WO2014084340A1 - Yogurt and production method therefor, production method for extracellular functional product of lactic acid bacteria, and production-increasing agent for extracellular functional product of lactic acid bacteria - Google Patents

Yogurt and production method therefor, production method for extracellular functional product of lactic acid bacteria, and production-increasing agent for extracellular functional product of lactic acid bacteria Download PDF

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WO2014084340A1
WO2014084340A1 PCT/JP2013/082154 JP2013082154W WO2014084340A1 WO 2014084340 A1 WO2014084340 A1 WO 2014084340A1 JP 2013082154 W JP2013082154 W JP 2013082154W WO 2014084340 A1 WO2014084340 A1 WO 2014084340A1
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lactic acid
yogurt
acid bacteria
buffer
functional product
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PCT/JP2013/082154
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French (fr)
Japanese (ja)
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圭介 古市
聖也 牧野
浩文 後藤
紘子 川嶋
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株式会社 明治
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Priority to JP2014549909A priority Critical patent/JP6392668B2/en
Priority to SG11201503729QA priority patent/SG11201503729QA/en
Priority to CN201380057872.5A priority patent/CN104780767B/en
Publication of WO2014084340A1 publication Critical patent/WO2014084340A1/en
Priority to HK15107263.8A priority patent/HK1206560A1/en

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    • 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; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
    • A23C9/1322—Inorganic compounds; Minerals, including organic salts thereof, oligo-elements; Amino-acids, peptides, protein-hydrolysates or derivatives; Nucleic acids or derivatives; Yeast extract or autolysate; Vitamins; Antibiotics; Bacteriocins
    • 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; MAKING 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
    • A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/137—Delbrueckii

Definitions

  • Lactobacillus delbruechii subsp. Bulgaricus (hereinafter also referred to as “Bulgaria bacterium”) is one of lactic acid bacteria used as a yogurt starter in the production of fermented milk.
  • Bactaria bacterium is one of lactic acid bacteria used as a yogurt starter in the production of fermented milk.
  • EPS exopolysaccharide
  • Patent Document 4 discloses that a high concentration of EPS is obtained in a culture solution by culturing lactic acid bacteria having a kefiran-producing ability such as Lactobacillus kefiranofaciens in a medium containing peptone, yeast extract, and an unsaturated fatty acid or an ester thereof. It is disclosed that it can be produced.
  • Patent Document 5 discloses that EPS derived from lactic acid bacteria is remarkably increased by adding whey protein or soybean protein to milk and fermenting with lactococcus lactis subspecies cremolith.
  • yogurt production method As a result of intensive studies by the inventors of the present application, as described in the above-mentioned yogurt production method, by adding a pH buffer to the yogurt raw material and then fermenting the yogurt raw material, the time in the pH region where lactic acid bacteria can grow is extended. As a result, it was revealed that the production amount of functional products derived from lactic acid bacteria in yogurt can be increased. That is, this yogurt manufacturing method can efficiently increase the production amount of functional products derived from lactic acid bacteria.
  • the yogurt raw material to which the pH buffer is added is fermented by lactic acid bacteria at a temperature within a range of 30 ° C. or higher and 40 ° C. or lower.
  • Yogurt ingredients include at least one of milk, dairy products, and milk proteins.
  • yogurt ingredients include animal milk such as milk, sheep and goats, processed products thereof, sterilized milk, skim milk, full fat milk powder, partially skimmed milk, skim milk powder, full fat concentrated milk, skim concentrated milk, cream, Butter, buttermilk, whey, whey protein concentrate (WPC), whey protein isolate (WPI), ⁇ -lactalbumin ( ⁇ -La), ⁇ -lactoglobulin ( ⁇ -Lg), ⁇ -casein, ⁇ - Casein, ⁇ -casein, milk raw materials such as non-protein nitrogen, sugar, sugar, modified starch (dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, sweetener, organic Examples include acids (malic acid, citric acid, lactic acid, tartaric acid, etc.), fragrances, and water.
  • the yogurt raw material can be obtained by mixing conventional raw materials as described above and dissolving them while heating. Gelling agents such as gelatin, agar, pectin, and carboxymethylcellulose (CMC), thickeners, and stabilizers may be added to the yogurt raw material.
  • a raw material for yogurt is prepared by preliminarily heating and dissolving a stabilizer such as gelatin in a solvent such as water and mixing this aqueous stabilizer solution with other components.
  • EPS-producing lactic acid bacteria such as Lactobacillus bulgaricus and Lactococcus.
  • -Lactococcus lactis ssp. Cremoris may be used in combination with Bulgarian bacteria.
  • the lactic acid bacterial extracellular functional product according to the present embodiment is produced through a pH buffer addition step and a fermentation step.
  • a pH buffer is added to the milk raw material.
  • the milk raw material to which the pH buffer is added is fermented by lactic acid bacteria that produce lactic acid bacteria extracorporeal functional products.
  • milk raw material here means animal milk such as cow's milk, sheep, goat, etc., processed products, sterilized milk, skim milk, whole milk powder, partially skimmed milk, skimmed milk powder, whole fat concentrated milk, skimmed milk Concentrated milk, cream, butter, buttermilk, whey, whey protein concentrate (WPC), whey protein isolate (WPI), ⁇ -lactalbumin ( ⁇ -La), ⁇ -lactoglobulin ( ⁇ -Lg), ⁇ -Casein, ⁇ -casein, ⁇ -casein, non-protein nitrogen, etc.
  • WPC whey protein concentrate
  • WPI whey protein isolate
  • ⁇ -La ⁇ -lactalbumin
  • ⁇ -Lg ⁇ -lactoglobulin
  • ⁇ -Casein ⁇ -casein
  • non-protein nitrogen etc.
  • the non-fat milk solid content (hereinafter referred to as “SNF”) of the milk raw material is preferably in the range of 8% by weight to 20% by weight, and in the range of 8.5% by weight to 18.5% by weight. More preferably, it is more preferably in the range of 9% by weight to 15% by weight, and particularly preferably in the range of 9.5% by weight to 14% by weight.
  • OLL1073R-1 strain Lactobacillus delbruechii subsp. bulgaricus OLL1073R-1 strain (hereinafter referred to as OLL1073R-1 strain) was issued on November 29, 2006 (contract date), National Institute of Advanced Industrial Science and Technology Patent Biology Center (1-1-1 Tsukuba, Tsukuba, Ibaraki Prefecture) Lactic acid bacteria are deposited internationally under the Budapest Treaty under the accession number FERM BP-10741 in the center center 6).
  • Streptococcus thermophilus OLS3059 strain was entrusted to the National Institute of Advanced Industrial Science and Technology Patent Biology Center (Tsukuba Center 1-1-1 Tsukuba Center Central 6th, Ibaraki Prefecture) on December 15, 2006 (contract date). It is a lactic acid bacterium that has been deposited internationally under the Budapest Treaty as FERM BP-10740.
  • the patent microorganisms deposit work of the National Institute of Advanced Industrial Science and Technology patent biological depository center was succeeded by the National Institute of Technology and Evaluation on April 1, 2012. As of April 1, 2013, the Biological Depositary Center has been relocated to the National Institute of Technology and Evaluation, 2-5-8, Kazusa Kamashika, Kisarazu City, Chiba Prefecture, Japan.
  • the sample 1-4 was added Na 2 HPO 4 and NaH 2 PO 4 as Na 2 HPO 4 and NaH 2 PO 4 is contained 0.5 wt% respectively of the total amount.
  • phosphate was not added to Sample 1-5 (control). Then, each said sample was immersed in a 43 degreeC thermostat and fermented. When the pH of each sample reached 4.4 to 4.5, the sample was cooled to 10 ° C. or lower, and the fermentation of the sample was stopped. About each sample, time to fermentation stop (fermentation time), acidity, EPS content, the number of Bulgaria bacteria, and the number of thermophilus bacteria were measured (refer Table 1).
  • PH was measured with a pH meter (TOA-HM50V, manufactured by Toa DKK Corporation) using a glass electrode.
  • the acidity was measured by the following procedure. 9.00 g was sampled from each sample, and 500 ⁇ L of phenolphthalein was added to the sample. Then, the preparative sample was titrated with 0.1N sodium hydroxide, and the end point was a time point at which the faint red color did not disappear for 30 seconds.
  • the mixture was allowed to stand overnight at a temperature of about 4 ° C., and then the mixture was centrifuged under the same conditions as described above. Thereafter, the supernatant of the mixture was discarded, and 10 mL of purified water was added to the precipitate to completely dissolve the precipitate in purified water.
  • 150 ⁇ L of the aqueous solution was injected into the HPLC using a syringe with a filter having a diameter of 0.45 ⁇ m.
  • the ratio of the “peak area of a single peak detected by the RI detector around 16 minutes after the start of injection” to the “total peak area” was taken as the EPS content.
  • the analytical operation conditions of HPLC are as follows.
  • HPLC system Aquity H-class (Waters) Column: OHpak 806HQ (Shodex) + SB-G (Shodex) Column temperature: 40 ° C Solvent: 0.2 M NaCl aqueous solution Flow rate: 0.5 mL / min Detector: RI detector 2414 (Waters), detection temperature 40 ° C Sample injection: 150 ⁇ L Analysis time: 50 min
  • yogurt starter which is a mixed culture of OLS 3059 and 1073R-1 strains, was inoculated into 500 g of yoghurt raw material mix A so as to contain 2% by weight of the lactic acid bacteria starter.
  • the yogurt raw material mix A was dispensed into 6 test tubes each 20 mL to prepare 6 samples 2-1, 2-2, 2-3, 2-4, 2-5, 2-6.
  • Na 2 HPO 4 was added so that 0.5% by weight of Na 2 HPO 4 was contained in the total amount.
  • the sample 2-2 was added K 2 HPO 4 as K 2 HPO 4 is contained 0.61 wt% of the total amount.
  • the molar concentration of K 2 HPO 4 in sample 2-2 is the same as the molar concentration of Na 2 HPO 4 in sample 2-1.
  • NaCl was added so that NaCl was contained at 0.21% by weight based on the total amount.
  • the molar concentration of NaCl in Sample 2-3 is the same as the molar concentration of Na 2 HPO 4 in Sample 2-1.
  • the sample 2-4 was added Na 2 HPO 4 as Na 2 HPO 4 is contained 0.3 wt% of the total amount.
  • the sample 2-5 was added Na 2 HPO 4 as Na 2 HPO 4 is contained 0.1% by weight based on the total amount. Note that phosphate was not added to Sample 2-6 (control). Then, each said sample was immersed in a 43 degreeC thermostat and fermented.
  • Sample 3-1 (control) received no phosphate.
  • the sample 3-2 was added Na 2 HPO 4 as Na 2 HPO 4 is contained 0.1 wt%.
  • Sample 3-3 Na 2 HPO 4 was added so that 0.3 wt% of Na 2 HPO 4 was contained.
  • the sample 3-4 was added Na 2 HPO 4 as Na 2 HPO 4 is contained 0.5 wt%.
  • the sample 3-5 was added Na 2 HPO 4 as Na 2 HPO 4 is contained 1.0 wt%.
  • Table 3 shows the fermentation time, EPS content, and CT results of each sample. As the amount of Na 2 HPO 4 added increased, the EPS content in the sample increased and the CT value decreased (ie the sample became softer).
  • the EPS content of Sample 4-1 was 46.0 mg / kg.
  • the EPS content of Sample 4-2 was 48.6 mg / kg. That is, the EPS content of Sample 4-2 was 1.1 times that of Sample 4-1. Therefore, it became clear that the EPS content in yogurt can be increased by using a high SNF yogurt raw material.
  • a yogurt raw material mix A was prepared as sample 6-1 (control).
  • the yogurt raw material mix B was divided into three to prepare three samples 6-2, 6-3, and 6-4.
  • each sample was inoculated with a lactic acid bacteria starter which is a mixed culture of Thermophilus bacteria and 1073R-1 strain generally used in the production of yogurt so that the lactic acid bacteria starter was contained at 2% by weight.
  • the sample 6-4 was further added Na 2 HPO 4 as Na 2 HPO 4 is contained 0.3 wt% of the total amount.
  • Samples 6-1 and 6-2 were then fermented at 43 ° C, and samples 6-3 and 6-4 were fermented at 37 ° C. When the acidity of each sample reached 0.8, the sample was cooled to 10 ° C. or lower, and the fermentation of the sample was stopped.
  • the EPS content in each sample was measured.
  • the EPS content was measured by the same method as in Example 1.
  • Table 4 shows SNF, fermentation temperature, phosphate (Na 2 HPO 4 ) addition amount, and EPS content in yogurt for each sample.
  • sample 6-2 high SNF content
  • sample 6-3 high SNF content, low fermentation temperature
  • the EPS content is increased to the control sample. It was increased to 1.19 times 6-1.
  • sample 6-4 high SNF content, low fermentation temperature, phosphate addition
  • the EPS content of yogurt was increased to 1.37 times that of control sample 6-1.

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Abstract

The present invention addresses the problem of providing a novel method with which the production amount of a lactic-acid-bacteria-derived functional product can be efficiently increased. A yogurt production method according to the present invention is provided with a pH-buffer-agent adding step, and a fermentation step. In the pH-buffer-agent adding step, a pH buffer agent is added to a yogurt starting material. In the fermentation step, lactic acid bacteria are used to cause fermentation of the yogurt starting material having the pH buffer agent added thereto.

Description

ヨーグルトおよびその製造方法、乳酸菌体外機能性産生物製造方法、ならびに乳酸菌体外機能性産生物増産剤Yogurt and method for producing the same, method for producing lactic acid bacteria extracellular functional product, and lactic acid bacteria extracellular functional product production agent
　本発明は、乳酸菌由来の機能性産生物の産生量を増加させるヨーグルト製造方法、及び、そのヨーグルト製造方法によって製造されるヨーグルトに関する。また、本発明は、乳酸菌の菌体外機能性産生物の産生量を増加させる乳酸菌体外機能性産生物製造方法、並びに、その方法に使用される乳酸菌体外機能性産生物増産剤に関する。 The present invention relates to a yogurt production method for increasing the production amount of a functional product derived from lactic acid bacteria, and a yogurt produced by the yogurt production method. In addition, the present invention relates to a method for producing a lactic acid bacterium extracellular functional product that increases the production amount of the lactic acid bacterium extracellular functional product, and a lactic acid bacterium extracellular functional product enhancer used in the method.
　乳酸菌および乳酸菌由来の機能性産生物を摂取することにより、消化管内の細菌叢を改善し、宿主に有益な作用（健康の維持・疾病リスクの低減）をすることが知られている。例えば、ラクトバチルス・デルブルエッキー・サブスピーシス・ブルガリクス（Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｄｅｌｂｒｕｅｃｈｉｉ　ｓｕｂｓｐ．　ｂｕｌｇａｒｉｃｕｓ）（以下「ブルガリア菌」とも称する）は、ヨーグルトスターターとして発酵乳の製造に用いられている乳酸菌のひとつである。このブルガリア菌の中には、菌体外多糖体(ｅｘｏｐｏｌｙｓａｃｃｈａｒｉｄｅ：ＥＰＳ)を産生する菌株も多く存在する。そして、このＥＰＳは、発酵乳製品の物性や安定性に寄与するのみならず、プロバイオティクス効果の享受にも寄与することが知られている。例えば、Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｄｅｌｂｒｕｅｃｈｉｉ　ｓｕｂｓｐ．　ｂｕｌｇａｒｉｃｕｓ　ＯＬＬ１０７３Ｒ－１株（以下「ＯＬＬ１０７３Ｒ－１株」とも称する）が産生するＥＰＳには、自己免疫疾患を予防する効果があることが知られている。また、本菌株を用いて製造された発酵乳には、ＮＫ細胞の活性化、感冒罹患の減少、抗インフルエンザ等の効果があることが知られている。（例えば、特許文献１～３等参照）。このように、ＥＰＳを産生する乳酸菌や、乳酸菌などの産生するＥＰＳを利用することにより、健康に寄与する機能性食品やサプリメントを提供することができることとなる。 It is known that by ingesting lactic acid bacteria and functional products derived from lactic acid bacteria, the bacterial flora in the gastrointestinal tract is improved and has beneficial effects on the host (maintenance of health and reduction of disease risk). For example, Lactobacillus delbruechii subsp. Bulgaricus (hereinafter also referred to as “Bulgaria bacterium”) is one of lactic acid bacteria used as a yogurt starter in the production of fermented milk. Among these Bulgarian bacteria, there are many strains that produce exopolysaccharide (EPS). And this EPS is known not only to contribute to the physical properties and stability of fermented milk products, but also to enjoy the probiotic effect. For example, Lactobacillus delbruechii subsp. EPS produced by Bulgaricus OLL1073R-1 strain (hereinafter also referred to as “OLL1073R-1 strain”) is known to have an effect of preventing autoimmune diseases. In addition, it is known that fermented milk produced using this strain has effects such as activation of NK cells, reduction of common cold, and anti-influenza. (For example, see Patent Documents 1 to 3). Thus, functional foods and supplements that contribute to health can be provided by using lactic acid bacteria that produce EPS or EPS that is produced by lactic acid bacteria or the like.
　ところで、このような機能性食品やサプリメントを効率的に製造するためには、同機能性食品中のＥＰＳ産生量を高める必要がある。 By the way, in order to efficiently produce such functional foods and supplements, it is necessary to increase the EPS production amount in the functional foods.
　ブルガリア菌に限らず乳酸菌由来のＥＰＳを高めるために、過去に様々な方法が提案されている。例えば、特許文献４には、ペプトン、酵母エキス、及び不飽和脂肪酸もしくはそのエステルを含む培地で、Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｋｅｆｉｒａｎｏｆａｃｉｅｎｓ等のケフィラン生産能を有する乳酸菌を培養することにより、培養液中に高濃度のＥＰＳを産生することができることが開示されている。また、特許文献５には、牛乳に乳清タンパクや大豆タンパクを加え、ラクトコッカス・ラクティス・サブスピーシーズ・クレモリスで発酵させることにより、乳酸菌由来のＥＰＳが顕著に増加することが開示されている。 In order to increase EPS derived from lactic acid bacteria as well as Bulgaria bacteria, various methods have been proposed in the past. For example, Patent Document 4 discloses that a high concentration of EPS is obtained in a culture solution by culturing lactic acid bacteria having a kefiran-producing ability such as Lactobacillus kefiranofaciens in a medium containing peptone, yeast extract, and an unsaturated fatty acid or an ester thereof. It is disclosed that it can be produced. Patent Document 5 discloses that EPS derived from lactic acid bacteria is remarkably increased by adding whey protein or soybean protein to milk and fermenting with lactococcus lactis subspecies cremolith.
特開２０００－２４７８９５号公報JP 2000-247895 A 特開２００５－１９４２５９号公報JP 2005-194259 A 国際公開パンフレットＷＯ２０１１／０６５３００International Publication Pamphlet WO2011 / 065300 特開２０１１－２５０７５６号公報JP 2011-250756 A 特開２０１１－５１２６８号公報JP 2011-51268 A
　本発明の課題は、乳酸菌由来の機能性産生物の産生量を効率的に増加させることができる新たな方法を提供することである。 An object of the present invention is to provide a new method capable of efficiently increasing the production amount of a functional product derived from lactic acid bacteria.
　本発明の一局面に係るヨーグルト製造方法は、ｐＨ緩衝剤添加工程および発酵工程を備える。ｐＨ緩衝剤添加工程では、ヨーグルト原料にｐＨ緩衝剤が添加される。発酵工程では、ｐＨ緩衝剤が添加されたヨーグルト原料（以下「ｐＨ緩衝剤添加ヨーグルト原料」という）が乳酸菌によって発酵させられる。なお、ヨーグルト原料への乳酸菌の添加はｐＨ緩衝剤の添加前であってもよいし、添加後であってもよい。 The yogurt manufacturing method according to one aspect of the present invention includes a pH buffer addition step and a fermentation step. In the pH buffer addition step, a pH buffer is added to the yogurt raw material. In the fermentation process, a yogurt raw material to which a pH buffer is added (hereinafter referred to as “pH buffer added yogurt raw material”) is fermented by lactic acid bacteria. In addition, the addition of lactic acid bacteria to the yogurt raw material may be before the addition of the pH buffering agent or after the addition.
　本願発明者らの鋭意検討の結果、上述のヨーグルト製造方法の通り、ヨーグルト原料にｐＨ緩衝剤を添加してからそのヨーグルト原料を発酵させることによって、乳酸菌が増殖できるｐＨ領域の時間を延長することができ、その結果、ヨーグルト中の乳酸菌由来の機能性産生物の産生量を高めることができることが明らかとなった。すなわち、このヨーグルト製造方法は、乳酸菌由来の機能性産生物の産生量を効率的に高めることができる。 As a result of intensive studies by the inventors of the present application, as described in the above-mentioned yogurt production method, by adding a pH buffer to the yogurt raw material and then fermenting the yogurt raw material, the time in the pH region where lactic acid bacteria can grow is extended. As a result, it was revealed that the production amount of functional products derived from lactic acid bacteria in yogurt can be increased. That is, this yogurt manufacturing method can efficiently increase the production amount of functional products derived from lactic acid bacteria.
　ところで、このヨーグルト製造方法において、乳酸菌は、少なくとも、乳酸菌体外多糖体産生能力を有する乳酸菌を含むことが好ましい。そして、この乳酸菌体外多糖体産生能力を有する乳酸菌は、少なくとも、ラクトバチルス・デルブルエッキー・サブスピーシス・ブルガリクス種の乳酸菌を含むことが好ましい。なお、かかる場合、乳酸菌体外多糖体産生能力を有する乳酸菌は、ラクトバチルス・デルブルエッキー・サブスピーシス・ブルガリクス種の乳酸菌単体であってもよいし、ラクトバチルス・デルブルエッキー・サブスピーシス・ブルガリクス種と他の乳酸菌を含む乳酸菌混合物であってもよい。 By the way, in this yogurt production method, the lactic acid bacteria preferably include at least a lactic acid bacterium having an ability to produce lactic acid bacteria exopolysaccharide. And it is preferable that the lactic acid bacteria which have the ability to produce lactic acid bacteria exopolysaccharides include at least Lactobacillus delbruecki subspice bulgaricus lactic acid bacteria. In such a case, the lactic acid bacteria having the ability to produce lactic acid bacteria exopolysaccharides may be Lactobacillus delbruecky subspice bulgaricus lactic acid bacteria alone, or Lactobacillus delbruecki subspice bulgaricus sp. And lactic acid bacteria mixture containing other lactic acid bacteria.
　また、このヨーグルト製造方法において、ｐＨ緩衝剤はリン酸塩であることが好ましい。乳酸菌として、ラクトバチルス・デルブルエッキー・サブスピーシス・ブルガリクス種の乳酸菌とストレプトコッカス・サーモフィルス（Ｓｔｒｅｐｔｏｃｏｃｃｕｓ　ｔｈｅｒｍｏｐｈｉｌｕｓ）種の乳酸菌（以下「サーモフィルス菌」とも称する）との混合物が使用される場合、サーモフィルス菌の増殖を抑制しつつラクトバチルス・デルブルエッキー・サブスピーシス・ブルガリクス種の乳酸菌の増殖を促進することができるからである。なお、このリン酸塩は、アルカリ金属塩であることが好ましい。さらに、リン酸塩は、リン酸水素二ナトリウムおよびリン酸水素二カリウムより成る群から選択される少なくとも一方のリン酸塩であることが好ましい。 In this method for producing yogurt, the pH buffering agent is preferably a phosphate. When a mixture of lactic acid bacteria of the Lactobacillus delbruecki subspice bulgaricus species and Streptococcus thermophilus lactic acid bacteria (hereinafter also referred to as “thermophilus bacteria”) is used as the lactic acid bacteria, This is because it is possible to promote the growth of Lactobacillus delbruecki, subspice bulgaricus lactic acid bacteria while suppressing the growth of the bacteria. In addition, it is preferable that this phosphate is an alkali metal salt. Furthermore, the phosphate is preferably at least one phosphate selected from the group consisting of disodium hydrogen phosphate and dipotassium hydrogen phosphate.
　さらに、このヨーグルト製造方法において、ヨーグルト原料には、８重量％以上２０重量％以下の範囲内の無脂乳固形分が含まれていることが好ましい。なお、ここで、ヨーグルト原料とはｐＨ緩衝剤添加前のものを意味する。 Furthermore, in this yogurt manufacturing method, it is preferable that the yogurt raw material contains non-fat milk solids in the range of 8 wt% to 20 wt%. Here, the yoghurt raw material means that before adding the pH buffering agent.
　加えて、このヨーグルト製造方法において、発酵工程では、ｐＨ緩衝剤添加ヨーグルト原料が３０℃以上４０℃以下の範囲内の温度下で乳酸菌によって発酵させられることが好ましい。 In addition, in this yogurt production method, in the fermentation step, it is preferable that the yogurt raw material to which the pH buffer is added is fermented by lactic acid bacteria at a temperature within a range of 30 ° C. or higher and 40 ° C. or lower.
　上述のヨーグルト製造方法によって製造されたヨーグルトには、乳酸菌およびｐＨ緩衝剤が含有される。なお、このヨーグルトでは、ｐＨ緩衝剤が均一に溶解されている。ここで、乳酸菌およびｐＨ緩衝剤は、上述の通りである。また、このヨーグルト中のｐＨ緩衝剤は、例えば、高速液体クロマトグラフィー等のクロマトグラフィー技術や、核磁気共鳴（ＮＭＲ）技術（例えば、Szlyk E, Hrynczyszyn P, 「Phosphate additives determination in meat products by 31-phosphorus nuclear magnetic resonance using new internal reference standard: hexamethylphosphoroamide phosphatesolution」, Talanta. 2011 Mar 15;84(1):199-203. doi: 10.1016/j.talanta.2010.12.046 参照）などによって検出することができる。 The yogurt produced by the above-mentioned yogurt production method contains lactic acid bacteria and a pH buffer. In this yogurt, the pH buffer is uniformly dissolved. Here, the lactic acid bacteria and the pH buffer are as described above. In addition, the pH buffer in this yogurt is, for example, a chromatographic technique such as high performance liquid chromatography or a nuclear magnetic resonance (NMR) technique (for example, Szlyk E, Hrynczyszyn P, “Phosphate additives determination in meat products by 31- phosphorus nuclear magnetic resonance using new internal reference standard: hexamethylphosphoroamide phosphatesolution ', Talanta. 2011 Mar 15; 84 (1): 199-203. doi: 10.1016 / j.talanta.2010.12.046).
　また、上述のヨーグルトは乳酸菌体外機能性産生物（例えば、乳酸菌体外多糖体など）をさらに含有することが好ましい。かかる場合、乳酸菌体外機能性産生物は、ヨーグルト全量に対して４０ｍｇ／ｋｇ以上１００ｍｇ／ｋｇ以下の範囲内の量含まれていることが好ましい。 Moreover, it is preferable that the above-mentioned yogurt further contains a lactic acid bacterial extracellular functional product (for example, a lactic acid bacterial extracellular polysaccharide). In such a case, it is preferable that the lactic acid bacterial extracellular functional product is contained in an amount within the range of 40 mg / kg to 100 mg / kg with respect to the total amount of yogurt.
　本発明の他の局面に係る乳酸菌体外機能性産生物製造方法は、ｐＨ緩衝剤添加工程および発酵工程を備える。ｐＨ緩衝剤添加工程では、乳原料にｐＨ緩衝剤が添加される。発酵工程では、ｐＨ緩衝剤が添加された乳原料が、乳酸菌体外機能性産生物を産生する乳酸菌（以下「菌体外機能性産生物産生乳酸菌」ともいう）によって発酵させられる。なお、乳原料への乳酸菌の添加はｐＨ緩衝剤の添加前であってもよいし、添加後であってもよい。 The method for producing a lactic acid bacteria extracellular functional product according to another aspect of the present invention includes a pH buffering agent addition step and a fermentation step. In the pH buffer addition step, a pH buffer is added to the milk raw material. In the fermentation process, the milk raw material to which the pH buffer is added is fermented by a lactic acid bacterium that produces a lactic acid bacteria extracellular functional product (hereinafter also referred to as “extracellular functional product-producing lactic acid bacteria”). The lactic acid bacterium may be added to the milk raw material before or after the addition of the pH buffering agent.
　本願発明者らの鋭意検討の結果、上述の乳酸菌体外機能性産生物製造方法の通り、乳原料にｐＨ緩衝剤を添加してからその乳原料を発酵させることによって、菌体外機能性産生物産生乳酸菌が増殖できるｐＨ領域の時間を延長することができ、その結果、乳酸菌由来の機能性産生物の産生量を高めることができることが明らかとなった。すなわち、この乳酸菌体外機能性産生物製造方法は、乳酸菌由来の機能性産生物の産生量を効率的に高めることができる。すなわち、本局面においてｐＨ緩衝剤は、乳酸菌体外機能性産生物増産剤の有効成分として機能する。 As a result of intensive studies by the inventors of the present application, the extracellular functional product is produced by adding a pH buffer to the milk raw material and then fermenting the milk raw material in accordance with the above-described method for producing an extracellular functional product of lactic acid bacteria. It has been clarified that the time in the pH region where the produced lactic acid bacteria can grow can be extended, and as a result, the production amount of the functional product derived from the lactic acid bacteria can be increased. That is, this lactic acid bacteria extracorporeal functional product manufacturing method can efficiently increase the production amount of functional products derived from lactic acid bacteria. That is, in this aspect, the pH buffer functions as an active ingredient of the lactic acid bacteria extracellular functional product increaser.
　なお、上述の乳酸菌体外機能性産生物製造方法は、別の観点から捉えると、「乳原料にｐＨ緩衝剤を配合（添加）して乳酸菌によりその乳原料を発酵させる乳酸菌体外機能性産生物の増産方法」、「乳原料にｐＨ緩衝剤を配合（添加）する乳酸菌体外機能性産生物の増産方法」、「乳酸菌体外機能性産生物を増産させるために乳原料にｐＨ緩衝剤を配合（添加）する方法」、「乳酸菌体外機能性産生物を増産させるための乳原料に対するｐＨ緩衝剤の使用（使用方法）」などとも表現することができる。 In addition, the above-mentioned method for producing an extracellular functional product of a lactic acid bacterium has a different viewpoint: “A lactic acid bacterial extracellular functional product in which a lactic acid bacterium is fermented by adding (adding) a pH buffer to the milk material. "Production method", "Production method of lactic acid bacteria functional product by adding (adding) a pH buffer to milk material", "Combination (addition of pH buffering agent to dairy material to increase production of lactic acid bacteria functional product" ) "And" use of pH buffering agent for milk raw material to increase production of lactic acid bacteria extracorporeal functional product (method of use) ".
　本発明の一局面に係るヨーグルト製造方法では、ヨーグルト原料にｐＨ緩衝剤を添加してからそのヨーグルト原料を発酵させることによって、乳酸菌が増殖できるｐＨ領域の時間を延長することができ、その結果、ヨーグルト中の乳酸菌由来の機能性産生物の産生量を高めることができる。すなわち、このヨーグルト製造方法は、乳酸菌由来の機能性産生物の産生量を効率的に高めることができる。 In the yogurt production method according to one aspect of the present invention, by adding a pH buffer to the yogurt raw material and then fermenting the yogurt raw material, the time in the pH region where the lactic acid bacteria can grow can be extended. The production amount of functional products derived from lactic acid bacteria in yogurt can be increased. That is, this yogurt manufacturing method can efficiently increase the production amount of functional products derived from lactic acid bacteria.
　また、本発明の他の局面に係る乳酸菌体外機能性産生物製造方法では、乳原料にｐＨ緩衝剤を添加してからその乳原料を発酵させることによって、乳酸菌が増殖できるｐＨ領域の時間を延長することができ、その結果、乳酸菌由来の機能性産生物の産生量を高めることができる。すなわち、この乳酸菌体外機能性産生物製造方法は、乳酸菌由来の機能性産生物の産生量を効率的に高めることができる。 In addition, in the method for producing a lactic acid bacteria extracellular functional product according to another aspect of the present invention, by adding a pH buffer to the milk material and then fermenting the milk material, the time in the pH region where the lactic acid bacteria can grow is extended. As a result, the production amount of the functional product derived from lactic acid bacteria can be increased. That is, this lactic acid bacteria extracorporeal functional product manufacturing method can efficiently increase the production amount of functional products derived from lactic acid bacteria.
　以下、本発明を実施するための形態を詳細に説明するが、本発明は、以下に述べる個々の実施の形態に限定されない。 Hereinafter, although the form for implementing this invention is demonstrated in detail, this invention is not limited to each embodiment described below.
－第１実施形態－
　本実施形態に係るヨーグルトは、ｐＨ緩衝剤添加工程および発酵工程を経て製造される。ｐＨ緩衝剤添加工程では、ヨーグルト原料にｐＨ緩衝剤が添加される。発酵工程では、ｐＨ緩衝剤が添加されたヨーグルト原料（以下「ｐＨ緩衝剤添加ヨーグルト原料」という）が乳酸菌によって発酵させられる。 -First embodiment-
The yogurt according to the present embodiment is manufactured through a pH buffer addition process and a fermentation process. In the pH buffer addition step, a pH buffer is added to the yogurt raw material. In the fermentation process, a yogurt raw material to which a pH buffer is added (hereinafter referred to as “pH buffer added yogurt raw material”) is fermented by lactic acid bacteria.
　本実施形態において、ヨーグルトとは、乳等省令で定義される「発酵乳」および「乳酸菌飲料」を指す。乳等省令における「発酵乳」とは、「乳またはこれと同等以上の無脂乳固形分を含む乳などを乳酸菌または酵母で発酵させ、糊状または液状にしたもの」または「これらを凍結したもの」と定義されている。発酵乳は、「容器充填後に発酵させて固化させたハードヨーグルト（固形状発酵乳、セットタイプヨーグルト）」、「発酵後にカードを粉砕し、容器充填したソフトヨーグルト（糊状発酵乳）」、「ソフトヨーグルトを均質機でさらに細かく砕き、液状の性質を高めたドリンクヨーグルト（液状発酵乳）」に大別される。また、乳等省令における「乳酸菌飲料」とは、「乳等を乳酸菌又は酵母で発酵させたものを加工し、又は主要原料とした飲料（発酵乳を除く。）」をいう。 In this embodiment, yogurt refers to “fermented milk” and “lactic acid bacteria beverage” as defined by a ministerial ordinance such as milk. “Fermented milk” in the ministerial ordinance of milk, etc. means “milk or milk containing non-fat milk solids equal to or higher than this, fermented with lactic acid bacteria or yeast to make paste or liquid” or “frozen Defined as "thing". Fermented milk is “hard yogurt fermented and solidified after filling in container (solid fermented milk, set type yogurt)”, “soft yogurt (fermented fermented milk) filled with container after fermentation and filled into containers”, “ Soft yogurt is roughly classified into drink yogurt (liquid fermented milk) that is further crushed with a homogenizer to enhance liquid properties. In addition, “lactic acid bacteria beverage” in a ministerial ordinance such as milk means “a beverage (excluding fermented milk) obtained by processing or fermenting milk or the like with lactic acid bacteria or yeast”.
　ヨーグルト原料には、乳、乳製品、乳タンパクの少なくとも一種以上が含まれる。ヨーグルト原料としては、例えば、牛乳、羊、ヤギ等の獣乳や、その加工品、殺菌乳、脱脂乳、全脂粉乳、部分脱脂乳、脱脂粉乳、全脂濃縮乳、脱脂濃縮乳、クリーム、バター、バターミルク、ホエイ、ホエイタンパク質濃縮物（ＷＰＣ）、ホエイタンパク質単離物（ＷＰＩ）、α－ラクトアルブミン（α－Ｌａ）、β－ラクトグロブリン（β－Ｌｇ）、α－カゼイン、β－カゼイン、κ－カゼイン、非たんぱく態窒素などの乳原料、砂糖、糖類、加工澱粉（デキストリンの他、可溶性澱粉、ブリティッシュスターチ、酸化澱粉、澱粉エステル、澱粉エーテル等）、食物繊維、甘味料、有機酸（リンゴ酸、クエン酸、乳酸、酒石酸など）、香料、水などを挙げることができる。なお、ヨーグルト原料は、上述のような常用の原料を混合し、加温させながら溶解させることで得られる。ヨーグルト原料には、ゼラチン、寒天、ペクチン、カルボキシメチルセルロース（ＣＭＣ）等のゲル化剤、増粘剤、安定剤が添加されてもよい。かかる場合、ゼラチン等の安定剤などを水などの溶媒に予め加温して溶解させておき、この安定剤水溶液を他の成分と混合することによって、ヨーグルト原料を調製する。 Yogurt ingredients include at least one of milk, dairy products, and milk proteins. Examples of yogurt ingredients include animal milk such as milk, sheep and goats, processed products thereof, sterilized milk, skim milk, full fat milk powder, partially skimmed milk, skim milk powder, full fat concentrated milk, skim concentrated milk, cream, Butter, buttermilk, whey, whey protein concentrate (WPC), whey protein isolate (WPI), α-lactalbumin (α-La), β-lactoglobulin (β-Lg), α-casein, β- Casein, κ-casein, milk raw materials such as non-protein nitrogen, sugar, sugar, modified starch (dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, sweetener, organic Examples include acids (malic acid, citric acid, lactic acid, tartaric acid, etc.), fragrances, and water. The yogurt raw material can be obtained by mixing conventional raw materials as described above and dissolving them while heating. Gelling agents such as gelatin, agar, pectin, and carboxymethylcellulose (CMC), thickeners, and stabilizers may be added to the yogurt raw material. In such a case, a raw material for yogurt is prepared by preliminarily heating and dissolving a stabilizer such as gelatin in a solvent such as water and mixing this aqueous stabilizer solution with other components.
　また、このヨーグルト原料の無脂乳固形分（以下「ＳＮＦ」という）は８重量％以上２０重量％以下の範囲内であることが好ましい。本実施形態に係るヨーグルト製造方法において、乳酸菌としてＥＰＳを産生するブルガリア菌とサーモフィルス菌を採用し、ｐＨ緩衝剤としてリン酸塩を採用した場合、ヨーグルトの風味を良好に保ちつつ、ヨーグルト中の菌体外多糖体（以下「ＥＰＳ」という）含有量を高めることができるからである。なお、ヨーグルト原料のＳＮＦは、８．５重量％以上１８．５重量％以下の範囲内であることがより好ましく、９重量％以上１５重量％以下の範囲内であることがさらに好ましく、９．５重量％以上１４重量％以下の範囲内であることが特に好ましい。 In addition, the non-fat milk solid content (hereinafter referred to as “SNF”) of the yogurt raw material is preferably in the range of 8 wt% to 20 wt%. In the yogurt production method according to the present embodiment, when Bulgarian bacteria and Thermophilus bacteria that produce EPS are employed as lactic acid bacteria and phosphate is employed as a pH buffering agent, while maintaining the flavor of yogurt in a good manner, This is because the exopolysaccharide content (hereinafter referred to as “EPS”) can be increased. The SNF of the yogurt raw material is more preferably in the range of 8.5% by weight to 18.5% by weight, still more preferably in the range of 9% by weight to 15% by weight. It is particularly preferable that the amount be in the range of 5% by weight to 14% by weight.
　本実施形態において、ｐＨ緩衝剤は、特に限定されないが、リン酸塩であることが特に好ましい。なお、本実施形態において、リン酸塩には水和物も含まれるものとする。リン酸塩としては、例えば、リン酸二水素一ナトリウム（ＮａＨ２ＰＯ４）、リン酸水素二ナトリウム（Ｎａ２ＨＰＯ４）、リン酸二水素一カリウム（ＫＨ２ＰＯ４）、リン酸水素二カリウム（Ｋ２ＨＰＯ４）等が挙げられる。なお、これらのリン酸塩は、単独で用いられてもよいし併用されてもよい。また、本実施形態に係るヨーグルト製造方法では、リン酸水素二ナトリウム（Ｎａ２ＨＰＯ４）、リン酸水素二カリウム（Ｋ２ＨＰＯ４）を単独あるいは組み合わせて用いることが好ましく、リン酸水素二ナトリウム（Ｎａ２ＨＰＯ４）を単独で用いることが特に好ましい。 In this embodiment, the pH buffer is not particularly limited, but is particularly preferably a phosphate. In the present embodiment, the phosphate includes a hydrate. Examples of the phosphate include monosodium dihydrogen phosphate (NaH 2 PO 4 ), disodium hydrogen phosphate (Na 2 HPO 4 ), dipotassium dihydrogen phosphate (KH 2 PO 4 ), and dihydrogen phosphate. potassium (K 2 HPO 4), and the like. In addition, these phosphates may be used independently and may be used together. In the yogurt production method according to the present embodiment, it is preferable to use disodium hydrogen phosphate (Na 2 HPO 4 ) and dipotassium hydrogen phosphate (K 2 HPO 4 ) alone or in combination, and disodium hydrogen phosphate. It is particularly preferred to use (Na 2 HPO 4 ) alone.
　また、本実施形態に係るヨーグルト製造方法のｐＨ緩衝剤添加工程において、ヨーグルト原料に対して０．０５重量％以上０．５５重量％以下の範囲内の量のｐＨ緩衝剤が添加されるのが好ましい。本実施形態に係るヨーグルト製造方法において、乳酸菌としてＥＰＳを産生するブルガリア菌とサーモフィルス菌を採用し、ｐＨ緩衝剤としてリン酸塩を採用した場合、ｐＨ緩衝剤の添加量がこの範囲内であれば、ヨーグルトのカード強度（カードテンション）や風味を良好に保ちつつ、ブルガリア菌の増殖を促進することができると共にブルガリア菌由来のＥＰＳの産生量を増強することができるからである。なお、ｐＨ緩衝剤添加量は、０．０８重量％以上０．４５重量％以下の範囲内であることがより好ましく、０．１重量％以上０．３５重量％以下の範囲内であることが特に好ましい。 In addition, in the pH buffer addition step of the yogurt production method according to this embodiment, an amount of the pH buffer in the range of 0.05 wt% or more and 0.55 wt% or less is added to the yogurt raw material. preferable. In the yogurt production method according to this embodiment, when Bulgarian bacteria and Thermophilus bacteria that produce EPS are employed as lactic acid bacteria and phosphate is employed as the pH buffering agent, the added amount of the pH buffering agent is within this range. This is because, while maintaining good yogurt card strength (card tension) and flavor, the growth of Bulgarian bacteria can be promoted and the production of EPS derived from Bulgarian bacteria can be enhanced. The addition amount of the pH buffering agent is more preferably in the range of 0.08 wt% or more and 0.45 wt% or less, and is preferably in the range of 0.1 wt% or more and 0.35 wt% or less. Particularly preferred.
　本実施形態において、乳酸菌は、菌体外機能性産生物、例えばＥＰＳを産生する乳酸菌（以下「菌体外機能性産生物産生乳酸菌」という）、例えば、ラクトバチルス・デルブルエッキー・サブスピーシス・ブルガリクス（Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｄｅｌｂｒｕｅｃｈｉｉ　ｓｕｂｓｐ．　ｂｕｌｇａｒｉｃｕｓ）種の乳酸菌（以下「ブルガリア菌」ともいう）である。本実施の形態では、乳酸菌は、菌体外機能性産生物産生乳酸菌であれば菌株を問わないが、ＥＰＳを産生するＬａｃｔｏｂａｃｉｌｌｕｓ　ｄｅｌｂｒｕｅｃｈｉｉ　ｓｕｂｓｐ．　ｂｕｌｇａｒｉｃｕｓ　ＯＬＬ１０７３Ｒ－１株（ＦＥＲＭ　ＢＰ－１０７４１）が好適である。なお、本実施形態では、ブルガリア菌を主体とすることが好ましいが、その他のＥＰＳを産生する乳酸菌（以下「ＥＰＳ産生乳酸菌」という）、例えば、ラクトバチルス・ブルガリクス（Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｂｕｌｇａｒｉｃｕｓ）や、ラクトコッカス・ラクティス・クレモリス（Ｌａｃｔｏｃｏｃｃｕｓ　ｌａｃｔｉｓ　ｓｓｐ．　ｃｒｅｍｏｒｉｓ）等をブルガリア菌と併用してもよい。 In the present embodiment, the lactic acid bacterium is an extracellular functional product such as lactic acid bacterium that produces EPS (hereinafter referred to as “extracellular functional product-producing lactic acid bacterium”), such as Lactobacillus delbruecki subspice bulgari. Lactobacillus species of Lactobacillus delbruechii subsp. Bulgaricus (hereinafter also referred to as “Bulgaria bacterium”). In the present embodiment, the lactic acid bacterium may be any strain as long as it is an extracellular functional product-producing lactic acid bacterium, but Lactobacillus delbruechii subsp. Bulgaricus OLL1073R-1 strain (FERM BP-10741) is preferred. In this embodiment, it is preferable to mainly use Bulgaria bacteria, but other lactic acid bacteria producing EPS (hereinafter referred to as “EPS-producing lactic acid bacteria”) such as Lactobacillus bulgaricus and Lactococcus. -Lactococcus lactis ssp. Cremoris may be used in combination with Bulgarian bacteria.
　また、ＥＰＳ産生乳酸菌以外の乳酸菌、例えば、サーモフィルス菌、ラクトバチルス・ガッセリ（Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｇａｓｓｅｒｉ）、ビフィズス菌、プロピオン酸菌などの発酵乳の製造に一般的に用いられるその他の乳酸菌を、ＥＰＳ産生乳酸菌と併用してもよい。また、上記乳酸菌と共に酵母を添加してもよい。なお、本実施形態に係るヨーグルト製造方法では、乳酸菌として、ＥＰＳを産生するブルガリア菌とサーモフィルス菌を併用することが特に好ましい。 In addition, lactic acid bacteria other than EPS-producing lactic acid bacteria, such as Thermophilus bacteria, Lactobacillus gasseri, Bifidobacterium, propionic acid bacteria, and other lactic acid bacteria generally used for the production of fermented milk are used. You may use together. Moreover, you may add yeast with the said lactic acid bacteria. In addition, in the yogurt manufacturing method which concerns on this embodiment, it is especially preferable to use together the Bulgarian bacterium and Thermophilus bacterium which produce EPS as a lactic acid bacterium.
　本実施形態に係るヨーグルト製造方法の発酵工程において、発酵温度を３０℃以上４０℃以下の範囲内とすることが好ましい。乳酸菌としてＥＰＳを産生するブルガリア菌とサーモフィルス菌を採用し、ｐＨ緩衝剤としてリン酸塩を採用した場合、ヨーグルト中のＥＰＳ含有量を高めることができるからである。なお、発酵温度は、３２℃以上３９℃以下の範囲内とすることがより好ましく、３４℃以上３８℃以下の範囲内とすることがさらに好ましい。 In the fermentation process of the yogurt production method according to the present embodiment, it is preferable that the fermentation temperature be in the range of 30 ° C. or higher and 40 ° C. or lower. This is because, when Bulgarian bacteria and Thermophilus bacteria that produce EPS are employed as lactic acid bacteria and phosphate is employed as a pH buffering agent, the EPS content in yogurt can be increased. The fermentation temperature is more preferably in the range of 32 ° C. or more and 39 ° C. or less, and further preferably in the range of 34 ° C. or more and 38 ° C. or less.
　本実施形態にかかる菌体外機能性産生物産生乳酸菌は、全量に対して４０ｍｇ／ｋｇ以上１００ｍｇ／ｋｇ以下の範囲内の量の菌体外機能性産生物を産生することが好ましい。例えば、乳酸菌としてＥＰＳを産生するブルガリア菌とサーモフィルス菌を採用し、ｐＨ緩衝剤としてリン酸塩を採用した場合、上記範囲内の量のＥＰＳが産生される場合においてｐＨ緩衝剤非添加時のＥＰＳの産生量に対するｐＨ緩衝剤添加時のＥＰＳの産生量の比が高くなる、すなわちｐＨ緩衝剤の添加効果が高くなることが認められるからである。なお、菌体外機能性産生物産生乳酸菌は、全量に対して４２ｍｇ／ｋｇ以上１００ｍｇ／ｋｇ以下の範囲内の量の菌体外機能性産生物を産生することが好ましく、４４ｍｇ／ｋｇ以上１００ｍｇ／ｋｇ以下の範囲内の量の菌体外機能性産生物を産生することがより好ましく、４８ｍｇ／ｋｇ以上１００ｍｇ／ｋｇ以下の範囲内の量の菌体外機能性産生物を産生することがさらに好ましく、５０ｍｇ／ｋｇ以上１００ｍｇ／ｋｇ以下の範囲内の量の菌体外機能性産生物を産生することが特に好ましい。 The extracellular functional product-producing lactic acid bacterium according to this embodiment preferably produces an extracellular functional product in an amount within the range of 40 mg / kg to 100 mg / kg with respect to the total amount. For example, when Bulgarian bacteria and Thermophilus bacteria producing EPS are used as lactic acid bacteria and phosphate is used as a pH buffering agent, when an amount of EPS within the above range is produced, the pH buffering agent is not added. This is because it is recognized that the ratio of the EPS production amount when the pH buffer agent is added to the EPS production amount is increased, that is, the effect of adding the pH buffer agent is enhanced. The extracellular functional product-producing lactic acid bacterium preferably produces an extracellular functional product in an amount in the range of 42 mg / kg to 100 mg / kg, and more than 44 mg / kg to 100 mg. It is more preferable to produce an extracellular functional product in an amount within the range of / kg or less, and to produce an extracellular functional product in an amount within the range of 48 mg / kg or more and 100 mg / kg or less. More preferably, it is particularly preferable to produce an extracellular functional product in an amount in the range of 50 mg / kg to 100 mg / kg.
　また、本実施形態に係るヨーグルト製造方法において、菌体外機能性産生物の産生量は、ｐＨ緩衝剤無添加時の産生量の１．０５倍以上であることが好ましく、１．１倍以上であることがより好ましく、１．２倍以上であることがさらに好ましく、１．２５倍以上であることが特に好ましい。例えば、乳酸菌としてＥＰＳを産生するブルガリア菌とサーモフィルス菌を採用し、ｐＨ緩衝剤としてリン酸塩を採用した場合、ｐＨ緩衝剤添加時のＥＰＳ産生量は、ｐＨ緩衝剤無添加時のＥＰＳ産生量の１．２５倍以上となる場合がある。 In the yogurt production method according to the present embodiment, the production amount of the extracellular functional product is preferably 1.05 times or more, 1.1 times or more of the production amount when no pH buffer is added. Is more preferably 1.2 times or more, and particularly preferably 1.25 times or more. For example, when Bulgarian bacteria and Thermophilus bacteria producing EPS are used as lactic acid bacteria, and phosphate is used as a pH buffering agent, the EPS production amount when adding a pH buffering agent is the EPS production when no pH buffering agent is added. It may be 1.25 times or more of the amount.
－第２実施形態－
　本実施形態に係る乳酸菌体外機能性産生物は、ｐＨ緩衝剤添加工程および発酵工程を経て製造される。ｐＨ緩衝剤添加工程では、乳原料にｐＨ緩衝剤が添加される。発酵工程では、ｐＨ緩衝剤が添加された乳原料が、乳酸菌体外機能性産生物を産生する乳酸菌によって発酵させられる。 -Second Embodiment-
The lactic acid bacterial extracellular functional product according to the present embodiment is produced through a pH buffer addition step and a fermentation step. In the pH buffer addition step, a pH buffer is added to the milk raw material. In the fermentation process, the milk raw material to which the pH buffer is added is fermented by lactic acid bacteria that produce lactic acid bacteria extracorporeal functional products.
　なお、本実施形態に係る乳酸菌体外機能性産生物は、ヨーグルト原料が乳原料とされること以外、第１実施形態に係るヨーグルト製造方法と同じである。また、本実施形態にいう「乳酸菌体外機能性産生物」は、第１実施形態にいう「菌体外機能性産生物」と同じものを示す。そして、ここにいう「乳原料」とは、牛乳、羊、ヤギ等の獣乳や、その加工品、殺菌乳、脱脂乳、全脂粉乳、部分脱脂乳、脱脂粉乳、全脂濃縮乳、脱脂濃縮乳、クリーム、バター、バターミルク、ホエイ、ホエイタンパク質濃縮物（ＷＰＣ）、ホエイタンパク質単離物（ＷＰＩ）、α－ラクトアルブミン（α－Ｌａ）、β－ラクトグロブリン（β－Ｌｇ）、α－カゼイン、β－カゼイン、κ－カゼイン、非たんぱく態窒素などである。また、この乳原料の無脂乳固形分（以下「ＳＮＦ」という）は８重量％以上２０重量％以下の範囲内であることが好ましく、８．５重量％以上１８．５重量％以下の範囲内であることがより好ましく、９重量％以上１５重量％以下の範囲内であることがさらに好ましく、９．５重量％以上１４重量％以下の範囲内であることが特に好ましい。 In addition, the lactic acid bacteria extracorporeal functional product which concerns on this embodiment is the same as the yogurt manufacturing method which concerns on 1st Embodiment except a yoghurt raw material being used as a milk raw material. Moreover, the “lactic acid extracellular functional product” referred to in the present embodiment is the same as the “extracellular functional product” referred to in the first embodiment. And the “milk raw material” here means animal milk such as cow's milk, sheep, goat, etc., processed products, sterilized milk, skim milk, whole milk powder, partially skimmed milk, skimmed milk powder, whole fat concentrated milk, skimmed milk Concentrated milk, cream, butter, buttermilk, whey, whey protein concentrate (WPC), whey protein isolate (WPI), α-lactalbumin (α-La), β-lactoglobulin (β-Lg), α -Casein, β-casein, κ-casein, non-protein nitrogen, etc. The non-fat milk solid content (hereinafter referred to as “SNF”) of the milk raw material is preferably in the range of 8% by weight to 20% by weight, and in the range of 8.5% by weight to 18.5% by weight. More preferably, it is more preferably in the range of 9% by weight to 15% by weight, and particularly preferably in the range of 9.5% by weight to 14% by weight.
　また、本実施形態に係る乳酸菌体外機能性産生物の産生量は、ｐＨ緩衝剤無添加時の乳酸菌体外機能性産生物の産生量の１．０５倍以上であることが好ましく、１．１倍以上であることがより好ましく、１．２倍以上であることがさらに好ましく、１．２５倍以上であることが特に好ましい。 Further, the production amount of the lactic acid bacteria extracellular functional product according to the present embodiment is preferably 1.05 times or more, 1.1 times the production amount of the lactic acid bacteria extracellular functional product when no pH buffer is added. More preferably, it is 1.2 times or more, more preferably 1.25 times or more.
＜実施例＞
　以下、本発明の好ましい実施例を説明するが、本発明は、前述した実施形態や以下の実施例に限定されることなく、特許請求の範囲の記載から把握される技術的範囲において種々に変更することが可能である。なお、実施例に用いられているＬａｃｔｏｂａｃｉｌｌｕｓ　ｄｅｌｂｒｕｅｃｈｉｉ　ｓｕｂｓｐ．　ｂｕｌｇａｒｉｃｕｓ　ＯＬＬ１０７３Ｒ－１株（以下、ＯＬＬ１０７３Ｒ－１株）は、２００６年１１月２９日付（受託日）で、独立行政法人産業技術総合研究所特許生物寄託センター（茨城県つくば市東１－１－１　つくばセンター　中央第６）に、受託番号でＦＥＲＭ　ＢＰ－１０７４１としてブタペスト条約に基づき国際寄託されている乳酸菌である。また、Ｓｔｒｅｐｔｃｏｃｃｕｓ　ｔｈｅｒｍｏｐｈｉｌｕｓＯＬＳ３０５９株は、２００６年１２月１５日付（受託日）で、独立行政法人産業技術総合研究所特許生物寄託センター（茨城県つくば市東１－１－１　つくばセンター　中央第６）に、受託番号でＦＥＲＭ　ＢＰ－１０７４０として、ブタペスト条約に基づき国際寄託されている乳酸菌である。なお、独立行政法人産業技術総合研究所特許生物寄託センターの特許微生物寄託業務は２０１２年４月１日をもって独立行政法人製品評価技術基盤機構に承継されており、独立行政法人製品評価技術基盤機構特許生物寄託センターは２０１３年４月１日をもって日本国千葉県木更津市かずさ鎌足２－５－８の独立行政法人製品評価技術基盤機構事業所内に移転している。 <Example>
Hereinafter, preferred examples of the present invention will be described, but the present invention is not limited to the above-described embodiments and the following examples, and various modifications are made within the technical scope grasped from the description of the scope of claims. Is possible. Note that Lactobacillus delbruechii subsp. bulgaricus OLL1073R-1 strain (hereinafter referred to as OLL1073R-1 strain) was issued on November 29, 2006 (contract date), National Institute of Advanced Industrial Science and Technology Patent Biology Center (1-1-1 Tsukuba, Tsukuba, Ibaraki Prefecture) Lactic acid bacteria are deposited internationally under the Budapest Treaty under the accession number FERM BP-10741 in the center center 6). Streptococcus thermophilus OLS3059 strain was entrusted to the National Institute of Advanced Industrial Science and Technology Patent Biology Center (Tsukuba Center 1-1-1 Tsukuba Center Central 6th, Ibaraki Prefecture) on December 15, 2006 (contract date). It is a lactic acid bacterium that has been deposited internationally under the Budapest Treaty as FERM BP-10740. In addition, the patent microorganisms deposit work of the National Institute of Advanced Industrial Science and Technology patent biological depository center was succeeded by the National Institute of Technology and Evaluation on April 1, 2012. As of April 1, 2013, the Biological Depositary Center has been relocated to the National Institute of Technology and Evaluation, 2-5-8, Kazusa Kamashika, Kisarazu City, Chiba Prefecture, Japan.
（製造例）
（１）ヨーグルト原料ミックスＡ（ＳＮＦ＝９．４重量％）の調製
　原料乳５０ｋｇ、脱脂粉乳５．４９ｋｇ、無塩バター１．５ｋｇおよび水４３．０１ｋｇを混合し、その混合物を９５℃で５分間加熱殺菌した後に３７℃前後まで冷却して、ヨーグルト原料ミックスＡを調製した。 (Production example)
(1) Preparation of yogurt raw material mix A (SNF = 9.4% by weight) 50 kg of raw milk, 5.49 kg of skim milk powder, 1.5 kg of unsalted butter and 43.01 kg of water were mixed, and the mixture was mixed at 95 ° C. for 5 After heat sterilization for minutes, the mixture was cooled to around 37 ° C. to prepare a yogurt raw material mix A.
（２）ヨーグルト原料ミックスＢ（ＳＮＦ＝１０．２重量％）の調製
　原料乳２５ｋｇ、脱脂濃縮乳１８．４ｋｇ、脱脂粉乳０．９６ｋｇ、無塩バター１．３１ｋｇおよび水４５．７５ｋｇを混合し、その混合物を９５℃で５分間加熱殺菌した後に３７℃前後まで冷却して、ヨーグルト原料ミックスＡよりも無脂乳固形分（ＳＮＦ）が高いヨーグルト原料ミックスＢを調製した。 (2) Preparation of yogurt raw material mix B (SNF = 10.2% by weight) 25 kg raw milk, 18.4 kg nonfat concentrated milk, 0.96 kg nonfat dry milk, 1.31 kg unsalted butter and 45.75 kg water are mixed, The mixture was sterilized by heating at 95 ° C. for 5 minutes and then cooled to around 37 ° C. to prepare a yogurt raw material mix B having a non-fat milk solid content (SNF) higher than that of the yogurt raw material mix A.
－ＥＰＳ産生量に及ぼすリン酸塩添加量の影響の検証－
　サーモフィルス菌ＯＬＳ３０５９株（Ｓｔｒｅｐｔｃｏｃｃｕｓ　ｔｈｅｒｍｏｐｈｉｌｕｓ　ＯＬＳ３０５９、以下ＯＬＳ３０５９株とも称する）、及び、多糖体を産生するラクトバチルス・デルブルエッキー・サブスピーシス・ブルガリクス種の乳酸菌である１０７３Ｒ－１株の混合培養物である乳酸菌スターター（ヨーグルトスターター）を、乳酸菌スターターが全量に対して２重量％含まれるようにヨーグルト原料ミックスＡに接種した後、その乳酸菌スターター入りヨーグルト原料ミックスＡを５本の試験管に２０ｍＬずつ分注して、５つのサンプル１－１、１－２、１－３、１－４、１－５を準備した。そして、サンプル１－１には、Ｎａ２ＨＰＯ４が全量に対して０．５重量％含まれるようにＮａ２ＨＰＯ４を添加した。サンプル１－２には、Ｎａ２ＨＰＯ４が全量に対して０．４重量％含み且つＮａＨ２ＰＯ４が全量に対して０．１重量％含まれるようにＮａ２ＨＰＯ４およびＮａＨ２ＰＯ４を添加した。サンプル１－３には、Ｎａ２ＨＰＯ４とＮａＨ２ＰＯ４が全量に対してそれぞれ０．２５重量％含まれるようにＮａ２ＨＰＯ４およびＮａＨ２ＰＯ４を添加した。サンプル１－４には、Ｎａ２ＨＰＯ４とＮａＨ２ＰＯ４が全量に対してそれぞれ０．５重量％含まれるようにＮａ２ＨＰＯ４およびＮａＨ２ＰＯ４を添加した。なお、サンプル１－５（対照）には、リン酸塩を添加しなかった。その後、上記各サンプルを４３℃の恒温水槽に浸漬して発酵させた。各サンプルのｐＨが４．４～４．５となった時点でサンプルを１０℃以下まで冷却し、そのサンプルの発酵を停止させた。各サンプルについて、発酵停止までの時間（発酵時間）、酸度、ＥＰＳ含有量、ブルガリア菌数、サーモフィラス菌数を測定した（表１参照）。 -Verification of the effect of phosphate addition on EPS production-
This is a mixed culture of Thermophilus OLS3059 strain (Streptcoccus thermophilus OLS3059, hereinafter also referred to as OLS3059 strain) and 1073R-1 strain, which is a lactic acid bacterium of Lactobacillus delbruecki subspice bulgaricus that produces polysaccharides. After inoculating the lactic acid bacteria starter (yoghurt starter) into the yogurt raw material mix A so that the lactic acid bacterium starter is contained in 2% by weight with respect to the total amount, dispense 20 mL of the yoghurt raw material mix A containing the lactic acid bacteria starter into 5 test tubes. Five samples 1-1, 1-2, 1-3, 1-4, and 1-5 were prepared. Then, the sample 1-1 was added Na 2 HPO 4 as Na 2 HPO 4 is contained 0.5 wt% of the total amount. The sample 1-2, Na 2 Na 2 HPO as HPO 4 comprises 0.4% by weight based on the total amount and NaH 2 PO 4 is contained 0.1% by weight based on the total amount 4 and NaH 2 PO 4 Was added. The Sample 1-3, was added Na 2 HPO 4 and NaH 2 PO 4 as Na 2 HPO 4 and NaH 2 PO 4 is contained 0.25 wt% respectively of the total amount. The sample 1-4 was added Na 2 HPO 4 and NaH 2 PO 4 as Na 2 HPO 4 and NaH 2 PO 4 is contained 0.5 wt% respectively of the total amount. In addition, phosphate was not added to Sample 1-5 (control). Then, each said sample was immersed in a 43 degreeC thermostat and fermented. When the pH of each sample reached 4.4 to 4.5, the sample was cooled to 10 ° C. or lower, and the fermentation of the sample was stopped. About each sample, time to fermentation stop (fermentation time), acidity, EPS content, the number of Bulgaria bacteria, and the number of thermophilus bacteria were measured (refer Table 1).
　ｐＨは、ガラス電極を用いたｐＨメーター（ＴＯＡ－ＨＭ５０Ｖ、東亜ディーケーケー社製）にて測定した。酸度は次の手順で測定した。各サンプルから９．００ｇを分取し、その分取サンプルに５００μＬのフェノールフタレインを添加した。そして、その分取サンプルを０．１Ｎの水酸化ナトリウムで滴定し、３０秒間、微紅色の消失しない時点を終点とした。 PH was measured with a pH meter (TOA-HM50V, manufactured by Toa DKK Corporation) using a glass electrode. The acidity was measured by the following procedure. 9.00 g was sampled from each sample, and 500 μL of phenolphthalein was added to the sample. Then, the preparative sample was titrated with 0.1N sodium hydroxide, and the end point was a time point at which the faint red color did not disappear for 30 seconds.
　また、ヨーグルトのＥＰＳ含有量は次の手順で測定した。まず、各サンプルを１０ｇずつ計り取ってそれぞれを５０ｍＬのチューブに入れ、そこに、１００％のトリクロロ酢酸１ｍＬを添加した。次に、その内容物を攪拌した後、その内容物を約４℃、約１０分で静置した。次いで、４℃の温度下２０分間、その内容物を１２０００×ｇの相対遠心力で遠心分離し、上清を新たな５０ｍＬのチューブに移した。そして、この上清を攪拌しながら、上清の２倍量の冷エタノールをその上清に徐々に添加して、上清と冷エタノールとを完全に混合した。続いて、その混合物を約４℃の温度下で一晩静置した後、前記と同様の条件下でその混合物を遠心分離した。その後、その混合物の上清を捨て、沈澱物に１０ｍＬの精製水を添加して、その沈殿物を精製水に完全に溶解させた。口径０．４５μｍのフィルター付きのシリンジを用いてその水溶液１５０μＬをＨＰＬＣに注入した。そして、「注入開始時点から１６分後付近においてＲＩ検出器によって検出される単一ピークのピーク面積」の「全ピーク面積」に対する割合をＥＰＳ含有量とした。ＨＰＬＣの分析操作条件は以下の通りである。
ＨＰＬＣシステム：　Ａｑｕｉｔｙ　Ｈ－ｃｌａｓｓ（Ｗａｔｅｒｓ）
カラム：　ＯＨｐａｋ　８０６ＨＱ（Ｓｈｏｄｅｘ）＋ＳＢ－Ｇ（Ｓｈｏｄｅｘ）
カラム温度：　４０℃
溶媒：　０．２Ｍ　ＮａＣｌ水溶液
流速：　０．５ｍＬ／ｍｉｎ
検出器：ＲＩ　ｄｅｔｅｃｔｏｒ　２４１４（Ｗａｔｅｒｓ）、検出温度４０℃
サンプルインジェクション：　１５０μＬ
分析時間：５０ｍｉｎ Moreover, the EPS content of yogurt was measured by the following procedure. First, 10 g of each sample was weighed and placed in a 50 mL tube, and 1 mL of 100% trichloroacetic acid was added thereto. Next, after the contents were stirred, the contents were allowed to stand at about 4 ° C. for about 10 minutes. The contents were then centrifuged at 12,000 × g relative centrifugal force for 20 minutes at a temperature of 4 ° C., and the supernatant was transferred to a new 50 mL tube. Then, while stirring this supernatant, cold ethanol twice the amount of the supernatant was gradually added to the supernatant, and the supernatant and cold ethanol were mixed thoroughly. Subsequently, the mixture was allowed to stand overnight at a temperature of about 4 ° C., and then the mixture was centrifuged under the same conditions as described above. Thereafter, the supernatant of the mixture was discarded, and 10 mL of purified water was added to the precipitate to completely dissolve the precipitate in purified water. 150 μL of the aqueous solution was injected into the HPLC using a syringe with a filter having a diameter of 0.45 μm. The ratio of the “peak area of a single peak detected by the RI detector around 16 minutes after the start of injection” to the “total peak area” was taken as the EPS content. The analytical operation conditions of HPLC are as follows.
HPLC system: Aquity H-class (Waters)
Column: OHpak 806HQ (Shodex) + SB-G (Shodex)
Column temperature: 40 ° C
Solvent: 0.2 M NaCl aqueous solution Flow rate: 0.5 mL / min
Detector: RI detector 2414 (Waters), detection temperature 40 ° C
Sample injection: 150 μL
Analysis time: 50 min
　ＥＰＳ含有量に及ぼすリン酸塩添加量の影響を表１に示す。リン酸塩を添加したサンプル１－１、１－２、１－３及び１－４では、対照サンプル１－５に比べて、発酵時間が長くなり、発酵終了時の酸度も１以上となった。リン酸塩の添加によって、ヨーグルトのＥＰＳ産生量が増加する結果となった。なお、リン酸塩としてＮａ２ＨＰＯ４のみを添加した場合（サンプル１－１）に、ＥＰＳ含有量が最も多かった。また、ブルガリア菌数はリン酸塩の添加によって増加したが、サーモフィルス菌はＮａ２ＨＰＯ４の濃度の上昇に伴って減少した。 Table 1 shows the influence of the phosphate addition amount on the EPS content. In Samples 1-1, 1-2, 1-3, and 1-4 to which phosphate was added, the fermentation time was longer than that of Control Sample 1-5, and the acidity at the end of fermentation was also 1 or more. . The addition of phosphate resulted in increased EPS production of yogurt. When only Na 2 HPO 4 was added as a phosphate (Sample 1-1), the EPS content was the highest. In addition, the number of Bulgarian bacteria increased with the addition of phosphate, whereas the number of Thermophilus decreased with increasing Na 2 HPO 4 concentration.
　これらの結果から、リン酸塩の添加によって乳酸菌が増殖することができるｐＨ域での発酵時間を延長することができ、この効果によってブルガリア菌の菌数が増加したと考えられる。さらに、リン酸塩、中でもＮａ２ＨＰＯ４の添加によってサーモフィルス菌の増殖低下が観察されたことから、サーモフィルス菌の増殖低下もブルガリア菌由来のＥＰＳ増加に寄与したものと考えられる。
Figure JPOXMLDOC01-appb-T000001
 From these results, the fermentation time in the pH range where lactic acid bacteria can grow can be extended by the addition of phosphate, and it is considered that the number of Bulgarian bacteria increased due to this effect. Furthermore, since a decrease in growth of Thermophilus was observed by the addition of phosphate, particularly Na 2 HPO 4 , it is considered that the decrease in growth of Thermophilus also contributed to an increase in EPS derived from Bulgaria.
Figure JPOXMLDOC01-appb-T000001
－ＥＰＳ産生量に及ぼすリン酸塩の種類の影響の検証－
　ＯＬＳ３０５９株及び１０７３Ｒ－１株の混合培養物である乳酸菌スターター（ヨーグルトスターター）を、乳酸菌スターターが全量に対して２重量％含まれるように５００ｇのヨーグルト原料ミックスＡに接種した後、その乳酸菌スターター入りヨーグルト原料ミックスＡを６本の試験管に２０ｍＬずつ分注して、６つのサンプル２－１、２－２、２－３、２－４、２－５、２－６を準備した。そして、サンプル２－１には、Ｎａ２ＨＰＯ４が全量に対して０．５重量％含まれるようにＮａ２ＨＰＯ４を添加した。サンプル２－２には、Ｋ２ＨＰＯ４が全量に対して０．６１重量％含まれるようにＫ２ＨＰＯ４を添加した。なお、サンプル２－２におけるＫ２ＨＰＯ４のモル濃度は、サンプル２－１におけるＮａ２ＨＰＯ４のモル濃度と同じである。サンプル２－３には、ＮａＣｌが全量に対して０．２１重量％含まれるようにＮａＣｌを添加した。なお、サンプル２－３におけるＮａＣｌのモル濃度は、サンプル２－１におけるＮａ２ＨＰＯ４のモル濃度と同じである。サンプル２－４には、Ｎａ２ＨＰＯ４が全量に対して０．３重量％含まれるようにＮａ２ＨＰＯ４を添加した。サンプル２－５には、Ｎａ２ＨＰＯ４が全量に対して０．１重量％含まれるようにＮａ２ＨＰＯ４を添加した。なお、サンプル２－６（対照）には、リン酸塩を添加しなかった。その後、上記各サンプルを４３℃の恒温水槽に浸漬して発酵させた。各サンプルのｐＨが４．４～４．５となった時点でサンプルを１０℃以下まで冷却し、そのサンプルの発酵を停止させた。各サンプルについて、発酵停止までの時間（発酵時間）、酸度、ＥＰＳ含有量、ブルガリア菌数、サーモフィルス菌数を測定した（表２参照）。なお、これらの物性値は実施例１と同様の方法で測定した。 -Verification of the effect of phosphate type on EPS production-
Lactic acid bacteria starter (yoghurt starter), which is a mixed culture of OLS 3059 and 1073R-1 strains, was inoculated into 500 g of yoghurt raw material mix A so as to contain 2% by weight of the lactic acid bacteria starter. The yogurt raw material mix A was dispensed into 6 test tubes each 20 mL to prepare 6 samples 2-1, 2-2, 2-3, 2-4, 2-5, 2-6. In Sample 2-1, Na 2 HPO 4 was added so that 0.5% by weight of Na 2 HPO 4 was contained in the total amount. The sample 2-2 was added K 2 HPO 4 as K 2 HPO 4 is contained 0.61 wt% of the total amount. The molar concentration of K 2 HPO 4 in sample 2-2 is the same as the molar concentration of Na 2 HPO 4 in sample 2-1. In Sample 2-3, NaCl was added so that NaCl was contained at 0.21% by weight based on the total amount. The molar concentration of NaCl in Sample 2-3 is the same as the molar concentration of Na 2 HPO 4 in Sample 2-1. The sample 2-4 was added Na 2 HPO 4 as Na 2 HPO 4 is contained 0.3 wt% of the total amount. The sample 2-5 was added Na 2 HPO 4 as Na 2 HPO 4 is contained 0.1% by weight based on the total amount. Note that phosphate was not added to Sample 2-6 (control). Then, each said sample was immersed in a 43 degreeC thermostat and fermented. When the pH of each sample reached 4.4 to 4.5, the sample was cooled to 10 ° C. or lower, and the fermentation of the sample was stopped. About each sample, time to fermentation stop (fermentation time), acidity, EPS content, the number of Bulgaria bacteria, and the number of thermophilus bacteria were measured (refer Table 2). These physical property values were measured in the same manner as in Example 1.
　ＥＰＳ含有量に及ぼすリン酸塩の種類の影響を表２に示す。Ｎａ２ＨＰＯ４のみを添加したサンプル２－１、２－４、２－５では、Ｎａ２ＨＰＯ４の添加量の増加に伴って発酵時間が長くなると共にＥＰＳ含有量が増加した。また、Ｎａ２ＨＰＯ４の添加量の増加に伴ってブルガリア菌の菌数は増加し、サーモフィルス菌の菌数は減少した。ここで、サーモフィルス菌の菌数の減少に影響する要因を調べた。具体的には、サンプル２－１、サンプル２－２及びサンプル２－３について、サーモフィルス菌の菌数などを対比した。サンプル２－２のサーモフィルス菌の菌数およびＥＰＳ含有量は、サンプル２－１のサーモフィルス菌の菌数およびＥＰＳ含有量とほぼ同等であった。サンプル２－３では、サンプル２－６（対照）とほぼ同様に、ＥＰＳ含有量の増加やサーモフィルス菌の菌数の減少は見られなかった。なお、これは、添加したＮａＣｌがｐＨ緩衝作用を発揮しなかったためであると思われる。したがって、リン酸塩の添加がサーモフィルス菌の増殖を抑制している可能性が示唆された。
Figure JPOXMLDOC01-appb-T000002
 Table 2 shows the influence of the type of phosphate on the EPS content. In Samples 2-1, 2-4, and 2-5 to which only Na 2 HPO 4 was added, the fermentation time was increased and the EPS content was increased as the amount of Na 2 HPO 4 added was increased. In addition, the number of Bulgarian bacteria increased and the number of Thermofilus bacteria decreased as the amount of Na 2 HPO 4 added increased. Here, the factors affecting the decrease in the number of thermophilus bacteria were examined. Specifically, the numbers of thermophilus bacteria were compared for sample 2-1, sample 2-2, and sample 2-3. The number and EPS content of Thermophilus bacteria in Sample 2-2 were almost the same as the number and EPS content of Thermophilus bacteria in Sample 2-1. In sample 2-3, as in sample 2-6 (control), no increase in EPS content or decrease in the number of thermophilus bacteria was observed. This seems to be because the added NaCl did not exhibit the pH buffering action. Therefore, it was suggested that the addition of phosphate may suppress the growth of Thermophilus bacteria.
Figure JPOXMLDOC01-appb-T000002
　－ＥＰＳ産生量およびカードテンションに及ぼすリン酸塩添加量の影響の検証－
　１００ｋｇのヨーグルト原料ミックスＡを２０ｋｇずつに分け、５つのサンプル３－１、３－２、３－３、３－４、３－５を準備した。サンプル３－１（対照）には、リン酸塩を添加しなかった。サンプル３－２には、Ｎａ２ＨＰＯ４が０．１重量％含まれるようにＮａ２ＨＰＯ４を添加した。サンプル３－３には、Ｎａ２ＨＰＯ４が０．３重量％含まれるようにＮａ２ＨＰＯ４を添加した。サンプル３－４には、Ｎａ２ＨＰＯ４が０．５重量％含まれるようにＮａ２ＨＰＯ４を添加した。サンプル３－５には、Ｎａ２ＨＰＯ４が１．０重量％含まれるようにＮａ２ＨＰＯ４を添加した。そして、ヨーグルトの製造に一般的に使用されるサーモフィラス菌及び１０７３Ｒ－１株の混合培養物である乳酸菌スターターを、乳酸菌スターターが全量に対して２重量％含まれるように各サンプルに接種した。その後、乳酸菌スターター入りの各サンプルをそれぞれ８５ｇ容量の容器に８５ｇ充填して４３℃の恒温室で発酵させた。各サンプルのｐＨが４．４になった時点でサンプルを１０℃以下まで冷却し、そのサンプルの発酵を停止させた。 -Verification of the effect of phosphate addition on EPS production and card tension-
100 kg of yogurt raw material mix A was divided into 20 kg, and five samples 3-1, 3-2, 3-3, 3-4, 3-5 were prepared. Sample 3-1 (control) received no phosphate. The sample 3-2 was added Na 2 HPO 4 as Na 2 HPO 4 is contained 0.1 wt%. In Sample 3-3, Na 2 HPO 4 was added so that 0.3 wt% of Na 2 HPO 4 was contained. The sample 3-4 was added Na 2 HPO 4 as Na 2 HPO 4 is contained 0.5 wt%. The sample 3-5 was added Na 2 HPO 4 as Na 2 HPO 4 is contained 1.0 wt%. Then, each sample was inoculated with a lactic acid bacteria starter which is a mixed culture of Thermophilus bacteria and 1073R-1 strain generally used in the production of yogurt so that the lactic acid bacteria starter was contained at 2% by weight. Thereafter, 85 g of each sample containing the lactic acid bacteria starter was filled in an 85 g capacity container and fermented in a thermostatic chamber at 43 ° C. When the pH of each sample reached 4.4, the sample was cooled to 10 ° C. or lower, and the fermentation of the sample was stopped.
　そして、各サンプルについて、発酵停止までの時間（発酵時間）、ＥＰＳ含有量、カードテンション（ＣＴ）を測定した（表３参照）。なお、各サンプル中のＥＰＳ含有量は、実施例１と同様に測定した。ＣＴは次の手順で測定した。各サンプルに１００ｇの錘をつけ、カードメーターマックス（ＭＥ－５００、飛鳥機器社製）によって各サンプルが破断に至るときの弾力性を測定した。そして、この弾性力をＣＴ指標値とした。 And about each sample, time to fermentation stop (fermentation time), EPS content, and card tension (CT) were measured (refer Table 3). The EPS content in each sample was measured in the same manner as in Example 1. CT was measured by the following procedure. A 100 g weight was attached to each sample, and the elasticity when each sample was broken was measured by Card Meter Max (ME-500, manufactured by Asuka Kikai Co., Ltd.). This elastic force was used as the CT index value.
　各サンプルの発酵時間、ＥＰＳ含有量、ＣＴの結果を表３に示す。Ｎａ２ＨＰＯ４の添加量が増加するに伴ってサンプル中のＥＰＳ含有量が増加し、ＣＴ値が減少した（すなわちサンプルが柔らかくなった）。
Figure JPOXMLDOC01-appb-T000003
 Table 3 shows the fermentation time, EPS content, and CT results of each sample. As the amount of Na 2 HPO 4 added increased, the EPS content in the sample increased and the CT value decreased (ie the sample became softer).
Figure JPOXMLDOC01-appb-T000003
　－ＥＰＳ産生量に及ぼすヨーグルト原料ミックスのＳＮＦ含有量の影響の検証－
　ＯＬＳ－３０５９株及び１０７３Ｒ－１株の混合培養物である乳酸菌スターター（ヨーグルトスターター）を、乳酸菌スターターが全量に対して２重量％含まれるようにヨーグルト原料ミックスＡ及びヨーグルト原料ミックスＢにそれぞれ接種した後、乳酸菌スターター入りの各ヨーグルト原料ミックスＡ，Ｂを８５ｇ容量の容器に８５ｇずつ分注して、２種類のサンプル４－１（ＳＮＦ＝９．４重量％）、４－２（ＳＮＦ＝１０．２重量％）を準備した。そして、これらのサンプル４－１，２を４３℃の恒温室で発酵させた。各サンプルの酸度が０．８になった時点でサンプルを１０℃以下まで冷却し、そのサンプルの発酵を停止させた。各サンプル中のＥＰＳ含有量を測定した。なお、ＥＰＳ含有量は実施例１と同様の方法で測定した。 -Verification of the effect of SNF content of yogurt raw material mix on EPS production-
Lactic acid bacteria starter (yogurt starter), which is a mixed culture of OLS-3059 strain and 1073R-1 strain, was inoculated into yogurt raw material mix A and yogurt raw material mix B so that the lactic acid bacteria starter was contained at 2% by weight with respect to the total amount. Thereafter, 85 g of each yoghurt raw material mix A and B containing lactic acid bacteria starter was dispensed into a 85 g capacity container, and two types of samples 4-1 (SNF = 9.4 wt%), 4-2 (SNF = 10 2% by weight). These samples 4-1 and 2 were fermented in a constant temperature room at 43 ° C. When the acidity of each sample reached 0.8, the sample was cooled to 10 ° C. or lower, and the fermentation of the sample was stopped. The EPS content in each sample was measured. The EPS content was measured by the same method as in Example 1.
　サンプル４－１のＥＰＳ含有量は４６．０ｍｇ／ｋｇであった。これに対し、サンプル４－２のＥＰＳ含有量は４８．６ｍｇ／ｋｇであった。すなわち、サンプル４－２のＥＰＳ含有量は、サンプル４－１の１．１倍であった。したがって、高ＳＮＦのヨーグルト原料を使用することによりヨーグルト中のＥＰＳ含有量を高めることができることが明らかとなった。 The EPS content of Sample 4-1 was 46.0 mg / kg. On the other hand, the EPS content of Sample 4-2 was 48.6 mg / kg. That is, the EPS content of Sample 4-2 was 1.1 times that of Sample 4-1. Therefore, it became clear that the EPS content in yogurt can be increased by using a high SNF yogurt raw material.
　－ＥＰＳ産生量に及ぼす発酵温度の影響の検証－
　ＯＬＳ－３０５９株及び１０７３Ｒ－１株の混合培養物である乳酸菌スターター（ヨーグルトスターター）を、乳酸菌スターターが全量に対して２重量％含まれるようにヨーグルト原料ミックスＡに接種した後、その乳酸菌スターター入りヨーグルト原料ミックスＡを８５ｇ容量の容器に８５ｇずつ分注して、２種類のサンプル５－１、５－２を準備した。そして、サンプル５－１を４３℃で発酵させ、サンプル５－２を３７℃で発酵させた。各サンプルの酸度が０．８になった時点でサンプルを１０℃以下まで冷却し、そのサンプルの発酵を停止させた。各サンプル中のＥＰＳ含有量を測定した。なお、ＥＰＳ含有量は実施例１と同様の方法で測定した。 -Verification of the effect of fermentation temperature on EPS production-
After inoculating the lactic acid bacteria starter (yogurt starter), which is a mixed culture of OLS-3059 and 1073R-1 strains, into the yogurt raw material mix A so that the lactic acid bacteria starter is contained in 2% by weight, the lactic acid bacteria starter is contained. Two types of samples 5-1 and 5-2 were prepared by dispensing 85 g of yoghurt raw material mix A into a 85 g capacity container. Sample 5-1 was then fermented at 43 ° C., and sample 5-2 was fermented at 37 ° C. When the acidity of each sample reached 0.8, the sample was cooled to 10 ° C. or lower, and the fermentation of the sample was stopped. The EPS content in each sample was measured. The EPS content was measured by the same method as in Example 1.
　サンプル５－１のＥＰＳ含有量は４３．４ｍｇ／ｋｇであった。これに対し、サンプル５－２のＥＰＳ含有量は５１．１ｍｇ／ｋｇであった。すなわち、サンプル５－２のＥＰＳ含有量は、サンプル５－１の１．２倍であった。したがって、発酵温度を低温にすることで、ヨーグルト中のＥＰＳ含有量を高めることができることが明らかとなった。 The EPS content of Sample 5-1 was 43.4 mg / kg. In contrast, the EPS content of Sample 5-2 was 51.1 mg / kg. That is, the EPS content of Sample 5-2 was 1.2 times that of Sample 5-1. Therefore, it became clear that the EPS content in yogurt can be increased by lowering the fermentation temperature.
　先ず、サンプル６－１（対照）としてヨーグルト原料ミックスＡを準備した。また、ヨーグルト原料ミックスＢを３つに分けて、３つのサンプル６－２、６－３、６－４を準備した。そして、ヨーグルトの製造に一般的に使用されるサーモフィラス菌及び１０７３Ｒ－１株の混合培養物である乳酸菌スターターを、乳酸菌スターターが全量に対して２重量％含まれるように各サンプルに接種した。また、サンプル６－４には、さらに、Ｎａ２ＨＰＯ４が全量に対して０．３重量％含まれるようにＮａ２ＨＰＯ４を添加した。そして、サンプル６－１および６－２を４３℃で発酵させ、サンプル６－３および６－４を３７℃で発酵させた。各サンプルの酸度が０．８になった時点でサンプルを１０℃以下まで冷却し、そのサンプルの発酵を停止させた。各サンプル中のＥＰＳ含有量を測定した。なお、ＥＰＳ含有量は実施例１と同様の方法で測定した。 First, a yogurt raw material mix A was prepared as sample 6-1 (control). The yogurt raw material mix B was divided into three to prepare three samples 6-2, 6-3, and 6-4. Then, each sample was inoculated with a lactic acid bacteria starter which is a mixed culture of Thermophilus bacteria and 1073R-1 strain generally used in the production of yogurt so that the lactic acid bacteria starter was contained at 2% by weight. Further, the sample 6-4 was further added Na 2 HPO 4 as Na 2 HPO 4 is contained 0.3 wt% of the total amount. Samples 6-1 and 6-2 were then fermented at 43 ° C, and samples 6-3 and 6-4 were fermented at 37 ° C. When the acidity of each sample reached 0.8, the sample was cooled to 10 ° C. or lower, and the fermentation of the sample was stopped. The EPS content in each sample was measured. The EPS content was measured by the same method as in Example 1.
　各サンプルのＳＮＦ、発酵温度、リン酸塩（Ｎａ２ＨＰＯ４）添加量、ヨーグルト中のＥＰＳ含有量を表４に示す。サンプル６－２（高ＳＮＦ含有量）ではＥＰＳ含有量が対照サンプル６－１の１．１５倍に高められ、サンプル６－３（高ＳＮＦ含有量，低発酵温度）ではＥＰＳ含有量が対照サンプル６－１の１．１９倍にまで高められた。さらに、サンプル６－４（高ＳＮＦ含有量，低発酵温度，リン酸塩添加）では、ヨーグルトのＥＰＳ含有量が対照サンプル６－１の１．３７倍にまで高められた。
Figure JPOXMLDOC01-appb-T000004
 Table 4 shows SNF, fermentation temperature, phosphate (Na 2 HPO 4 ) addition amount, and EPS content in yogurt for each sample. In sample 6-2 (high SNF content), the EPS content is increased to 1.15 times that of the control sample 6-1, and in sample 6-3 (high SNF content, low fermentation temperature), the EPS content is increased to the control sample. It was increased to 1.19 times 6-1. Furthermore, in sample 6-4 (high SNF content, low fermentation temperature, phosphate addition), the EPS content of yogurt was increased to 1.37 times that of control sample 6-1.
Figure JPOXMLDOC01-appb-T000004
　本発明に係るヨーグルトの製造方法は、乳酸菌由来の機能性産生物の含有量を効率的に増加させることができるため、乳酸菌由来の機能性産生物（多糖体など）が有する健康増進作用を高めたヨーグルトや、有効量の乳酸菌由来の機能性産生物を十分含有した小容量のヨーグルトを効率よく製造することができる。 Since the method for producing yogurt according to the present invention can efficiently increase the content of functional products derived from lactic acid bacteria, it enhances the health promoting action of functional products derived from lactic acid bacteria (polysaccharides, etc.). Yogurt and a small volume of yogurt sufficiently containing an effective amount of a functional product derived from lactic acid bacteria can be efficiently produced.

Claims (12)

  1. 　ヨーグルト原料にｐＨ緩衝剤を添加するｐＨ緩衝剤添加工程と、
    　前記ｐＨ緩衝剤が添加された前記ヨーグルト原料（以下「ｐＨ緩衝剤添加ヨーグルト原料」という）を乳酸菌によって発酵させる発酵工程と
    を備える、ヨーグルト製造方法。     A pH buffering agent adding step of adding a pH buffering agent to the yogurt raw material;
    A yogurt production method comprising: a fermentation step of fermenting the yogurt material to which the pH buffer is added (hereinafter referred to as “pH buffer added yogurt material”) with lactic acid bacteria.
  2. 　前記乳酸菌は、少なくとも、菌体外多糖体産生能力を有する乳酸菌を含む
    請求項１に記載のヨーグルト製造方法。     The method for producing yogurt according to claim 1, wherein the lactic acid bacterium includes at least a lactic acid bacterium having an exopolysaccharide-producing ability.
  3. 　前記菌体外多糖体産生能力を有する乳酸菌は、少なくとも、ラクトバチルス・デルブルエッキー・サブスピーシス・ブルガリクス種の乳酸菌を含む
    請求項２に記載のヨーグルト製造方法。     The method for producing yogurt according to claim 2, wherein the lactic acid bacteria having the ability to produce exopolysaccharides include at least a lactic acid bacterium of Lactobacillus delbruecky subspices bulgaricus species.
  4. 　前記ｐＨ緩衝剤は、リン酸塩である
    請求項１から３のいずれかに記載のヨーグルト製造方法。     The yogurt production method according to any one of claims 1 to 3, wherein the pH buffer is a phosphate.
  5. 　前記リン酸塩は、リン酸水素二ナトリウムおよびリン酸水素二カリウムより成る群から選択される少なくとも一方のリン酸塩である
    請求項４に記載のヨーグルト製造方法。     The method for producing yogurt according to claim 4, wherein the phosphate is at least one phosphate selected from the group consisting of disodium hydrogen phosphate and dipotassium hydrogen phosphate.
  6. 　前記ヨーグルト原料には、８重量％以上２０重量％以下の範囲内の無脂乳固形分が含まれている
    請求項１から５のいずれか１項に記載のヨーグルト製造方法。     The yogurt manufacturing method according to any one of claims 1 to 5, wherein the yogurt raw material contains a non-fat milk solid content in a range of 8 wt% to 20 wt%.
  7. 　前記発酵工程では、前記ｐＨ緩衝剤添加ヨーグルト原料が３０℃以上４０℃以下の範囲内の温度下で前記乳酸菌によって発酵させられる
    請求項１から６のいずれか１項に記載のヨーグルト製造方法。     The method for producing yogurt according to any one of claims 1 to 6, wherein, in the fermentation step, the yogurt raw material to which the pH buffer is added is fermented by the lactic acid bacteria at a temperature within a range of 30 ° C or higher and 40 ° C or lower.
  8. 　請求項１から７のいずれか１項に記載のヨーグルト製造方法によって得られるヨーグルト。 Yogurt obtained by the method for producing yogurt according to any one of claims 1 to 7.
  9. 　乳酸菌と、
    　ｐＨ緩衝剤と
    を含有するヨーグルト。     With lactic acid bacteria,
    Yogurt containing a pH buffer.
  10. 　前記乳酸菌は、乳酸菌体外機能性産生物を産生する乳酸菌であり、
    　全量に対して４０ｍｇ／ｋｇ以上１００ｍｇ／ｋｇ以下の範囲内の量の乳酸菌体外機能性産生物をさらに含有する
    請求項９に記載のヨーグルト。     The lactic acid bacterium is a lactic acid bacterium that produces a lactic acid bacterium extracorporeal functional product,
    The yoghurt of Claim 9 which further contains the lactic-acid-bacteria extracellular functional product of the quantity within the range of 40 mg / kg or more and 100 mg / kg or less with respect to the whole quantity.
  11. 　乳原料にｐＨ緩衝剤を添加するｐＨ緩衝剤添加工程と、
    　前記ｐＨ緩衝剤が添加された前記乳原料を、乳酸菌体外機能性産生物を産生する乳酸菌によって発酵させる発酵工程と
    を備える、乳酸菌体外機能性産生物製造方法。     Adding a pH buffering agent to the milk material;
    A method for producing a lactic acid bacterial extracellular functional product, comprising a fermentation step of fermenting the milk material to which the pH buffer is added with a lactic acid bacterium producing a lactic acid bacterial extracellular functional product.
  12. 　ｐＨ緩衝剤を有効成分とする乳酸菌体外機能性産生物増産剤。 Lactic acid bacteria extra-functional product enhancer with pH buffer as active ingredient.
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