electrode into the fermentation product during fermentation. In the following experiments, the pH was measured in this manner unless otherwise specified.
• a pH sensor pH Sensor SE 555, Knick
• a pH monitoring device Horiba, Ltd.
• the delay rate of the induction phase (the time (minutes) required to change the pH of the fermentation medium by 0.2 at the beginning of the culture, i.e., in this example, the delay rate for the pH of the fermentation medium to change from pH 6.5 to pH 6.3) was as shown in the table below.
• Delay rate of induction phase [(Time required for treated lactic acid bacteria to change the pH by 0.2 at the beginning of cultivation (min)) - (Time required for untreated lactic acid bacteria to change the pH by 0.2 at the beginning of cultivation (min))] / (Time required for untreated lactic acid bacteria to change the pH by 0.2 at the beginning of cultivation (min))
• Example 2 Increase in polysaccharide production by L. delbrueckii by heat treatment (synthetic medium)]
• a skim milk powder medium was used, assuming yogurt fermentation.
• Example 2 assuming that polysaccharides produced by lactic acid bacteria are produced and added to various products as ingredients, the amount of polysaccharides produced by four strains of L. delbrueckii was examined when a synthetic medium (MRS broth) was used.
• Fermentation medium MRS broth (Difco), pH 6.1
• Fermentation conditions 1% frozen bacteria was inoculated into the fermentation medium, fermented at 40°C, and fermentation was stopped after 24 hours by placing on ice.
• Polysaccharide concentration measurement method Phenol-sulfuric acid method (described in Example 1)
• the EPS content of each fermentation product after 24 hours of fermentation is shown in the table below and Figure 2. It was revealed that the polysaccharide content after 24 hours of fermentation was increased for the four strains of L. delbrueckii even in synthetic medium by heat-treating them at 60°C for 10 minutes before fermentation.
• the delay rate of the induction phase (the time (minutes) required to change the pH of the fermentation medium by 0.2 at the beginning of the culture, i.e., the delay rate of the time it took for the pH of the fermentation medium to change from pH 6.1 to pH 5.9 in this example) was as shown in the table below.
• Example 3 Effect of heat treatment on polysaccharide production of lactic acid bacteria species used in fermented milk
• S. thermophilus and B. breve which are species used in fermented milk other than L. delbrueckii.
• Two strains of S. thermophilus Streptococcus thermophilus OLS3618 (NITE BP-01815) and Streptococcus thermophilus OLS 3290 (FERM BP-19638)
• JCM1192 T Two strains of S. thermophilus (Streptococcus thermophilus OLS3618 (NITE BP-01815) and Streptococcus thermophilus OLS 3290 (FERM BP-19638)) and one type strain of B. breve (JCM1192 T ) were used.
• Methods Activation medium M17 broth (Gifco) for S. thermophilus, GAM broth (Nissui Pharmaceutical Co., Ltd.) for B. breve
• Activation culture conditions 1% frozen bacteria was added to the activation medium, and the activation liquid was added to the activation medium at 1% and cultured at 37°C for 16 hours.
• Heat treatment conditions 250 ⁇ L of the activation liquid was placed in a 1.5 mL tube and heated at 60°C for 10 minutes in a heat block.
• Fermentation medium 10% reduced skim milk medium (sterilized at 95°C) was prepared, and casein peptide CMA500 (casein protein hydrolysate, product name: Hyvital Casein CMA 500 Protein Hydrolysate, manufacturer: FrieslandCampina Domo, standard component values: protein: 86.4%, ash: 6.0%, moisture: ⁇ 5%, fat: ⁇ 0.1%, lactose: ⁇ 0.1%, average molecular weight: 267 daltons, decomposition method: enzymatic decomposition) was added to a final concentration of 0.1% for the fermentation of S. thermophilus, and B.
• casein peptide CMA500 casein protein hydrolysate, product name: Hyvital Casein CMA 500 Protein Hydrolysate, manufacturer: FrieslandCampina Domo, standard component values: protein: 86.4%, ash: 6.0%, moisture: ⁇ 5%, fat: ⁇ 0.1%, lactose: ⁇ 0.1%, average molecular weight: 267 dalton
• the EPS content of each fermentation product after 24 hours of fermentation is shown in the table below. It was also revealed that the polysaccharide content after 24 hours of fermentation was increased for two strains of S. thermophilus and one type strain of B. breve by heat treatment at 60°C for 10 minutes before fermentation.
• the delay rate of the induction phase (the time (minutes) required to change the pH from the initial stage of culture by 0.2, i.e., the delay rate of the time it takes for the pH of the fermentation medium to change from pH 6.5 to pH 6.3 in this example) was as shown in the table below.
• Example 4 Increase in polysaccharide production by L. delbrueckii due to differences in heat treatment conditions (skim milk medium)]
• Methods OLL1073R-1 was heat-treated at 60°C for 3 minutes, 60°C for 5 minutes, 60°C for 10 minutes, 80°C for 1 minute, and 80°C for 3 minutes, and the viable cell count after the treatment was measured.
• the heat-treated OLL1073R-1 was inoculated at 1% into a 10% skim milk powder medium (containing 1 mM inosinic acid, pH 6.5) that had been sterilized at 95°C, and fermented at 40°C. After 24 hours of fermentation, a sample was taken, and the viable cell count after fermentation and the EPS concentration were measured (phenol-sulfuric acid method, as described in Example 1).
• the polysaccharide concentration per log10 was higher than without heat treatment.
• the delay rate of the induction phase (the time (minutes) required to change the pH from the initial stage of cultivation by 0.2, i.e., the delay rate for the pH of the fermentation medium to change from pH 6.5 to pH 6.3 in this example) under the conditions of 60°C for 3 minutes, 60°C for 5 minutes, 60°C for 10 minutes, and 80°C for 1 minute, where an increase in polysaccharide concentration was observed, was as shown in the table below.
• Example 5 Increase in polysaccharide production by L. delbrueckii by salt (osmotic pressure) treatment (skim milk medium)
• OLL1073R-1 was mixed with frozen bacteria to achieve final NaCl concentrations of 0.8%, 5%, or 10%, and allowed to stand at 37°C for 3 or 6 hours (hyperosmotic pressure treatment).
• 2% of the treated OLL1073R-1 bacteria solution was inoculated into a 10% skim milk powder medium (with 1 mM inosinic acid added, pH 6.5) sterilized at 95°C, and fermented at 40°C. Sampling was performed 24 hours after fermentation, and the viable cell count after fermentation and EPS concentration were measured (phenol-sulfuric acid method, described in Example 1).
• Example 6 Increase in polysaccharide production by L. delbrueckii by salt (osmotic pressure) treatment (skim milk medium)]
• OLL1073R-1 was mixed with frozen bacteria so that the final NaCl concentrations were 0%, 0.8%, 5%, and 10%, and the mixture was left to stand at 37° C. for 3 hours (hyperosmotic pressure treatment).
• 4% of the treated OLL1073R-1 bacteria solution (2% bacteria) was inoculated into a 10% skim milk powder medium (1 mM inosinic acid added, pH 6.5) sterilized at 95° C., and fermented at 40° C. Sampling was performed 24 hours after fermentation, and the viable cell count after fermentation and EPS concentration were measured (phenol-sulfuric acid method, described in Example 1).
• Example 7 Increase in polysaccharide production by L. delbrueckii by high pH treatment
• Methods OLL1073R-1 was added at 0.3% to the medium shown in the table below (autoclaved, filter-sterilized 2 mol/L DL-malic acid was added at 0.2% (final 4 mM) based on the medium), and the initial culture pH was adjusted to pH 6.0 (control, no pH adjustment), pH 6.7, and pH 6.9 with potassium carbonate. Culture was continued at 37°C for 24 hours at pH 5.4 while neutralizing with potassium carbonate.
• Example 8 Increase in polysaccharide production by Lactococcus lactis spp. lactis by heat treatment (MRS medium) Methods Frozen Lactococcus lactis spp. lactis OLS3789 (NITE BP-1387) was added to MRS broth at 1% and cultured at 37°C for 16 hours. The activation liquid was then added to the activation medium (MRS) at 1% and cultured at 37°C for 16 hours (activation culture).
• MRS medium Lactococcus lactis spp. lactis by heat treatment
• Example 9 Increase in polysaccharide production by Lactococcus lactis spp. lactis by salt (osmotic pressure) treatment (skim milk medium) Methods Frozen Lactococcus lactis spp. lactis OLS3789 (NITE BP-1387) was added to MRS broth at 1% and cultured at 37°C for 16 hours. The activation liquid was then added to the activation medium (MRS) at 1% and cultured at 37°C for 16 hours (activation culture).
• salt osmotic pressure
• Skim milk medium skim milk medium
• the activated culture solution was mixed with frozen bacteria to a final NaCl concentration of 2%, and left to stand at 37°C for 3 hours. After that, 2% of the salt-treated bacteria solution of OLS3789 (1% bacteria) was inoculated into a medium consisting of 10% skim milk powder medium and 0.1% Meast P2G (yeast extract), and fermentation was carried out at 30°C, and fermentation was stopped after 24 hours by placing on ice.
• the polysaccharide concentration was measured using the phenol-sulfuric acid method, as described in Example 1.
• Example 10 EPS production during fermentation of heat-treated L. delbrueckii (skim milk medium) The effect of heat treatment of L. delbrueckii on polysaccharide production during fermentation of skim milk powder was examined.
• Heat treatment conditions 250 ⁇ L of each bacterial solution (bacteria cultured in a specified medium containing protein components (skim milk powder, lactose monohydrate, yeast extract, fish extract, SMO (Sunsoft Q17S), water). Bacterial count approximately 5.8 x 109 cfu/g) was placed in a 1.5 mL tube and heated in a heat block at 60°C for 5 minutes and 80°C for 1 minute. The heat-treated bacterial solutions were frozen and stored as necessary.
• a specified medium containing protein components skim milk powder, lactose monohydrate, yeast extract, fish extract, SMO (Sunsoft Q17S), water.
• Bacterial count approximately 5.8 x 109 cfu/g
• Fermentation medium 10% reduced skim milk medium (10% aqueous solution of skim milk powder (fat 1%, protein 34%, lactose 54%, ash 8%, non-fat solids 96%) (Meiji Co., Ltd.) to which inosinic acid was added to a final concentration of 1 mM. Lactose content: 5.4%, non-fat solids: 9.6%) in the medium, sterilized at 95°C, pH 6.5
• the 10% reconstituted skim milk medium means a medium containing 10% (W/W) skim milk components (reconstituted skim milk).
• Fermentation conditions 1% of the bacterial solution (frozen bacteria) was inoculated into the fermentation medium, fermentation was performed at 40°C, and the fermentation was stopped after 24 hours by placing it on ice. Sampling was also performed at pH 5.2 and pH 4.6. - Method for measuring polysaccharide concentration: See Example 1 - Method for measuring pH: See Example 1
• a method for producing exopolysaccharides includes the steps of treating various exopolysaccharide-producing bacteria under conditions that lengthen the induction period, particularly by 10% or more, and culturing the treated exopolysaccharide-producing bacteria in a medium to produce exopolysaccharides.
• the present invention provides a method for producing exopolysaccharides derived from exopolysaccharide-producing bacteria, and a method for producing foods containing exopolysaccharides, which prevent or improve diseases or conditions that can be improved by the functions of exopolysaccharides, and support the maintenance and improvement of the health of people who are not yet ill.
• it is possible to provide a food composition that maintains and improves people's health, and a method for producing foods.
• the present invention can improve the nutrition of various people, ensure healthy lives, and promote welfare.

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