JP6282446B2 - Bacteriostatic or antibacterial agent and method for producing the same - Google Patents
Bacteriostatic or antibacterial agent and method for producing the same Download PDFInfo
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- JP6282446B2 JP6282446B2 JP2013236175A JP2013236175A JP6282446B2 JP 6282446 B2 JP6282446 B2 JP 6282446B2 JP 2013236175 A JP2013236175 A JP 2013236175A JP 2013236175 A JP2013236175 A JP 2013236175A JP 6282446 B2 JP6282446 B2 JP 6282446B2
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- bacteriostatic
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- 239000006273 synthetic pesticide Substances 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- STCOOQWBFONSKY-UHFFFAOYSA-N tributyl phosphate Chemical compound CCCCOP(=O)(OCCCC)OCCCC STCOOQWBFONSKY-UHFFFAOYSA-N 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- DQWPFSLDHJDLRL-UHFFFAOYSA-N triethyl phosphate Chemical compound CCOP(=O)(OCC)OCC DQWPFSLDHJDLRL-UHFFFAOYSA-N 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 239000010457 zeolite Substances 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Description
本発明は、乳またはホエーに含まれる低分子の静菌または抗菌剤及びその製造方法に関する。また、当該静菌または抗菌剤を含有することを特徴とする植物病原菌防除剤、飲食品、栄養組成物または、飼料等に関する。 The present invention relates to a low-molecular bacteriostatic or antibacterial agent contained in milk or whey and a method for producing the same. The present invention also relates to a phytopathogenic fungus control agent, food and drink, nutritional composition, feed, etc., characterized by containing the bacteriostatic or antibacterial agent.
チーズ製造において副産物として生成するホエーには、良質な水溶性のタンパク質のほか、乳糖やミネラルなど様々な栄養素が豊富に含まれているが、近年はその有効利用のため、様々な処理工程を経て多様なホエー加工品が生産されている。特に、ホエーより乳糖及びミネラルを限外ろ過膜などにより除去した後、噴霧乾燥し、非タンパク質物質を最小化するように加工したホエータンパク質コンセントレート（ＷＰＣ）またはホエータンパク質アイソレート（ＷＰＩ）などは世界的に製造量・流通量の多い製品であり、ホエーをそのまま粉末化したホエー粉よりもタンパク質含量が多く、良好な溶解性、泡だち性、ゲル化能といった機能的な特性を備えているものもある。
ホエーの限外ろ過膜処理の際に副産物として生成する膜通過画分、ホエーの限外ろ過パーミエイトは乳糖が主要な成分であり、この乳糖もまた濃縮・結晶化の工程を経て回収され、そのまままたは精製された後、製品化されている。しかしながら、ホエーの限外ろ過パーミエイトから乳糖を除いた溶液については全く活用されることなく、廃液として処理されているのが現状である。
Whey produced as a by-product in cheese manufacture is rich in high-quality water-soluble proteins and various nutrients such as lactose and minerals. A variety of processed whey products are produced. In particular, whey protein concentrate (WPC) or whey protein isolate (WPI) processed to minimize non-protein substances after removing lactose and minerals from whey with an ultrafiltration membrane, etc. It is a product with a large amount of production and distribution worldwide, and it has higher protein content than whey powder that is powdered whey as it is, and has functional characteristics such as good solubility, foaminess, and gelling ability. Some are.
Membrane-passed fraction produced as a by-product during whey ultrafiltration membrane treatment, whey ultrafiltration permeate is the main component of lactose, and this lactose is also recovered through the concentration and crystallization process, Or it is commercialized after being purified. However, at present, the solution obtained by removing lactose from the whey ultrafiltration permeate is not used at all and is treated as a waste liquid.
一方、野菜等の植物に発生する病気には、多種多様な病原性菌が関与していることが知られている。例えば真菌であれば、マメ類、ムギ類、樹木類、果樹類に被害をもたらす担子菌類の銹病菌は世界で約５，０００種、わが国では約８７０種が知られている。また、多くの植物体に感染する子嚢菌類のうどんこ病菌には7属の病原かびがあり、野菜類や果樹類に病気をおこす不完全菌類の黒かび病菌(Aspergillus niger)等も昔から知られている。わが国の農業で最も重要なイネの真菌病として、いもち病菌(Pyricularia oryzae)や紋枯病菌(Rhizoctonia solani)がある。また、シュードモナス属、キサントモナス属、エルビニア属、アグロバクテリウム属、コリネバクテリウム属、ストレプトマイセス属、スピロプラスマ属のほかマイコプラスマ様細菌など多くの病原性細菌も知られている。特に我が国で問題となるイネの細菌病には、褐条病菌(Pseudomonas syringae)、籾枯病菌(Pseudomonas glumae)、葉鞘褐変病菌(Pseudomonas marginalis)及び白葉枯病菌(Xanthomonas campestris pv.orizae)がある。これらを防除するための農薬はあるが、たとえば収穫直前のブロッコリーの花蕾のように化学合成農薬を散布すると残留が問題となる場合などについては依然として防除する手立てが無い。よって、これらの病原菌を抑制する食品由来の静菌または抗菌性物質があれば、散布後もそのまま摂食することが可能であり、残留による健康への影響も無いため、有機農業にも活用することができる。
また、これらの病原菌は土壌伝染性であるため、事前に土壌中のこれらの病原菌を抑制することで、野菜などの病害予防が可能になると考えられる。
On the other hand, it is known that various pathogenic bacteria are involved in diseases occurring in plants such as vegetables. For example, in the case of fungi, there are about 5,000 mania of basidiomycetous fungi that cause damage to legumes, wheat, trees, fruit trees, and about 870 in Japan. In addition, there are 7 genera of fungal powdery mildew that infects many plant bodies, and incomplete fungi such as Aspergillus niger that cause diseases in vegetables and fruit trees have long been used. Are known. Rice fungi (Pyricularia oryzae) and blight fungus (Rhizoctonia solani) are the most important rice fungal diseases in agriculture in Japan. Further, many pathogenic bacteria such as Pseudomonas, Xanthomonas, Erbinia, Agrobacterium, Corynebacterium, Streptomyces, Spiroplasma, and Mycoplasma-like bacteria are also known. Rice bacterial diseases particularly problematic in Japan include Pseudomonas syringae, Pseudomonas glumae, Pseudomonas marginalis, and Xanthomonas campestris pv. Orizae. There are pesticides for controlling them, but there is still no way to control them when, for example, chemical synthetic pesticides become a problem when sprayed with, for example, a broccoli flower just before harvesting. Therefore, if there are bacteriostatic or antibacterial substances derived from foods that suppress these pathogens, they can be eaten as they are after spraying, and there is no health impact due to residues, so they are also used for organic agriculture be able to.
Moreover, since these pathogenic bacteria are soil infectious, it is thought that disease prevention of vegetables etc. becomes possible by suppressing these pathogenic bacteria in soil beforehand.
畜産分野において、家畜の感染症予防・治療に用いられる抗生物質量はヒトへの使用量の2倍以上と言われており、多薬耐性菌の出現等の問題が深刻化している。そのため、抗生物質を可能な限り使用せずに飼育することが強く望まれている。そこで、病原性菌に対して抑制効果のある食品副産物があれば、飼料に加えて投与することにより感染症を予防し、安心、安全かつ経済的な家畜の飼育が可能となる。 In the field of livestock, the amount of antibiotics used for the prevention and treatment of infectious diseases in livestock is said to be more than twice the amount used for humans, and problems such as the emergence of multidrug-resistant bacteria are becoming more serious. Therefore, it is strongly desired to breed without using antibiotics as much as possible. Therefore, if there is a food by-product that has an inhibitory effect on pathogenic bacteria, it can be administered in addition to feed to prevent infectious diseases, and safe, safe and economical livestock breeding becomes possible.
乳中には多数のタンパク性抗菌物質が含まれていることが知られており、例えば、乳清ホエータンパク質と水溶性銀化合物を混和した繊維の抗菌加工方法（特許文献１）、病原菌に対する抗体を含む乳を用いた感染症予防食品添加用組成物（特許文献２）、動物の血液及び乳汁中のグロブリン含有物を抗菌剤として利用した豚用飼料（特許文献３）等が開示されている。また、ニューロキノン系抗菌剤と乳由来ラクトフェリン類の加水分解物またはラクトフェリン類の加水分解物由来の抗菌性ペプチドを有効成分として含有する抗菌剤（特許文献４）、水不溶性銀含有乳清蛋白質加水分解物の製造法（特許文献５）等も開示されている。更に、乳タンパク質以外の抗菌成分として、ホエー由来の遊離脂肪酸、主に酪酸、カプロン酸、カプリル酸、カプリン酸及びラウリン酸の一つ又は複数から選ばれる遊離脂肪酸で構成される抗菌性組成物（特許文献６）も開示されている。 It is known that milk contains many protein antibacterial substances. For example, an antibacterial processing method for fibers mixed with whey whey protein and a water-soluble silver compound (Patent Document 1), antibodies against pathogenic bacteria Infectious disease preventive food additive composition using milk containing milk (patent document 2), pig feed using globulin-containing substances in animal blood and milk as an antibacterial agent (patent document 3) and the like are disclosed . In addition, an antibacterial agent (Patent Document 4) containing a neuroquinone antibacterial agent and a lactoferrin hydrolyzate or an antibacterial peptide derived from a hydrolyzate of lactoferrin as active ingredients, water-insoluble silver-containing whey protein hydrolyzate A method for producing a decomposition product (Patent Document 5) and the like are also disclosed. Furthermore, as an antibacterial component other than milk protein, an antibacterial composition comprising free fatty acids derived from whey, mainly free fatty acids selected from one or more of butyric acid, caproic acid, caprylic acid, capric acid and lauric acid ( Patent document 6) is also disclosed.
上述したように、乳タンパク質、脂肪酸の抗菌性については報告があるものの、特許文献１〜６については、用いる原料や製造工程が煩雑であったり、十分な効果が得られない等の様々な問題が認められる。
また、特許文献５では、乳タンパク質の加水分解物を抗菌剤として用いているが、乳成分単独の効果ではなく、さらに乳成分を加水分解する工程を有するため製造工程が煩雑である等の問題が認められる。
さらに、特許文献６の遊離脂肪酸の抗菌剤は、乳化剤として乳タンパク等が必要なこと、遊離脂肪酸の乳化組成物はそれらの合同融点未満の温度ではほとんどまたは全く抗菌効力を持たないことが開示されており、これらの点から実用化においては問題点が多い。
一方で、乳タンパク質、脂肪酸以外の低分子物質の静菌または抗菌性については全く報告がされていない。また、乳やホエーの限外ろ過パーミエイトについて静菌または抗菌性の効果を有する報告はなされていない。
As described above, although there are reports on the antibacterial properties of milk proteins and fatty acids, for Patent Documents 1 to 6, there are various problems such as complicated raw materials and manufacturing processes, and insufficient effects. Is recognized.
Moreover, in patent document 5, although the hydrolyzate of milk protein is used as an antibacterial agent, since it has the process of hydrolyzing a milk component further rather than the effect of a milk component single, problems, such as a complicated manufacturing process, are included. Is recognized.
Furthermore, it is disclosed that the free fatty acid antibacterial agent of Patent Document 6 requires milk protein or the like as an emulsifier, and that the free fatty acid emulsion composition has little or no antibacterial efficacy at temperatures below their combined melting point. From these points, there are many problems in practical use.
On the other hand, there has been no report on bacteriostatic or antibacterial properties of low molecular weight substances other than milk proteins and fatty acids. In addition, no report has been made on the ultrafiltration permeate of milk or whey having a bacteriostatic or antibacterial effect.
したがって、本発明は、乳またはホエーに含まれる低分子画分の有効利用を目的とし、乳またはホエーより静菌または抗菌性物質を分離する方法を提供すること、及び当該静菌または抗菌物質による植物病原菌除去、腸内環境改善等を課題とする。特に、農薬を使用できない野菜の病原菌防除や、抗生物質の使用を控えたい家畜の感染症予防等を課題とする。 Therefore, the present invention aims at effective use of a low molecular fraction contained in milk or whey, and provides a method for separating bacteriostatic or antibacterial substances from milk or whey, and the bacteriostatic or antibacterial substances. The subjects are removal of plant pathogens and improvement of the intestinal environment. In particular, the subjects are the control of pathogens in vegetables that cannot use pesticides and the prevention of infectious diseases in domestic animals that want to refrain from using antibiotics.
上記課題を解決するため鋭意検討した所、本発明者らは、ホエーの限外ろ過パーミエイトを更に分画し、その有効利用を目指して各種のスクリーニングを実施した結果、ホエーの限外ろ過パーミエイトから乳糖等の水溶性分子を除いた画分に、土壌細菌に対する高い静菌または抗菌活性があることを見出した。さらに、ホエーの限外ろ過パーミエイトを合成吸着剤樹脂（例えば、HP20）に吸着させた後、含水有機溶媒で溶出される画分に、植物病原性細菌や食中毒細菌などに対して静菌または抗菌活性があることを明らかにした。
本発明は、以下のような静菌または抗菌剤及びその製造方法を提供するものである。
〔1〕乳またはホエーより得られる成分を有効成分とする静菌または抗菌剤であって、前記乳またはホエーより得られる成分が以下の性質を有することを特徴とする静菌または抗菌剤。
（１）分画分子量２０，０００Ｄａ未満の限外ろ過膜を通過する性質
（２）乳糖含量が１０％以下である
（３）脂肪含量が１％以下である
（４）遊離アミノ酸含量が１％以下である
（５）有機酸含量が１％以下である
〔2〕前記乳またはホエーより得られる成分がさらに以下の性質を有する〔1〕に記載の静菌または抗菌剤。
（６）合成吸着樹脂に吸着する性質
（７）吸着した前記合成吸着樹脂から含水有機溶媒で溶出される性質
〔3〕有機溶媒がエタノール、メタノール、プロパノール、イソプロパノール、アセトン、アセトニトリルまたはジメチルスルホキシドのいずれかである〔2〕に記載の静菌または抗菌剤。
〔4〕合成吸着樹脂がスチレン−ジビニルベンゼン系合成吸着剤である〔2〕または〔3〕のいずれかに記載の静菌または抗菌剤。
〔5〕前記静菌または抗菌の対象とする微生物が、Escherichia属、シュードモナス属のいずれかである〔1〕〜〔4〕のいずれかに記載の静菌または抗菌剤。
〔6〕前記静菌または抗菌の対象とする微生物が大腸菌（Escherichia coli）、シュードモナス プチダ（Pseudomonas putida）、シュードモナス アエルギノーザ（Pseudomonas aeruginosa）、シュードモナス フルオレッセンス（Pseudomonas fluoressence）のいずれかであることを特徴とする〔5〕に記載の静菌または抗菌剤。
〔7〕前記乳またはホエーより得られる成分が、乳またはホエーの限外ろ過パーミエートから乳糖を除去することにより得られるものである、〔1〕〜〔6〕のいずれかに記載の静菌または抗菌剤。
〔8〕前記乳またはホエーより得られる成分が、以下の工程により得られるものである〔1〕〜〔6〕のいずれかに記載の静菌または抗菌剤。
（ａ）乳またはホエーより脂肪及びタンパク質成分を除去する工程と、
（ｂ）前記（ａ）の工程により得られた溶液を合成吸着樹脂と接触させる工程と、
（ｃ）前記（ｂ）の工程により得られた合成吸着樹脂を含水有機溶媒で溶出し、有効成分を得る工程
〔9〕前記乳またはホエーより得られる成分が、ｐＨ３．５以下で加水分解工程を経ることなく得られるものである〔1〕〜〔8〕のいずれかに記載の静菌または抗菌剤。
〔10〕乳またはホエーより得られる成分を有効成分とする静菌または抗菌剤の製造方法であって、以下の工程を有する製造方法。
（ａ）乳またはホエーより脂肪及びタンパク質成分を除去する工程
（ｂ）前記（ａ）の工程により得られた溶液を合成吸着樹脂と接触させる工程
（ｃ）前記（ｂ）の工程により得られた合成吸着樹脂を含水有機溶媒で溶出し、有効成分を得る工程
〔11〕前記の有機溶媒がエタノール、メタノール、プロパノール、イソオプロパノール、アセトン、アセトニトリルまたはジメチルスルホキシドのいずれかである〔10〕に記載の製造方法。
〔12〕合成吸着樹脂がスチレン−ジビニルベンゼン系合成吸着剤である〔10〕または〔11〕に記載の製造方法。
〔13〕前記（ａ）のタンパク工程は分画分子量２０，０００Ｄａ未満の限外ろ過膜を用いてタンパク質を除去する工程である〔10〕〜〔12〕のいずれかに記載の製造方法。
〔14〕タンパク質成分の加水分解工程を含まない〔10〕〜〔13〕のいずれかに記載の製造方法。
〔15〕〔1〕〜〔9〕のいずれかに記載の静菌または抗菌剤を含有することを特徴とする植物病原菌防除剤。
〔16〕〔1〕〜〔9〕のいずれかに記載の静菌または抗菌剤を含有することを特徴とする飲食品、栄養組成物または飼料。
As a result of diligent investigations to solve the above problems, the present inventors further fractionated whey ultrafiltration permeate and conducted various screenings for its effective use. It was found that the fraction excluding water-soluble molecules such as lactose has a high bacteriostatic or antibacterial activity against soil bacteria. Furthermore, after adsorbing whey ultrafiltration permeate to a synthetic adsorbent resin (eg, HP20), the fraction eluted with a water-containing organic solvent is bacteriostatic or antibacterial against phytopathogenic bacteria and food poisoning bacteria. It was revealed that there was activity.
The present invention provides the following bacteriostatic or antibacterial agents and methods for producing the same.
[1] A bacteriostatic or antibacterial agent comprising a component obtained from milk or whey as an active ingredient, wherein the component obtained from milk or whey has the following properties.
(1) A property of passing through an ultrafiltration membrane having a fractional molecular weight of less than 20,000 Da (2) Lactose content is 10% or less (3) Fat content is 1% or less (4) Free amino acid content is 1% The following (5) The organic acid content is 1% or less [2] The bacteriostatic or antibacterial agent according to [1], wherein the component obtained from the milk or whey further has the following properties.
(6) Properties to be adsorbed on the synthetic adsorption resin (7) Properties to be eluted from the adsorbed synthetic adsorption resin with a water-containing organic solvent [3] Any of the organic solvents is ethanol, methanol, propanol, isopropanol, acetone, acetonitrile or dimethyl sulfoxide The bacteriostatic or antibacterial agent according to [2].
[4] The bacteriostatic or antibacterial agent according to any one of [2] or [3], wherein the synthetic adsorption resin is a styrene-divinylbenzene synthetic adsorbent.
[5] The bacteriostatic or antibacterial agent according to any one of [1] to [4], wherein the microorganism to be bacteriostatic or antibacterial is either the genus Escherichia or the genus Pseudomonas.
[6] The microorganism to be bacteriostatic or antibacterial is Escherichia coli, Pseudomonas putida, Pseudomonas aeruginosa, or Pseudomonas fluoressence. The bacteriostatic or antibacterial agent according to [5].
[7] The bacteriostatic or bacterium according to any one of [1] to [6], wherein the component obtained from the milk or whey is obtained by removing lactose from the ultrafiltration permeate of milk or whey Antibacterial agent.
[8] The bacteriostatic or antibacterial agent according to any one of [1] to [6], wherein the component obtained from the milk or whey is obtained by the following steps.
(A) removing fat and protein components from milk or whey;
(B) contacting the solution obtained by the step (a) with a synthetic adsorption resin;
(C) A step of eluting the synthetic adsorption resin obtained in the step (b) with a water-containing organic solvent to obtain an active ingredient [9] A hydrolysis step wherein the component obtained from the milk or whey is pH 3.5 or less The bacteriostatic or antibacterial agent according to any one of [1] to [8], which is obtained without passing through the above.
[10] A method for producing a bacteriostatic or antibacterial agent comprising a component obtained from milk or whey as an active ingredient, the method comprising the following steps.
(A) Step of removing fat and protein components from milk or whey (b) Step of contacting the solution obtained by the step (a) with a synthetic adsorption resin (c) Obtained by the step of (b) A step of eluting the synthetic adsorption resin with a water-containing organic solvent to obtain an active ingredient [11] The organic solvent is any one of ethanol, methanol, propanol, isopropanol, acetone, acetonitrile, or dimethyl sulfoxide. Production method.
[12] The production method according to [10] or [11], wherein the synthetic adsorption resin is a styrene-divinylbenzene synthetic adsorbent.
[13] The production method according to any one of [10] to [12], wherein the protein step (a) is a step of removing protein using an ultrafiltration membrane having a fractional molecular weight of less than 20,000 Da.
[14] The production method according to any one of [10] to [13], which does not include a protein component hydrolysis step.
[15] A plant pathogen control agent comprising the bacteriostatic or antibacterial agent according to any one of [1] to [9].
[16] A food, beverage, nutritional composition, or feed comprising the bacteriostatic or antibacterial agent according to any one of [1] to [9].
本発明によれば、乳またはホエーより得られた低分子画分を有効成分とする静菌または抗菌剤を提供することができる。本発明により得られたホエーの低分子静菌または抗菌性物質は食品由来の天然物であり、ブロッコリーの花蕾等の農薬を散布できない野菜に対して用いることができるほか、土壌散布によっても植物病原菌を抑制することが可能である。さらには、ヒトや家畜動物等で問題となる病原性大腸菌等に対して効果が期待できるため、安全な腸内病原菌の抑制剤としても利用できる。 ADVANTAGE OF THE INVENTION According to this invention, the bacteriostatic or antibacterial agent which uses the low molecular fraction obtained from milk or whey as an active ingredient can be provided. The low molecular weight bacteriostatic or antibacterial substance of whey obtained by the present invention is a food-derived natural product, and can be used for vegetables that cannot be sprayed with agricultural chemicals such as broccoli florets. Can be suppressed. Furthermore, since it can be expected to be effective against pathogenic Escherichia coli, which is a problem in humans and livestock animals, it can also be used as a safe inhibitor of enteric pathogens.
以下、本発明の静菌または抗菌剤及びその製造方法について説明する。
本発明の静菌または抗菌剤は、乳またはホエーより脂肪成分、タンパク質成分、乳糖成分を除去する工程により得ることができ、望ましくは、乳またはホエーより脂肪及びタンパク質成分を除去する工程と、前記脂肪及びタンパク質を除去した溶液を合成吸着剤樹脂に吸着させる工程と、前記合成吸着樹脂に吸着させた溶液を、含水有機溶媒で抽出することにより得ることができる。
Hereinafter, the bacteriostatic or antibacterial agent of the present invention and the production method thereof will be described.
The bacteriostatic or antibacterial agent of the present invention can be obtained by a step of removing a fat component, a protein component, or a lactose component from milk or whey, and preferably the step of removing a fat and protein component from milk or whey, It can be obtained by adsorbing a solution from which fat and protein have been removed to a synthetic adsorbent resin and extracting the solution adsorbed on the synthetic adsorption resin with a water-containing organic solvent.
本発明において使用する原料としては、生乳や脱脂乳等の乳又は、ホエー等を用いることができる。
乳は、牛乳、人乳、ヤギ乳、羊乳等の乳を用いることができる。また、該乳は、液状でもよいし、粉末化したものを還元して用いることもできる。
ホエーは、酸や乳酸菌で分離した酸ホエーでも良いし、レンネットを用いたチーズ製造時に二次的に生成するホエーであっても良く、液状でもよいし、粉末化したものを還元して用いることもできる。ところで、乳に含まれるラクトフェリンから抗菌ペプチドであるラクトフェリシンを製造する工程においては、ラクトフェリンが非常に安定なタンパク質であるため、ラクトフェリンを含む溶液をｐＨ３．０以下の酸性下におき、ペプシンやキモシンなどの加水分解酵素と反応させるといった煩雑な工程が含まれる（非特許文献１）。本発明で用いるホエーとは、通常のチーズ製造時に産生する副産物でよく、ｐＨが３．５以上の段階でカードと分離したホエーであれば、どのようなチーズのホエーでもかまわない。例えばゴーダチーズ、チェダーチーズ、モツアレラチーズなどから得られるホエーを使用する事が望ましい。なお、ｐＨが３程度となっても、キモシンなどの加水分解酵素を含まずに製造されるホエーであれば、使用することが出来ることはいうまでもない。脱脂乳からミルクタンパク質コンセントレート（MPC）を製造する際に副産物として生成する限外ろ過パーミエイトを用いても良い。なお、副産物の有効利用という観点から、ホエーやその限外ろ過パーミエイト等を使用することが望ましい。
As a raw material used in the present invention, milk such as raw milk and skim milk, whey and the like can be used.
As milk, milk such as cow's milk, human milk, goat milk, and sheep milk can be used. In addition, the milk may be liquid, or powdered product can be reduced and used.
The whey may be acid whey separated by acid or lactic acid bacteria, may be whey that is secondarily produced during cheese production using rennet, may be liquid, or is reduced to a powdered product. You can also By the way, in the process of producing lactoferrin which is an antibacterial peptide from lactoferrin contained in milk, since lactoferrin is a very stable protein, a solution containing lactoferrin is placed under an acidic pH of 3.0 or less, and pepsin and A complicated process of reacting with a hydrolase such as chymosin is included (Non-patent Document 1). The whey used in the present invention may be a by-product produced during normal cheese production, and any cheese whey can be used as long as it is separated from the curd at a pH of 3.5 or higher. For example, it is desirable to use whey obtained from gouda cheese, cheddar cheese, mozzarella cheese or the like. Needless to say, even if the pH is about 3, it can be used as long as it is produced without containing a hydrolase such as chymosin. You may use the ultrafiltration permeate produced | generated as a by-product when manufacturing milk protein concentrate (MPC) from skim milk. From the viewpoint of effective use of by-products, it is desirable to use whey and its ultrafiltration permeate.
乳またはホエーより脂肪成分を除去する方法としては、例えば、クリームセパレーター等を用いて遠心分離により除去する方法を挙げることができるが、特に限定されず、脂肪成分を除く一般的な方法を用いることができる。 Examples of a method for removing a fat component from milk or whey include a method for removing by centrifugation using a cream separator or the like, but are not particularly limited, and a general method for removing a fat component is used. Can do.
乳またはホエーよりタンパク質成分を除去する方法としては、限外ろ過膜を用いることもできるし、溶液中の塩濃度を高めて析出させる（塩析）方法も用いることができるが、特に限定されず、タンパク質を除去する一般的な方法を用いることができる。なお、限外ろ過膜としては、乳またはホエー中のタンパク質成分を除去できれば特に限定されないが、乳タンパク質の大きさを考慮すると、分画分子量が２０，０００Ｄａ未満のものが適当であり、１５，０００以下、１０，０００以下がさらに望ましい。２０，０００Ｄａ以上であると不要なタンパク質を十分に除去することができないためである。
一方、除去効率を考慮すると、膜の分画分子量は、２，０００Ｄａ以上が望ましく、より多くの静菌または抗菌効果を有する低分子画分を捉えるためにさらに望ましくは３，０００Ｄａ以上である。後述する実施例では、このような観点より分画分子量１０，０００Ｄａの限外ろ過膜を用いて、静菌・抗菌効果の高い分画を多数得ている。
なお、本発明の製造方法では、乳またはホエーを加水分解等することなく、タンパク質成分を除去する工程を有していることから、本発明の有効成分には、ラクトフェリンの加水分解物が含まれることはない。
As a method of removing protein components from milk or whey, an ultrafiltration membrane can be used, and a method of depositing by increasing the salt concentration in the solution (salting out) can be used, but is not particularly limited. A general method for removing proteins can be used. The ultrafiltration membrane is not particularly limited as long as the protein component in milk or whey can be removed, but considering the size of the milk protein, those having a fractional molecular weight of less than 20,000 Da are suitable. More preferably, it is 000 or less and 10,000 or less. This is because unnecessary protein cannot be sufficiently removed when the viscosity is 20,000 Da or more.
On the other hand, considering the removal efficiency, the molecular weight cutoff of the membrane is preferably 2,000 Da or more, and more preferably 3,000 Da or more in order to capture a low molecular fraction having more bacteriostatic or antibacterial effects. In the examples described later, many fractions having a high bacteriostatic / antibacterial effect are obtained by using an ultrafiltration membrane having a fractional molecular weight of 10,000 Da from such a viewpoint.
In addition, since the production method of the present invention includes a step of removing protein components without hydrolyzing milk or whey, the active ingredient of the present invention includes a hydrolyzate of lactoferrin. There is nothing.
本発明の製造工程における乳またはホエーより脂肪及びタンパク質成分を除去する工程とは、脂肪成分を除去する工程とタンパク質成分を除去する工程がそれぞれ異なる手段で別々に行われる工程でもよく、同一工程で一度に除去される工程でもよい。 The step of removing fat and protein components from milk or whey in the production process of the present invention may be a step in which the step of removing the fat component and the step of removing the protein component are performed separately by different means. It may be a process that is removed at a time.
乳またはホエーより乳糖を除去する方法、あるいは乳またはホエーよりタンパク質を除去した溶液から乳糖を除去する方法としては、合成吸着樹脂を用いて、必要成分の方を吸着させ、その後溶出することにより必要成分を得る方法が挙げられる。
このように用いられる合成吸着樹脂としては、一定の疎水性度があり、乳糖やミネラルを吸着しにくいものであれば特に限定されないが、工業的規模の利用とした場合には、例えばスチレン−ジビニルベンゼン系合成吸着剤が挙げられ、市販されているものとしては、ＤＩＡＩＯＮ（登録商標、以下同じ）ＨＰ２０（三菱化学製）等の吸着樹脂用いることができる。
これらの脂肪成分除去工程、タンパク質成分除去工程、乳糖除去工程の順番は、特に問わないが、脂肪成分とタンパク質成分の除去を先に行い、その後乳糖を除去することが効率の上から望ましい。
It is necessary to remove lactose from milk or whey, or to remove lactose from a solution from which protein has been removed from milk or whey, by adsorbing the necessary components using a synthetic adsorption resin and then eluting it. The method of obtaining an ingredient is mentioned.
The synthetic adsorption resin used in this way is not particularly limited as long as it has a certain degree of hydrophobicity and is difficult to adsorb lactose and minerals. However, when used on an industrial scale, for example, styrene-divinyl Examples of the commercially available benzene-based synthetic adsorbent include DIAION (registered trademark, the same shall apply hereinafter) HP20 (manufactured by Mitsubishi Chemical).
The order of the fat component removal step, the protein component removal step, and the lactose removal step is not particularly limited, but it is desirable from the viewpoint of efficiency that the fat component and the protein component are removed first and then the lactose is removed.
合成吸着樹脂に吸着させた溶液を溶出するために、含水有機溶媒を用いることができる。該溶媒としては、溶出力があるものであれば特に限定されずに用いることができ、例えばメタノール、エタノール、イソプロパノール、アセトニトリルまたはジメチルスルホキシド等を用いることができる。なお、樹脂に吸着した静菌または抗菌性成分は、水では溶出されにくいが、有機溶媒が含まれていれば溶出されるため、含水有機溶媒であれば濃度は特に限定されないが、溶出効率等を考慮すると、１０％以上の濃度の有機溶媒を用いることが好ましい。 A water-containing organic solvent can be used to elute the solution adsorbed on the synthetic adsorption resin. The solvent can be used without particular limitation as long as it has a soluble power, and for example, methanol, ethanol, isopropanol, acetonitrile, dimethyl sulfoxide, or the like can be used. The bacteriostatic or antibacterial components adsorbed on the resin are not easily eluted in water, but are eluted if an organic solvent is contained, so the concentration is not particularly limited as long as it is a water-containing organic solvent, elution efficiency, etc. In view of the above, it is preferable to use an organic solvent having a concentration of 10% or more.
このようにして得られた分画成分は、所定の分画分子量の限外濾過膜を通過する性質、乳糖含量が０〜１０％、脂肪およびアミノ酸および有機酸含量が１％以下の成分であり、各種の製品に添加する際に扱いやすい組成となっている。また、さらに望ましくは合成吸着樹脂に吸着する性質、吸着した前記合成吸着樹脂から含水有機溶媒で溶出される性質をも有している。
なお、このような組成の分画物は、上述のように、乳やホエーを原料として、脂肪成分除去工程、タンパク成分除去工程、乳糖成分除去工程をそれぞれ、あるいは同時に経て取得することもできるが、原料として中間物質や、廃棄物質を利用した場合には、それらの工程の一部を省略できることはいうまでもない。すなわち、ホエーより限外ろ過膜（前述の分画分子量の膜）を使用して、ＷＰＣやＷＰＩを濃縮した場合のホエーの限外ろ過膜通過画分（限外ろ過パーミエイト）を利用した場合には、脂肪成分除去工程、タンパク成分除去工程が不要であり、乳糖成分除去工程により本発明の前記組成の分画成分を得ることができる。また、脱脂乳を利用する場合は、脂肪成分除去工程が不要であり、限外ろ過膜によるタンパク成分除去工程と乳糖除去工程があればよい。
The fractionated component thus obtained is a component having a property of passing through an ultrafiltration membrane having a predetermined fractional molecular weight, a lactose content of 0 to 10%, and fat, amino acid and organic acid content of 1% or less. The composition is easy to handle when added to various products. Further, it desirably has a property of adsorbing to the synthetic adsorption resin and a property of being eluted from the adsorbed synthetic adsorption resin with a water-containing organic solvent.
In addition, as described above, a fraction having such a composition can be obtained by using milk or whey as a raw material, respectively, or through a fat component removal step, a protein component removal step, or a lactose component removal step. Needless to say, when intermediate materials or waste materials are used as raw materials, some of these steps can be omitted. That is, when the ultrafiltration membrane (the membrane with the above-mentioned molecular weight cut off) is used from whey and the fraction passing through ultrafiltration membrane (ultrafiltration permeate) of whey when WPC or WPI is concentrated is used. The fat component removing step and the protein component removing step are unnecessary, and the fraction component of the composition of the present invention can be obtained by the lactose component removing step. In addition, when skim milk is used, the fat component removing step is unnecessary, and there may be a protein component removing step and a lactose removing step using an ultrafiltration membrane.
本発明の静菌または抗菌剤は、そのまま静菌または抗菌剤として用いることもできるし、該剤に含まれる有効成分の精製度を上げるため、常法に従ってさらに溶媒抽出や結晶化等を行っても良い。例えば、酢酸エチル、塩酸、水飽和n−ブタノール等の有機溶媒を用いてさらに精製度を上げた画分を得ることができる。
噴霧乾燥や凍結乾燥におり粉末化しても、タブレット等の錠剤、顆粒剤、カプセル等の形態にしても用いることができるし、液状の状態のままでも用いることができる。
The bacteriostatic or antibacterial agent of the present invention can be used as it is as a bacteriostatic or antibacterial agent, and in order to increase the purity of the active ingredient contained in the agent, further solvent extraction or crystallization is carried out according to a conventional method. Also good. For example, a fraction with a further increased degree of purification can be obtained using an organic solvent such as ethyl acetate, hydrochloric acid, or water-saturated n-butanol.
It can be used in the form of tablets such as tablets, granules, capsules, etc., even if it is powdered by spray drying or freeze drying, or it can be used in a liquid state.
本発明の静菌または抗菌剤は、Escherichia属、シュードモナス属等に対して静菌または抗菌効果を有する。Escherichia属としては、大腸菌（Escherichia coli）、シュードモナス属としては、シュードモナス プチダ（Pseudomonas putida）、シュードモナス アエルギノーザ（Pseudomonas aeruginosa）、シュードモナス フルオレッセンス（Pseudomonas fluoressence）等を挙げることができる。したがって、本発明の静菌または抗菌剤を、後述する植物病原菌防除剤として用いる場合は、Escherichia属、シュードモナス属に属する病原菌に対して防除効果を奏する。そのような病原菌としては、例えば、褐条病菌(Pseudomonas syringae)、籾枯病菌(Pseudomonas glumae)、葉鞘褐変病菌(Pseudomonas marginalis)、花蕾病菌（Pseudomonas putida）などを挙げることができる。 The bacteriostatic or antibacterial agent of the present invention has a bacteriostatic or antibacterial effect against Escherichia, Pseudomonas and the like. Examples of Escherichia include Escherichia coli, and examples of Pseudomonas include Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas fluoressence, and the like. Therefore, when the bacteriostatic or antibacterial agent of the present invention is used as a phytopathogenic fungi controlling agent described later, it exerts a controlling effect on pathogenic fungi belonging to the genus Escherichia and Pseudomonas. Examples of such pathogenic bacteria include brown fungus (Pseudomonas syringae), bacterial wilt (Pseudomonas glumae), leaf sheath browning fungus (Pseudomonas marginalis), and flower rot (Pseudomonas putida).
本発明の静菌または抗菌剤は、植物病原菌防除剤として用いることができるが、該用途として用いる場合、例えば液状の物質に溶解して単独で散布しても良いし、他の農薬等の散布時に薬剤と混和して同時に散布することもできる。例えば、粉末化した該剤を水や他の液剤に約０．１〜１０%溶解して散布することにより、植物病原菌防除剤としての効果が期待できる。植物体表面への付着性を高めるため、農業上通常用いられる展着剤、界面活性剤を補助剤として添加することも可能である。例えば、ポリオキシエチレンアルキルエーテル、ポリオキシエチレンアルキルアリールエーテル、ポリオキシエチレン脂肪酸エステル、ソルビタン脂肪酸エステル、ポリオキシエチレンソルビタン脂肪酸エステル、オキシエチレンポリマー、オキシプロピレンポリマー、ポリオキシエチレンアルキルリン酸エステル、脂肪酸塩、アルキル硫酸エステル塩、アルキルスルホン酸塩、アルキルアリールスルホン酸塩、アルキルリン酸塩、アルキルリン酸エステル塩、ポリオキシエチレンアルキル硫酸エステル、第四級アンモニウム塩、オキシアルキルアミン、レシチン、サポニン等が挙げられる。また、必要に応じてゼラチン、カゼイン、デンプン、寒天、ポリビニルアルコール、アルギン酸ソーダなどを補助剤として用いることが出来る。また、土壌中の病原菌防除の目的では、粉末状で直接散布することもできるが、ゼオライト等の土壌改良資材に混和あるいは吸着させた後、土壌にすき込んで使用することも可能であるし、肥料に混和して用いることも可能である。
植物体への散布部位は、病原菌の発症、繁殖可能部位であればどこにでも用いることができ、花、茎、葉、根および種子などが挙げられる。
The bacteriostatic or antibacterial agent of the present invention can be used as a phytopathogenic agent, but when used as the application, for example, it may be dissolved in a liquid substance and sprayed alone, or sprayed with other agricultural chemicals, etc. Sometimes mixed with drugs and sprayed at the same time. For example, the effect as a phytopathogenic fungi control agent can be expected by dissolving about 0.1 to 10% of the powdered agent in water or other liquid and spraying it. In order to enhance the adhesion to the plant body surface, it is possible to add a spreading agent or surfactant usually used in agriculture as an auxiliary agent. For example, polyoxyethylene alkyl ether, polyoxyethylene alkyl aryl ether, polyoxyethylene fatty acid ester, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, oxyethylene polymer, oxypropylene polymer, polyoxyethylene alkyl phosphate ester, fatty acid salt Alkyl sulfate salt, alkyl sulfonate salt, alkyl aryl sulfonate salt, alkyl phosphate salt, alkyl phosphate ester salt, polyoxyethylene alkyl sulfate ester, quaternary ammonium salt, oxyalkylamine, lecithin, saponin, etc. Can be mentioned. If necessary, gelatin, casein, starch, agar, polyvinyl alcohol, sodium alginate and the like can be used as auxiliary agents. In addition, for the purpose of controlling pathogenic bacteria in the soil, it can be sprayed directly in powder form, but it can also be used by mixing in or adsorbing to soil improvement materials such as zeolite, It is also possible to mix them with fertilizers.
The site of application to the plant body can be used anywhere as long as it is a site where pathogenic bacteria can develop and propagate, and examples thereof include flowers, stems, leaves, roots and seeds.
本発明の静菌または抗菌剤は、ヒトの感染症予防を目的として用いることができる。例えば、飲料や乳製品、健康食品類等の様々な飲食品に利用することができる。本発明の静菌または抗菌剤は、飲食品の製造工程中に原材料として添加することもできる。本発明の静菌または抗菌剤は、通常飲食品中に０．０１〜９９％配合することができる。
また、本発明の静菌または抗菌剤は、家畜・ペット等の感染症予防を目的として用いることができる。動物用途であれば、固形飼料・餌に混和することもできるし、飲料水に溶解して摂取させることもできる。また、粉末を飼料・餌に直接振りかけて与えることもできる。粉末化した本剤を豚または鶏用の固形飼料に０．１〜５％程度混和して給与することで、感染症予防効果が期待できる。
The bacteriostatic or antibacterial agent of the present invention can be used for the purpose of preventing human infectious diseases. For example, it can be used for various foods and beverages such as beverages, dairy products, and health foods. The bacteriostatic or antibacterial agent of the present invention can also be added as a raw material during the production process of food and drink. The bacteriostatic or antibacterial agent of the present invention can be usually blended in an amount of 0.01 to 99% in food and drink.
The bacteriostatic or antibacterial agent of the present invention can be used for the purpose of preventing infectious diseases such as livestock and pets. For animal use, it can be mixed with solid feed and feed, or can be dissolved in drinking water and ingested. In addition, powder can be sprinkled directly on feed / food. Infectious disease prevention effect can be expected by mixing about 0.1 to 5% of this powdered product with pig or chicken solid feed.
本発明の実施により、ホエーの限外ろ過パーミエイト等の乳副産物の付加価値を向上し、有効に活用することが可能となる。乳糖を除去する工程を経るため粉末化がしやすく、各種製品への添加が容易なうえ保存性も良いので汎用性が高い。また、乳糖を資化可能な細菌の増殖を抑制できるという効果を有する。 By carrying out the present invention, it is possible to improve the added value of milk by-products such as whey ultrafiltration permeate and effectively utilize it. Since it goes through the process of removing lactose, it is easy to be powdered, and it is easy to add to various products. In addition, it has an effect of suppressing the growth of bacteria capable of assimilating lactose.
当該静菌または抗菌性物質は食品由来の天然物であるため、例えばブロッコリーの花蕾等、そのまま食するため農薬を散布できない野菜に対しても使用することができる。その結果、これらの野菜類の病害を抑え、安定的に農業生産を行うことができる。更に、本発明の静菌または抗菌性物質を土壌散布することで、植物病原菌の繁殖、感染などの抑制効果も期待できる。また、種子を本発明の静菌または抗菌性物質に浸漬しておいたり、発症前の植物体に散布しておくことで、植物病原菌の発症を予防することも期待できる。また、ヒトや家畜動物などで問題となる病原性大腸菌等に対しても効果が期待できるため、食品や家畜飼料として摂食することにより、安全に腸内病原菌を防除することもできる。
以下、本発明を実施例によりさらに具体的に説明するが、本発明の範囲は下記の実施例
に限定されるものではない。
Since the bacteriostatic or antibacterial substance is a food-derived natural product, it can also be used for vegetables that cannot be sprayed with pesticides because they are eaten as they are, for example, broccoli florets. As a result, diseases of these vegetables can be suppressed and agricultural production can be stably performed. Furthermore, by inhibiting the bacteriostatic or antibacterial substance of the present invention to be applied to the soil, it is also possible to expect the effect of suppressing the propagation and infection of phytopathogenic bacteria. Moreover, it can also be expected to prevent the onset of phytopathogenic fungi by immersing the seeds in the bacteriostatic or antibacterial substance of the present invention or spraying the seeds on the plant body before the onset. In addition, since it can be expected to be effective against pathogenic Escherichia coli and the like which are problematic in humans and livestock animals, enteric pathogens can be safely controlled by eating them as food or livestock feed.
EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example.
（ホエーからの静菌または抗菌成分の分画）
ゴーダタイプチーズの製造工程で排出されたチーズホエー２５Ｌを８，０００ｇ×３０分間、４℃で遠心分離後、脂肪層を除いた上清を分画分子量が１０，０００Ｄａの限外ろ過膜（3838 HFK-131、Koch Membrane Systems Inc.製）に供し、通過した画分をホエー限外ろ過パーミエイトとした（約２０Ｌ）。合成吸着樹脂であるＤＩＡＩＯＮ ＨＰ２０（三菱化学製）をメタノールで膨潤後、蒸留水で洗浄し、オープンカラム（内径４５ｍｍ×長さ５５０ｍｍ）に充填した。ホエー限外ろ過パーミエイトをこの樹脂に供し、蒸留水にて１回洗浄を行った後、１０％、３０％、５０％のエタノール溶液各３Ｌで段階的に溶出して、溶出液をエバポレーターで濃縮し、濃縮液を凍結乾燥した。２０Ｌのホエー限外ろ過パーミエイトより、それぞれ５．９３ｇ、４．４１ｇ及び４．４９ｇの抽出画分を回収した。ぞれぞれ、以下１０％溶出画分（実施品１）、３０％溶出画分（実施品２）、５０％溶出画分（実施品３）という。これらの画分について一般成分を分析した結果を表１に示す。脂肪はレーゼゴットリーブ法で、窒素量（窒素）はケルダール法で、遊離アミノ酸はトリクロロ酢酸にて除タンパクしたサンプル溶液を、日立高速アミノ酸分析計L-8800（（株）日立ハイテクノロジーズ）にて測定して算出した。乳糖はＦ−キット乳糖／Ｄ−ガラクトース（Roche Diagnostics GmbH製)で分析した。該抽出画分はそのまま本発明の静菌または抗菌剤として用いることができる。
(Fraction of bacteriostatic or antibacterial components from whey)
The cheese whey 25L discharged in the Gouda type cheese production process was centrifuged at 8,000 g × 30 minutes at 4 ° C., and then the supernatant was removed from the supernatant, and the ultrafiltration membrane (3838) with a molecular weight cutoff of 10,000 Da. HFK-131 (manufactured by Koch Membrane Systems Inc.), and the passed fraction was used as whey ultrafiltration permeate (about 20 L). DIAION HP20 (Mitsubishi Chemical), which is a synthetic adsorption resin, was swollen with methanol, washed with distilled water, and packed into an open column (inner diameter 45 mm × length 550 mm). Use whey ultrafiltration permeate for this resin, wash once with distilled water, elute stepwise with 3 L each of 10%, 30% and 50% ethanol solutions, and concentrate the eluate with an evaporator. The concentrated solution was lyophilized. Extracted fractions of 5.93 g, 4.41 g, and 4.49 g were collected from 20 L of whey ultrafiltration permeate, respectively. These are hereinafter referred to as a 10% elution fraction (practical product 1), a 30% elution fraction (practical product 2), and a 50% elution fraction (practical product 3), respectively. Table 1 shows the results of analysis of general components for these fractions. Fat sample measured by the Rosette Gottlieb method, nitrogen content (nitrogen) by Kjeldahl method, free amino acids deproteinized by trichloroacetic acid, measured with Hitachi High Speed Amino Acid Analyzer L-8800 (Hitachi High-Technologies Corporation) And calculated. Lactose was analyzed with F-kit lactose / D-galactose (Roche Diagnostics GmbH). The extracted fraction can be used as it is as a bacteriostatic or antibacterial agent of the present invention.
（試験例１）
（ホエー溶出画分のシュードモナス属細菌の抑制効果）
北海道別海町の土壌を滅菌水に懸濁後、ＰＤＢ培地（パールコア標準寒天培地、栄研化学製）に塗布し、２７℃で1日培養した。生育したコロニーの一つを釣菌し、その16S rRNAの遺伝子配列を解読したところ、当株がPseudomonas sp.であることが確認された。この分離株を以降の抗菌性試験に用いた。
分離したシュードモナス属細菌（Pseudomonas sp.）をＰＤＢ培地（パールコア標準寒天培地、栄研化学製）に塗布し、２７℃で1日培養した。生育した菌体を生理食塩水に懸濁し、その１００μｌを標準寒天培地に塗布した。前記実施例１で得られたホエーの溶出画分（実施品１〜３）を１％水溶液とし、滅菌した直径１０ｍｍのペーパーディスク（ＡＤＶＡＮＴＥＣ製）に各々１００μl吸収させ、寒天培地上に等間隔に並べた。寒天培地を３０℃で２日間培養した後、ペーパーディクス周辺で菌の生育を阻害している範囲（阻止円）の大きさを測定した。その結果、実施品１〜３の全てで阻止円を形成した（図１、表２）。なお、蒸留水を滅菌したペーパーディスクに１００μｌ吸収させたものを寒天培地上に置いて同様に培養したが（対照区）、阻止部分は形成されなかった（阻止円の大きさは１０ｍｍ）。
以上の結果から、ホエー溶出画分には植物病原性菌の一つであるシュードモナス属細菌に対して静菌または抗菌効果があることが示された。
(Test Example 1)
(Inhibition effect of Pseudomonas bacteria in whey elution fraction)
The soil of Betsukai-cho, Hokkaido was suspended in sterilized water, applied to PDB medium (Pearl Core Standard Agar, manufactured by Eiken Chemical Co., Ltd.), and cultured at 27 ° C. for 1 day. When one of the grown colonies was picked and the 16S rRNA gene sequence was decoded, it was confirmed that the strain was Pseudomonas sp. This isolate was used for subsequent antibacterial tests.
The separated Pseudomonas bacterium (Pseudomonas sp.) Was applied to a PDB medium (Pearl Core Standard Agar, manufactured by Eiken Chemical Co., Ltd.) and cultured at 27 ° C. for 1 day. The grown cells were suspended in physiological saline, and 100 μl thereof was applied to a standard agar medium. The whey elution fraction obtained in Example 1 (Examples 1 to 3) was made into a 1% aqueous solution, and 100 μl each was absorbed on a sterilized 10 mm diameter paper disc (manufactured by ADVANTEC), and equidistant on the agar medium. Lined up. After culturing the agar medium at 30 ° C. for 2 days, the size of the range (inhibition circle) in which the growth of the bacteria was inhibited around the paper disc was measured. As a result, all of the products 1 to 3 formed a blocking circle (FIG. 1, Table 2). Although 100 μl of distilled water absorbed on a paper disk was placed on an agar medium and cultured in the same manner (control group), a blocking portion was not formed (the size of the blocking circle was 10 mm).
From the above results, it was shown that the whey elution fraction has a bacteriostatic or antibacterial effect against Pseudomonas bacteria, which is one of the phytopathogenic bacteria.
（試験例２）
（ホエー溶出画分のブロッコリー花蕾腐病の抑制効果）
北海道江別市のブロッコリー圃場で採取した花蕾腐病の発生部位を滅菌水に懸濁後、ＰＤＢ培地（パールコア標準寒天培地、栄研化学製）に塗布し、２７℃で1日培養した。生育したコロニーの一つを釣菌し、その16S rRNAの遺伝子配列を解読したところ、Pseudomonas putidaであると同定した。この菌株はＰＤＢ培地で３７℃で１日培養した。培養液を生理食塩水で希釈して病原菌液として以下の試験に用いた。
市販のブロッコリーを花茎の部分を切り戻し、蒸留水の入ったビーカーに茎をつけ保持した。実施例１により得られた実施品１〜３を蒸留水で２５０倍希釈し、さらに展着剤アプローチＢＩ（（株）花王製）を０．１％添加したものを各々花蕾部分に霧吹きで数回噴霧した。対照区としては蒸留水に展着剤のみを添加した液を噴霧した。蛍光灯下、室温にて１日静置することによって噴霧した処理液を乾燥した。その後、前記病原菌液を花蕾部分に霧吹きで数回噴霧して病原菌を接種した。湿度１００％、３７℃にて２４時間培養した後、花蕾部分を上面から撮影し（図２参照）、病原菌の繁殖により変色した部分（病変部位）の全体に対する割合（面積比率）を画像解析ソフトImageJ（http://rsb.info.nih.gov/ij/）にて数値化した（表３参照）。その結果、病原菌を接種した対照区のブロッコリーでは、花蕾部分の５１．１％が腐敗・変色していたのに対し、１０％、５０％エタノール溶出画分（実施品１、３）で処理した処理区のブロッコリーでは変色部分が全く見られなかった。また、３０％エタノール溶出画分（実施品２）で処理したブロッコリーでは花蕾部分の２６．９％が腐敗・変色していたが、対照区よりも抑制効果が見られた。
以上の結果から、ホエー由来溶出画分にはPseudomonas putidaへの高い静菌または抗菌効果があることが示された。
(Test Example 2)
(Inhibiting effect of broccoli flower rot of whey elution fraction)
A flower rot occurrence site collected in a broccoli field in Ebetsu, Hokkaido was suspended in sterilized water, applied to a PDB medium (Pearl Core Standard Agar, manufactured by Eiken Chemical), and cultured at 27 ° C. for 1 day. One of the grown colonies was fished, and when the 16S rRNA gene sequence was decoded, it was identified as Pseudomonas putida. This strain was cultured in PDB medium at 37 ° C. for 1 day. The culture solution was diluted with physiological saline and used as a pathogen solution in the following tests.
A commercially available broccoli was cut back at the flower stem, and the stem was placed in a beaker containing distilled water and held. Examples 1 to 3 obtained in Example 1 were diluted 250-fold with distilled water, and 0.1% of a spreader approach BI (manufactured by Kao Corporation) was added to each floret part by spraying. Sprayed twice. As a control, a liquid obtained by adding only a spreading agent to distilled water was sprayed. The sprayed treatment liquid was dried by allowing it to stand at room temperature for 1 day under a fluorescent lamp. Thereafter, the pathogenic bacteria solution was sprayed on the flower buds several times by spraying to inoculate the pathogenic bacteria. After culturing at 100% humidity and 37 ° C for 24 hours, the flower part is photographed from the top (see Fig. 2), and the ratio (area ratio) of the part (lesion site) discolored due to the propagation of pathogenic bacteria to the whole image analysis software It was digitized by ImageJ (http://rsb.info.nih.gov/ij/) (see Table 3). As a result, in the broccoli in the control group inoculated with the pathogenic bacteria, 51.1% of the floret part was spoiled and discolored, whereas it was treated with the 10% and 50% ethanol elution fractions (Examples 1 and 3). No discoloration was observed in the broccoli in the treated area. In addition, in broccoli treated with the 30% ethanol-eluted fraction (Example product 2), 26.9% of the floret part was spoiled and discolored, but the inhibitory effect was seen as compared with the control group.
From the above results, it was shown that the whey-derived elution fraction has a high bacteriostatic or antibacterial effect on Pseudomonas putida.
（ホエーからの静菌または抗菌成分の粗精製）
ゴーダタイプチーズの製造工程で排出されたチーズホエー２０Ｌを９，０００ｇ×３０分、４℃で遠心分離後、脂肪層を除いた上清を分画分子量が１０，０００Ｄａの限外ろ過膜（3838 HFK-131、Koch Membrane Systems Inc.）に供し、通過した画分をホエー限外ろ過パーミエイトとして１８Ｌ得た。合成吸着樹脂（ＤＩＡＩＯＮ ＨＰ２０、三菱化学製）をエタノールで膨潤後、蒸留水で洗浄し、オープンカラム（内径４５ｍｍ×長さ５５０ｍｍ）に充填した。前記ホエー限外ろ過パーミエイトを該樹脂に供し、蒸留水にて１回洗浄を行った後、５０％のエタノール溶液で溶出して、溶出液をエバポレーターで乾固した。５００ｍｌの蒸留水に溶解した後、各種溶媒による段階的な抽出を行った。
まず、水酸化ナトリウム（和光純薬製）にてｐＨを８．０に調製した後、酢酸エチル（和光純薬製）５００ｍｌで３回溶媒抽出を行った。水相を回収し、塩酸（和光純薬製）にてｐＨを２．５に調製した後、酢酸エチル５００ｍｌで３回の溶媒抽出を行った。回収した酢酸エチル相は無水硫酸マグネシウムで乾燥し、エバポレーターで乾固後、蒸留水２０ｍｌに溶解し、ＡＥ画分とした。水相は水酸化ナトリウムにてｐＨを８．０に調製した後、水飽和ｎ−ブタノール（和光純薬）５００ｍｌで３回の溶媒抽出を行った。回収したｎ−ブタノール相をエバポレーターで乾固後、蒸留水２０ｍｌに溶解し、ＮＢ画分とした。水相は塩酸にてｐＨを２．５に調製した後、水飽和ｎ−ブタノール５００ｍｌで３回の溶媒抽出を行った。回収したｎ−ブタノール相をエバポレーターで乾固後、蒸留水２０ｍｌに溶解し、ＡＢ画分とした。最終的な水相は、含まれる塩類を除くため、先に用いたＤＩＡＩＯＮ ＨＰ２０樹脂に一度吸着させた後、蒸留水にて１回洗浄を行なった。５０％のエタノールで溶出した溶液をエバポレーターで乾固し、蒸留水２０ｍｌに溶解してＡＱ画分とした。該抽出画分はそのまま本発明の静菌または抗菌剤として用いることができる。
以上のように分画された粗精製画分は最終的に１０ｍｌとなるように含水エタノールで溶解し、以下の抗菌試験を実施した。
(Rough purification of bacteriostatic or antibacterial ingredients from whey)
After centrifuging the cheese whey 20L discharged in the Gouda-type cheese production process at 9,000 g × 30 minutes at 4 ° C., the supernatant excluding the fat layer was subjected to ultrafiltration membrane with a molecular weight cutoff of 10,000 Da (3838 HFK-131, Koch Membrane Systems Inc.), and 18 L of the passed fraction was obtained as a whey ultrafiltration permeate. A synthetic adsorption resin (DIAION HP20, manufactured by Mitsubishi Chemical) was swollen with ethanol, washed with distilled water, and packed into an open column (inner diameter 45 mm × length 550 mm). The whey ultrafiltration permeate was applied to the resin, washed once with distilled water, eluted with 50% ethanol solution, and the eluate was dried with an evaporator. After dissolving in 500 ml of distilled water, stepwise extraction with various solvents was performed.
First, after adjusting the pH to 8.0 with sodium hydroxide (manufactured by Wako Pure Chemical Industries), solvent extraction was performed three times with 500 ml of ethyl acetate (manufactured by Wako Pure Chemical Industries). The aqueous phase was recovered and adjusted to pH 2.5 with hydrochloric acid (manufactured by Wako Pure Chemical Industries, Ltd.), and then extracted with 500 ml of ethyl acetate three times. The recovered ethyl acetate phase was dried over anhydrous magnesium sulfate, dried to dryness with an evaporator, and then dissolved in 20 ml of distilled water to obtain an AE fraction. The aqueous phase was adjusted to pH 8.0 with sodium hydroxide and then subjected to solvent extraction three times with 500 ml of water saturated n-butanol (Wako Pure Chemical Industries). The recovered n-butanol phase was dried with an evaporator and then dissolved in 20 ml of distilled water to obtain an NB fraction. The aqueous phase was adjusted to pH 2.5 with hydrochloric acid, and then extracted three times with 500 ml of water-saturated n-butanol. The recovered n-butanol phase was dried with an evaporator and then dissolved in 20 ml of distilled water to obtain an AB fraction. The final aqueous phase was once adsorbed by the previously used DIAION HP20 resin and then washed once with distilled water in order to remove the contained salts. The solution eluted with 50% ethanol was dried with an evaporator and dissolved in 20 ml of distilled water to obtain an AQ fraction. The extracted fraction can be used as it is as a bacteriostatic or antibacterial agent of the present invention.
The crudely purified fraction fractionated as described above was dissolved in water-containing ethanol so that the final fraction was 10 ml, and the following antibacterial test was performed.
（試験例３）
（ホエー抽出画分の土壌微生物の抑制効果）
北海道・別海町の土壌サンプルを生理食塩水で１００倍に希釈した後、標準寒天培地（パールコア標準寒天培地、栄研化学製）に該土壌希釈液１００μｌを塗布した。滅菌した直径８ｍｍのペーパーディスク（ＡＤＶＡＮＴＥＣ製）に実施例２で得られた抽出画分ＡＥ画分、ＮＢ画分、ＡＢ画分、ＡＱ画分（実施品４〜７）を各々５０μl吸収させ、該寒天培地上に等間隔に並べた。ペーパーディスクを並べた培地を３０℃で２日間培養した後、ペーパーディクス周辺で菌の生育を阻害している範囲（阻止円）の大きさを測定した。なお、蒸留水を滅菌したペーパーディスクに５０μｌ吸収させたものを対照区とした。
その結果、ＡＥ画分、ＮＢ画分、ＡＢ画分、ＡＱ画分で阻止円を形成したことから（表４）、ホエー抽出画分には土壌微生物に対しても高い静菌または抗菌効果があり、多数の成分に静菌または抗菌効果があることが示された。
(Test Example 3)
(Suppressing effect of soil microorganisms in whey extract fraction)
A soil sample in Betsukai-cho, Hokkaido was diluted 100 times with physiological saline, and then 100 μl of the soil dilution was applied to a standard agar medium (Pearl Core Standard Agar, manufactured by Eiken Chemical Co., Ltd.). 50 μl each of the extracted fraction AE fraction, NB fraction, AB fraction, and AQ fraction (Examples 4 to 7) obtained in Example 2 was absorbed on a sterilized 8 mm diameter paper disc (manufactured by ADVANTEC) They were arranged on the agar medium at regular intervals. After culturing the medium on which the paper disks were arranged at 30 ° C. for 2 days, the size of the range (inhibition circle) in which the growth of the bacteria was inhibited around the paper disk was measured. In addition, 50 μl of distilled water was absorbed in a sterilized paper disk as a control group.
As a result, the AE fraction, NB fraction, AB fraction, and AQ fraction formed a blocking circle (Table 4), so the whey extract fraction has a high bacteriostatic or antibacterial effect against soil microorganisms. And many ingredients have been shown to have bacteriostatic or antibacterial effects.
（試験例４）
（ホエー抽出画分のシュードモナスの抑制効果）
土壌より分離したシュードモナス属細菌（Pseudomonas sp）を標準寒天培地（パールコア標準寒天培地、栄研化学製）に塗布し、３０℃で1日培養した。生育した菌体を生理食塩水に懸濁し、その１００μｌを標準寒天培地に塗布した。滅菌した直径８ｍｍのペーパーディスク（ＡＤＶＡＮＴＥＣ製）に実施例２で得られた抽出画分、ＡＥ画分、ＮＢ画分、ＡＢ画分、ＡＱ画分（実施品４〜７）を各々５０μl吸収させ、寒天培地上に等間隔に並べた。ペーパーディスクを並べた培地を３０℃で1日間培養した後、ペーパーディクス周辺で菌の生育を阻害している範囲（阻止円）の大きさを測定した。
その結果、ＡＥ画分、ＮＢ画分、ＡＢ画分、ＡＱ画分で阻止円を形成した（図３、表５）。なお、蒸留水を滅菌したペーパーディスクに５０μｌ吸収させたものを寒天培地上に置いて同様に培養したが（対照区）、阻止部分は形成されなかった（阻止円の大きさは８ｍｍ）。
以上の結果から、抽出画分には植物病原性菌の一つであるシュードモナス属細菌に対しても高い静菌または抗菌効果があることが判明し、静菌または抗菌効果のある多数の成分を含んでいることが明らかとなった。
(Test Example 4)
(Inhibiting effect of Pseudomonas on whey extract)
Pseudomonas sp. Isolated from soil was applied to a standard agar medium (Pearl Core Standard Agar, manufactured by Eiken Chemical Co., Ltd.) and cultured at 30 ° C. for 1 day. The grown cells were suspended in physiological saline, and 100 μl thereof was applied to a standard agar medium. Absorb 50 μl each of the extracted fraction, AE fraction, NB fraction, AB fraction, and AQ fraction (practical products 4 to 7) obtained in Example 2 on a sterilized 8 mm diameter paper disc (manufactured by ADVANTEC). They were arranged at regular intervals on an agar medium. After culturing the medium on which the paper disks were arranged at 30 ° C. for 1 day, the size of the range (inhibition circle) in which the growth of the bacteria was inhibited around the paper disc was measured.
As a result, a blocking circle was formed in the AE fraction, the NB fraction, the AB fraction, and the AQ fraction (FIG. 3, Table 5). Although 50 μl of distilled water absorbed on a sterilized paper disk was placed on an agar medium and cultured in the same manner (control group), a blocking portion was not formed (the size of the blocking circle was 8 mm).
From the above results, it was found that the extracted fraction has a high bacteriostatic or antibacterial effect against Pseudomonas bacteria, which is one of the phytopathogenic bacteria. It became clear that it included.
（試験例５）
（ホエー抽出画分の大腸菌の抑制効果）
子牛の糞便より分離した大腸菌（Escherichia coli）を標準寒天培地（パールコア標準寒天培地、栄研化学製）に塗布し、３７℃で1日培養した。生育した菌体を生理食塩水に懸濁し、その１００μｌを標準寒天培地に塗布した。滅菌した直径１０ｍｍのペーパーディスク（ＡＤＶＡＮＴＥＣ製）に実施例２で得られたＡＥ画分を５０μl吸収させ、寒天培地上に置いた。ペーパーディスクを置いた培地を３７℃で1日間培養した後、ペーパーディクス周辺で菌の生育を阻害している範囲（阻止円）の大きさを測定した。
その結果、２２．０ｍｍの阻止円を形成しており（図４）、以上の結果から、抽出画分には動物の感染症原因菌である大腸菌に対しても高い静菌または抗菌効果があることが判明した。なお、ヒトの糞便より分離した大腸菌（Escherichia coli）についても同様の静菌または抗菌効果が認められた。なお、蒸留水を滅菌したペーパーディスクに５０μｌ吸収させたものを寒天培地上に置いて同様に培養したが（対照区）、阻止部分は形成されなかった（阻止円の大きさは１０ｍｍ）。
(Test Example 5)
(Inhibition effect of Escherichia coli in whey extract)
Escherichia coli isolated from calf feces was applied to a standard agar medium (Pearl Core Standard Agar, manufactured by Eiken Chemical Co., Ltd.) and cultured at 37 ° C. for 1 day. The grown cells were suspended in physiological saline, and 100 μl thereof was applied to a standard agar medium. 50 μl of the AE fraction obtained in Example 2 was absorbed on a sterilized paper disk having a diameter of 10 mm (manufactured by ADVANTEC) and placed on an agar medium. After culturing the medium on which the paper disc was placed at 37 ° C. for 1 day, the size of the range (inhibition circle) in which the growth of the bacteria was inhibited around the paper disc was measured.
As a result, a blocking circle of 22.0 mm was formed (FIG. 4). From the above results, the extracted fraction has a high bacteriostatic or antibacterial effect also against E. coli, which is an infectious agent of animals. It has been found. The same bacteriostatic or antibacterial effect was also observed for Escherichia coli isolated from human feces. Although 50 μl of distilled water absorbed on a sterilized paper disk was placed on an agar medium and cultured in the same manner (control group), a blocking portion was not formed (the size of the blocking circle was 10 mm).
（脱脂乳からの静菌または静菌または抗菌成分の分画）
１２Ｌの１０％還元脱脂乳を８０００ｇで３０分間、４℃で遠心分離し、その上清を分画分子量が１０，０００Ｄａの限外ろ過膜（3838 HFK-131、Koch Membrane Systems Inc.製）に供した。通過した画分を乳限外ろ過パーミエイト（１０Ｌ）とした。合成吸着樹脂（ＤＩＡＩＯＮ ＨＰ２０、三菱化学製）をエタノールで膨潤後、蒸留水で洗浄し、オープンカラムに充填した。乳限外ろ過パーミエイトをこの樹脂に供し、蒸留水にて１回洗浄を行った後、５０％のエタノール溶液で溶出した。１０Ｌの乳限外ろ過パーミエイトより５．３ｇの抽出画分が得ることができた。該抽出画分はそのまま本発明の静菌または抗菌剤として用いることができる。
(Bacteriostatic or bacteriostatic or antibacterial fraction from skim milk)
12 L of 10% reduced skim milk was centrifuged at 8000 g for 30 minutes at 4 ° C., and the supernatant was applied to an ultrafiltration membrane (3838 HFK-131, Koch Membrane Systems Inc.) with a molecular weight cut off of 10,000 Da. Provided. The passed fraction was designated as milk ultrafiltration permeate (10 L). A synthetic adsorption resin (DIAION HP20, manufactured by Mitsubishi Chemical) was swollen with ethanol, washed with distilled water, and packed into an open column. A milk ultrafiltration permeate was applied to this resin, washed once with distilled water, and then eluted with a 50% ethanol solution. An extracted fraction of 5.3 g could be obtained from 10 L of milk ultrafiltration permeate. The extracted fraction can be used as it is as a bacteriostatic or antibacterial agent of the present invention.
（植物病原菌防除剤）
上記実施例２で得られた静菌または抗菌剤（実施品６）１０ｇをポリオキシエチレンノニルフェニルエーテルリン酸アンモニウム１９０ｇ、ポリオキシエチレンノニルフェニルエーテル６０ｇ、リン酸トリエチル２８０ｇ、リン酸トリブチル４７０ｇと均一に混和し、乳剤を調製し、植物病原菌防除剤を製造した。
(Plant pathogen control agent)
10 g of the bacteriostatic or antibacterial agent obtained in Example 2 (Example 6) was uniformly with 190 g of polyoxyethylene nonylphenyl ether ammonium phosphate, 60 g of polyoxyethylene nonylphenyl ether, 280 g of triethyl phosphate, and 470 g of tributyl phosphate. To prepare an emulsion and produce a plant pathogen control agent.
（家畜用感染症予防飼料）
上記実施例２で得られた静菌または抗菌剤（実施品６）２００ｇを９９．８ｋｇの脱イオン水に溶解し、５０℃まで加熱後、ＴＫホモミクサー（MARK II 160型；特殊機化工業社製）にて、３６００ｒｐｍで４０分間撹拌混合した。この混合溶液に大豆粕１２ｋｇ、脱脂粉乳１４ｋｇ、大豆油４ｋｇ、コーン油２ｋｇ、パーム油２３．２ｋｇ、トウモロコシ澱粉１４ｋｇ、小麦粉９ｋｇ、ふすま２ｋｇ、ビタミン混合物５ｋｇ、セルロース２．８ｋｇ、ミネラル混合物２ｋｇを配合し、１２０℃、４分間殺菌して、本発明の家畜用感染症予防飼料１００ｋｇを製造した。
(Infectious disease prevention feed for livestock)
200 g of the bacteriostatic or antibacterial agent (Example 6) obtained in Example 2 above was dissolved in 99.8 kg of deionized water, heated to 50 ° C., and then TK homomixer (MARK II 160 type; Tokushu Kika Kogyo Co., Ltd.). The mixture was stirred and mixed at 3600 rpm for 40 minutes. This mixed solution contains 12 kg soybean meal, 14 kg nonfat dry milk, 4 kg soybean oil, 2 kg corn oil, 23.2 kg palm oil, 14 kg corn starch, 9 kg wheat flour, 2 kg bran, 5 kg vitamin mixture, 2.8 kg cellulose, and 2 kg mineral mixture. And sterilized at 120 ° C. for 4 minutes to produce 100 kg of an infectious disease prevention feed for livestock of the present invention.
（ヒト用感染症予防剤）
上記実施例１で得られた静菌または抗菌剤（実施品１）１０ｇ、ラクトース１４０ｇ、シュガーエステル８ｇ、セルロース２ｇを混合し、圧縮錠剤機により圧縮して、本発明品のヒト用感染症予防剤を製造した。
(Infectious disease prevention agent for humans)
10 g of the bacteriostatic or antibacterial agent (Example 1) obtained in Example 1 above, 140 g of lactose, 8 g of sugar ester and 2 g of cellulose are mixed and compressed by a compression tablet machine to prevent infection of the present invention for humans. An agent was produced.
本発明は、乳またはホエー等から静菌または抗菌成分含有画分を抽出する製造方法、当該静菌または抗菌成分を含む植物病原菌防除剤、感染症予防剤に関する。本発明は、食品分野、医薬分野のみならず、酪農畜産分野、農業分野への利用が可能である。特に、農薬を使用できない野菜の病原菌防除や、抗生物質の使用を控えたい家畜の感染症予防、合成保存料を使用できない食品の保存性向上に大きく貢献することが期待できる。 The present invention relates to a production method for extracting a bacteriostatic or antibacterial component-containing fraction from milk, whey or the like, a phytopathogenic fungus control agent containing the bacteriostatic or antibacterial component, and an infection prevention agent. The present invention can be used not only in the food field and the pharmaceutical field, but also in the dairy and livestock field and the agricultural field. In particular, it can be expected to greatly contribute to the control of pathogens in vegetables that cannot use pesticides, the prevention of infectious diseases in livestock that refrain from the use of antibiotics, and the improvement in the preservability of foods that cannot use synthetic preservatives.
Claims (4)
（ａ）ホエーより脂肪及びタンパク質成分を除去する工程であって、タンパク質成分の除去は、タンパク質成分を加水分解することなく、分画分子量２，０００Ｄａ以上２０，０００Ｄａ未満の限外ろ過膜を用いて除去する工程である、前記工程
（ｂ）前記（ａ）の工程により得られた溶液をスチレン−ジビニルベンゼン系合成吸着剤と接触させる工程
（ｃ）前記（ｂ）の工程により得られたスチレン−ジビニルベンゼン系合成吸着剤を含水有機溶媒で溶出し、有効成分を得る工程 A method of manufacturing a bacteriostatic or antibacterial agent as an active ingredient component obtained from E er, said manufacturing method comprising the following steps.
(A) a step of removing the fat and protein components than e er, removal of the protein component, the protein component without hydrolysis, an ultrafiltration membrane less than a fractional molecular weight 2,000Da more 20,000Da The step obtained by the step (b) is a step of bringing the solution obtained by the step (b) and the step (a) into contact with a styrene-divinylbenzene synthetic adsorbent . Elution of styrene-divinylbenzene synthetic adsorbent with water-containing organic solvent to obtain active ingredients
（ａ）ホエーより脂肪及びタンパク質成分を除去する工程であって、タンパク質成分の除去は、タンパク質成分を加水分解することなく、分画分子量２，０００Ｄａ以上２０，０００Ｄａ未満の限外ろ過膜を用いてタンパク質を除去する工程である、前記工程
（ｂ）前記（ａ）の工程により得られた溶液をスチレン−ジビニルベンゼン系合成吸着剤と接触させる工程
（ｃ）前記（ｂ）の工程により得られたスチレン−ジビニルベンゼン系合成吸着剤を含水有機溶媒で溶出し、有効成分を得る工程 A method of manufacturing a plant pathogen control agents as an active ingredient component obtained from E er, said manufacturing method comprising the following steps.
(A) a step of removing the fat and protein components than e er, removal of the protein component, the protein component without hydrolysis, an ultrafiltration membrane less than a fractional molecular weight 2,000Da more 20,000Da A step of removing a protein using the step (b), a step of bringing the solution obtained in the step (a) into contact with a styrene-divinylbenzene synthetic adsorbent, and (c) a step obtained in the step (b). Of the obtained styrene-divinylbenzene synthetic adsorbent with a water-containing organic solvent to obtain an active ingredient
（ａ）ホエーの限外ろ過パーミエートをスチレン−ジビニルベンゼン系合成吸着剤と接触させる工程
（ｂ）前記（ａ）の工程により得られたスチレン−ジビニルベンゼン系合成吸着剤を含水有機溶媒で溶出し、有効成分を得る工程 A method of manufacturing a plant pathogen control agents as an active ingredient component obtained from E er, said manufacturing method comprising the following steps.
(A) styrene ultrafiltration permeate ho er - eluting divinylbenzene based synthetic adsorbent with aqueous organic solvent - contacting divinylbenzene based synthetic adsorbent (b) the resulting styrene obtained by the steps of (a) And obtaining an active ingredient
（ａ）ホエーより脂肪及びタンパク質成分を除去する工程であって、タンパク質成分の除去は、タンパク質成分を加水分解することなく、分画分子量２，０００Ｄａ以上２０，０００Ｄａ未満の限外ろ過膜を用いてタンパク質を除去する工程である、前記工程
（ｂ）前記（ａ）の工程により得られた溶液をスチレン−ジビニルベンゼン系合成吸着剤と接触させる工程
（ｃ）前記（ｂ）の工程により得られたスチレン−ジビニルベンゼン系合成吸着剤を含水有機溶媒で溶出する工程
（ｄ）（ｃ）の工程に得られる溶出物を飲食品、栄養組成物または飼料に添加する工程 Food or beverage containing a component obtained from E er, nutritional composition, a process for the preparation of feed, the manufacturing method comprising the following steps.
(A) a step of removing the fat and protein components than e er, removal of the protein component, the protein component without hydrolysis, an ultrafiltration membrane less than a fractional molecular weight 2,000Da more 20,000Da A step of removing a protein using the step (b), a step of bringing the solution obtained in the step (a) into contact with a styrene-divinylbenzene synthetic adsorbent, and (c) a step obtained in the step (b). A step of eluting the obtained styrene-divinylbenzene-based synthetic adsorbent with a water-containing organic solvent in steps (d) and (c) and adding the eluate obtained in the food, beverage, nutritional composition or feed
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