JP2004506403A5 - - Google Patents

Description

【００４７】
実施例２
本発明の組成物のインビボの効力を実証するためにホットドッグにＬｉｓｔｅｒｉａ ｍｏｎｏｃｙｔｏｇｅｎｅｓを接種する。ホットドッグを、（１）Ｎｏｖａｓｉｎ（商標）、リゾチーム、およびＢＨＡ、または（２）Ｎｏｖａｓｉｎ（商標）およびＢＨＡのいずれかを含有する懸濁液中に浸漬し、次いでその表面にＬ． ｍｏｎｏｃｙｔｏｇｅｎｅｓを接種した。次いでホットドッグを無菌の袋に詰め、１０℃で１３日間保った。各試料採取日にホットドッグを無菌の生理的食塩水で洗浄し、Ｌｉｓｔｅｒｉａ菌数を得るために樹脂をＬｉｓｔｅｒｉａ選択寒天の上に塗布する。リンス液１ ｍｌをＢＨＩブイヨンに移し、２４時間インキュベートし、続いてＬｉｓｔｅｒｉａ選択寒天の上に塗布することによりホットドッグの強化を行なう。強化後Ｌｉｓｔｅｒｉａ選択寒天の上でカウント零を示す処理物を陰性とみなし、全処理試料に対する分数として記録する。
【００４８】
表２は、Ｎｏｖａｓｉｎ（商標）およびＢＨＡの浸漬がホットドッグ上の最初のＬｉｓｔｅｒｉａ ｍｏｎｏｃｙｔｏｇｅｎｅｓの数をかなり減少させるのに対し、３成分の組み合わせは直接平板培養または強化回復法のどちらによっても事実上検出されないレベルまでＬｉｓｔｅｒｉａ ｍｏｎｏｃｙｔｏｇｅｎｅｓの数を減少させることを示している。この３成分の組み合わせでは、少数の損傷した細胞を回復させるために用いられる感受性強化法を適用した後の陰性の結果（３／３）によって立証されるように、増殖能力のあるＬｉｓｔｅｒｉａ ｍｏｎｏｃｙｔｏｇｅｎｅｓの生存は観察されなかった。新奇な３成分の組み合わせで処理された試料はすべて、強化後でさえＬｉｓｔｅｒｉａ ｍｏｎｏｃｙｔｏｇｅｎｅｓに関して陰性であったという発見は、本出願の最も予期せざる発見である。このような発見が、これら抗菌性成分の任意の１つを用いて本明細書に記載の安全かつ適切な使用レベルで報告されたことは未だかってない。
【００４９】
【表２】

【００５０】
実施例３
表３に示すように、Ｎｏｖａｓｉｎ、リゾチーム、およびβホップ酸の単独または組み合わせの阻害効果を示すために桿菌属の２種類のグラム陽性の芽胞菌をトリプチカーゼダイズブイヨン、ｐＨ ６．０中で、３０℃、４８時間試験した。試験は、芽胞菌に対するＮｏｖａｓｉｎ、リゾチーム、およびβホップ酸（ＢＨＡ）の驚くべき相乗効果を実証する。表３は、同じ濃度のＮｏｖａｓｉｎ、リゾチーム、およびＢＨＡの組み合わせが、いずれかの成分単独または３種類のうち２種類の成分のみよりも著しく優れた阻害効果を有することを示す。３成分方式の組み合わせは接種されたすべての芽胞を完全に殺すのに、単一成分または２成分で処理した場合はかなりの除去が観察されるが完全ではない。したがってＮｏｖａｓｉｎ、リゾチーム、およびＢＨＡの組成物が芽胞菌に対して相乗的な殺菌活性を示すことがここに結論として実証される。
【００５１】
【表３】

【００５２】
実施例４
熱を加えられたハムに対する本発明の組成物の有効性を表４で実証する。熱を加えられたハムの表面にＬｉｓｔｅｒｉａ ｍｏｎｏｃｙｔｏｇｅｎｅｓを接種し、次いでＮｏｖａｓｉｎ（商標）、リゾチーム、およびＢＨＡ（処理Ａ）、またはリゾチームおよびＢＨＡ（処理Ｂ）のいずれかを表面に噴霧することにより局所的な処理を行なった。次いでハムを収縮包装し、無菌の袋に入れて真空包装し、華氏４０度（４℃）で６０日間貯蔵した。各試料採取日にハムを無菌の緩衝液で洗浄し、樹脂をＬｉｓｔｅｒｉａ選択寒天の上に塗布して増殖能力のあるＬｉｓｔｅｒｉａ菌数を決定する。リンス液１ ｍｌを脳心臓浸出液（ＢＨＩ）のブイヨンに移し、２４時間インキュベートし、続いてＬｉｓｔｅｒｉａ選択寒天の上に塗布することにより検出可能な平板菌数未満のリステリア菌数の試料に対して強化法を実施した。強化後のＬｉｓｔｅｒｉａ選択寒天の上で否定的（１ ｍｌ 当たり１個未満）な菌数を有する処理物は陰性とみなし、全処理試料に対する分数として記録する。
【００５３】
表４は、対照群が１０，０００倍（対数４）に増加したのに、３成分の組み合わせは接種されたＬｉｓｔｅｒｉａ ｍｏｎｏｃｙｔｏｇｅｎｅｓを直接平板培養または強化回復法のどちらによっても検出されないレベルまで減少したことを示している。２成分方式の「リゾチームプラスＢＨＡ」の組み合わせは阻害効果は示したが、殺菌性としてではなかった。３成分の組み合わせにおいては、強化、すなわちきわめて少数の増殖能力のある細胞または損傷した細胞を回復させるために用いられる手法を適用した後の陰性の結果によって立証されるように、増殖能力のあるＬｉｓｔｅｒｉａ ｍｏｎｏｃｙｔｏｇｅｎｅｓの生存は観察されなかった。したがってこのデータは、好ましい３成分組成物の局所的な塗布により、熱を加えられたハムなど高脂肪の加工食料品中のＬｉｓｔｅｒｉａ ｍｏｎｏｃｙｔｏｇｅｎｅｓを完全に除去することができることを実証している。
【００５４】
【表４】

【００５５】
実施例５
フランクフルトソーセージをＬｉｓｔｅｒｉａから保護するための本発明の組成物の有効性。ホットドッグのエマルションを含有するセルロース製包装容器の中に組成物を組み込むことにより、調理の間にＮｏｖａｓｉｎおよびＢＨＡの組成物をフランクフルトソーセージの表面に送達した。調理ののち包装容器を剥ぎ、次いで仕上がったフランクフルトソーセージの表面にＬｉｓｔｅｒｉａ ｍｏｎｏｃｙｔｏｇｅｎｅｓを接種し、無菌の袋に真空包装した。包装され接種されたフランクフルトソーセージを華氏４０度（４℃）で６０日間以上貯蔵し、各試料採取の時点で新鮮な包装容器を開封した。各試料採取時に無菌の緩衝液を用いてフランクフルトソーセージをその包装容器と一緒に洗浄し、次いでＬｉｓｔｅｒｉａ選択寒天の上に塗布して増殖能力のあるＬｉｓｔｅｒｉａの菌数を決定した。
【００５６】
表５は、２成分の組み合わせが６１日を超える期間Ｌｉｓｔｅｒｉａ ｍｏｎｏｃｙｔｏｇｅｎｅｓの増殖を阻害し、インキュベーションの終わりには対照と比較して対数４（１０，０００倍）を超すリステリアの減少をもたらしたことを示す。このデータは、好ましい２成分組成物の塗布を送達する包装容器がフランクフルトソーセージ上のＬｉｓｔｅｒｉａ ｍｏｎｏｃｙｔｏｇｅｎｅｓの生長を妨ぎ、組成物を送達し、それによってフランクフルトソーセージおよびソーセージの安全性を向上する実用的な方法である可能性があることを実証する。より重要なことはこの表面塗布の方法が、典型的なホットドッグエマルションの高脂肪の環境においてさえ、成分の活性を十分に実現させることを可能にする。これとは対照的に、この組成物を調理前のホットドッグエマルション中に成分として使用する場合には抗菌活性は劇的に低下し（データは示さない）、これは有意な量の脂肪を含有する食物中でのナイシンの活性の喪失（Ｍｕｒｉａｎａ， Ｐ． Ｍ．およびＫａｎａｃｈ， Ｌ．の論文、Ｕｓｅ ｏｆ Ｎｉｓａｐｌｉｎ ｔｏ Ｉｎｈｉｂｉｔ Ｓｐｏｉｌａｇｅ Ｂａｃｔｅｒｉａ ｉｎ Ｂｕｔｔｅｒｍｉｌｋ Ｒａｎｃｈ Ｄｒｅｓｓｉｎｇ， Ｊ． Ｆｏｏｄ Ｐｒｏｔｅｃｔｉｏｎ， Ｖｏｌ． ５８， Ｎｏ．１０， １９９５）およびホップ酸の活性の喪失（Ｊｏｈｎｓｏｎ等の論文、１９９６年）を実証した以前の著者達による観察と一致している。したがって開示した表面塗布の方法は、高い脂肪量（４ ｗ／ｗ％を超す）を含有する食物中で本発明の組成物の効力を最大にする驚くべき手段を明らかにする。
【００５７】
【表５】

【００５８】
本発明を詳細かつその好ましい実施形態を参照して記述したが、これにより特許請求の範囲の領域を逸脱することなく修正形態および変形形態が可能であることは明らかであろう。[Document name] statement
Antibacterial composition for controlling gram positive bacteria used in foodstuffs
[Claim of claim]
(A) at least one gram-positive selected from the group consisting of bacteriocins of the lantibiotics (lantibiotics), pediocin (pediocin), and lacticin (lacticin) classes and / or degrading enzymes A first component comprising a bacteriostatic or bactericidal compound, and (b) at least one compound selected from the group consisting of hops acids, hops acid derivatives, hops resins, and hops resins derivatives An antimicrobial composition comprising two components.
2. A first component comprising at least one gram-positive bacteriostatic or bactericidal compound selected from the group consisting of (a) lantibiotics and degradative enzymes,
(B) An antimicrobial composition comprising a second component comprising at least one compound selected from the group consisting of hop acids, hop acid derivatives, hop resins, and hop resin derivatives.
3. An antimicrobial composition comprising: (a) nisin, b) lysozyme, and (c) beta hop acid or beta hop acid derivative.
4. The composition according to claim 1, containing about 38.5 to 99.8 parts by weight of the first component and about 61.5 to 0.2 parts by weight of the second component, all parts by weight of the first component. The antimicrobial composition according to claim 1, which is based on the total of and the second component.
5. The first component comprises about 1.0 to 2.5 parts by weight of lantibiotics and about 37.5 to 97.3 parts by weight of degrading enzymes, and the second component comprises hop acids and hop acid derivatives. At least one compound selected from the group consisting of about 61.5 to 0.2 parts by weight, all parts by weight of which based on the sum of the first and second components of the composition. The antimicrobial composition as described in.
6. A method for reducing Gram-positive bacteria in food, comprising: (a) at least one member selected from the group consisting of bacteriocins of the lantibiotics, pediocin and lacticin classes, and degrading enzymes A second component comprising a first component comprising a gram-positive bacteriostatic or bactericidal compound and (b) at least one compound selected from the group consisting of (b) hop acids, hop acid derivatives, hop resins, and hop resin derivatives Treating the food surface of the food product with a bacteriostatic or germicidally effective amount of the composition comprising the ingredients.
7. A method for reducing gram positive bacteria in food, comprising:
(A) a first component comprising at least one gram-positive bacteriostatic or bactericidal compound selected from the group consisting of lantibiotics and degrading enzymes,
(B) a bacteriostatic or bactericidally effective composition comprising a second component comprising at least one compound selected from the group consisting of hop acids, hop acid derivatives, hop resins, and hop resin derivatives; Treating the food surface of the food.
8. The method according to claim 7, wherein the composition comprises (a) nisin, (b) lysozyme, and (c) beta hop acid or beta hop acid derivative.
9. About 38.5-99.8 parts by weight of the first component, and about 61.5-0.2 parts by weight of the second component, all parts by weight of the first component comprising the first component The method according to claim 6, which is based on the sum of the second component and the second component.
10. The first component comprises about 1.0 to 2.5 parts by weight of lantibiotics and about 37.5 to 97.3 parts by weight of degrading enzyme, and the second component comprises hop acid and hop acid derivative 7. The method according to claim 6, comprising about 61.5 to 0.2 parts by weight of at least one compound selected from the group consisting of: all parts by weight based on the sum of the first and second components of the composition. The method described in.
11. A method according to claim 6, wherein the food is a solid containing greater than 4% fat content.
12. The method of claim 6, wherein the food product is processed meat.
13. The treating step comprises the steps of: (a) coating the composition on the surface of the packaging container, film or packaging material; and (b) subsequently adhering the surface coated with the composition to the food. The method of claim 6, comprising the steps.
14. The method according to claim 6, wherein said gram positive bacteria is of the genus Listeria.
15. The method according to claim 14, wherein the Listeria is Listeria monocytogenes or Listeria inocua.
16. The method according to claim 6, wherein said gram positive bacterium is a spore.
17. The method according to claim 16, wherein the spore fungus is Bacillus (Bacillus · bacillus) spp.
18. The method of claim 16, wherein said spore fungus is Clostridium spp.
Detailed Description of the Invention
[0001]
Background of the invention
1. Field of the Invention
The invention relates to one or more species from the category of lantibiotics, pediocins, bacteriocins of the class of lacticins, and / or degrading enzymes, in addition to one or more types of hops acid extract or modified hop acid extract. Disclosed are methods of inhibiting or retarding the growth of bacteria on food by treating with a composition comprising a safe, suitable gram-positive, bacteriostatic or bactericidal preparation. More particularly, the method comprises nisin, and / or lysozyme and beta-hop acid to reduce or eliminate gram-positive rot or pathogenic bacteria, more particularly all strains of harmful pathogenic Listeria Monocytogenes Use the composition as an ingredient or apply to a food surface. An important public health problem is that pathogenic Listeria species, in particular Listeria Monocytogenes, have the ability to grow at commercial freezing temperatures, which usually store processed food for an extended period of time. This ability to grow under standard conditions of distribution makes Listeria Monocytogenes one of the greatest public health hazards associated with raw and processed foods today. Any new antimicrobial system, its formulation and temperature conditions must be effective for commercial food products considering the practical implementation. The novel compositions of the present invention are effective at various food products at risk of Listeria contamination, particularly at frozen storage and transport temperatures common to food products.
[0002]
2. Description of technology
The degree of food transmission and food poisoning in the United States is quantitatively recorded in the CAST report of 1994 (Foodborne Pathogens: Risks and Consequences. Task Force Report No. 122, Counsil for Agricultural Science and Technology, Washington DC). Also, over the past few years, it has been extensively characterized by better reporting systems and programs (CDC. 1988c. 1997 Final FoodNet Surveillance Report. U. S. Department of Health and Human Services, October 1998). Various studies have been conducted to reduce the prevalence of listeriosis and other food transmissions, and compositions have been developed which act as antimicrobial components for foodstuffs. The individual compounds disclosed by this study are inherently ineffective in many cases with a single compound or are commercially expensive or use too expensive to use in food processing and formulations There was little if any. Even today, Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Clostridium botulinum, C., which pose significant health risks to consumers. There is a need for better control of Gram-positive pathogens such as perfringens. In addition, other gram-positive rot bacteria such as lactobacilli, streptococci, bacilli, enterococci, and micrococci species are known to cause decay, although they are not normally ill, and often the shelf life of certain foodstuffs And the main causative agent that reduces freshness.
[0003]
Both pathogens and spoilage bacteria are often found in raw foodstuffs, but heat processing helps to dramatically reduce bacterial load. After processing, when most food products can be exposed to germs in the food handling environment, there is a risk of recontamination before packaging, distribution, and final consumption. Even in the cleanest processing facilities, certain pathogens can usually contaminate already processed foodstuffs at very low levels. Then, in the case of cold-resistant pathogens, i.e. mainly of various Listeria species, they grow uninhibited on food throughout distribution and storage until final consumption. The more such pathogens grow in food, the higher the risk of transmitting the food to the consumer. For meat and everyday products that do not require cooking, such foods are particularly problematic because they are not reheated or reprocessed by the user prior to consumption. The most likely risk in such an example is from Listeria species that grows well under freezing. It is recognized that high intake of any pathogens increases the risk of transmission, particularly among infants, the elderly, pregnant women, and individuals with any immune deficiency.
[0004]
It is estimated that about 500 deaths have occurred in the United States probably due to food-borne listeriosis in 1998. Among the major foodborne pathogens, Listeriosis exhibits the highest mortality rate, exceeding 20% according to Meade et al. (Food-related Illness and Death in the United States, CDC 5: 5, September-October 1999). . The urgent need for a system to reduce the growth of Listeria in foodstuffs in light of its risk level and large social costs is the United States Department of Public Health, the Food and Drug Administration (FDA), and the United States Department of Agriculture (USDA) Recognized by). The subject matter of the present invention is a novel, Food Product (Generally Recognized as Safe), flavorless composition that targets Listeria in a real food product system, in particular in a meat product that does not require processed cooking. It is. The desired effect of such compositions is to kill or reduce the number of Listeria in foods that survive processing or are at risk of contamination with such species. In addition, the risks associated with the above-mentioned spoilage types and other gram-positive bacteria, including Corynebacteria, Diplococci, Mycobacteria, Streptococci, and less common strains of the genus Streptomyces are also highlighted as food problems, such as It may also benefit from the method or composition.
[0005]
U.S. Pat. Nos. 5,096,718 and 5,260,061 in 1992 and 1993, the metabolism of propionibacteria in certain foodstuffs to improve the shelf life of processed foods It was disclosed to use the product. These metabolites have proven to be highly potent against gram negative bacteria but unfortunately are not effective against gram positive bacteria.
[0006]
U.S. Pat. No. 5,217,950 suggested using a composition of nisin as a germicide. Nisin is a polypeptide with antibacterial properties that is naturally produced by lantibiotics, more particularly by various strains of Lactococcus lactis. Although nisin is primarily effective against gram positive bacteria, the common gram positive pathogen Listeria monocytogenes is more resistant to nisin than most other species of gram positive bacteria. The need to increase the activity of nisin against Listeria monocytogenes is well recognized and also explains why nisin itself is not commercially used as an anti-Listeria agent. Thus, the 5,217,950 patent describes nisin, which produces a broad spectrum of activity against both Listeria species and certain gram-negative bacteria, and disodium ethylenediaminetetraacetate (EDTA) or other acetates or citrates. We propose a combination with chelating agents such as acid salts.
[0007]
U.S. Pat. Nos. 5,573,797, 5,593,800, and 5,573,801 disclose bacteriocins derived from Streptococcus or Pediococcus or synthetic corresponding antibacterial agents and chelating agents. An antimicrobial composition comprising a combination is disclosed. The composition is applied to the surface of the foodstuff to be treated by direct application to the foodstuff or by incorporating the composition onto a flexible film container placed in intimate contact with the surface of the foodstuff. . Chelating agents capture free divalent cations in the outer membrane of gram negative cells to improve the permeability of the antimicrobial agent. Although the example of the 5,573,801 patent discloses applying nisin alone to a cooked meat surface, it has been adopted commercially because of the limited efficacy of this single component. Not.
[0008]
U.S. Pat. No. 5,458,876 proposes a combination of lantibiotics (such as nisin) and lysozyme as antimicrobial agents. In this example, lysozyme degrades the cell wall and weakens the structural integrity of the target cell such that the antimicrobial agent is more effective in damaging or killing the bacterial cells. In particular, it has been found that this combination improves the antibacterial effect of nisin against Listeria Monocytogenes and results in a significant reduction of Listeria at safe and adequate doses.
[0009]
European Patent Publication No. 0 466 244 discloses a composition with improved antimicrobial properties comprising a mixture of at least one of the following groups of compounds. That is, (i) cell wall lysing substances or salts thereof, (ii) antimicrobial compounds, and (iii) food, or salts of organic acids or salts thereof that are acceptable for use in preparations for cosmetic or hygienic use, phosphorus An adjuvant selected from acid salts and condensed phosphates or various corresponding acids, and other sequestering agents. Preferably (i) lysozyme, (ii) possibly bacteriocin (e.g. nisin or pediocin), and (iii) possibly acetic acid, sodium diacetate, lactic acid, citric acid, propionic acid, tartaric acid, orthophosphate, hexametaphosphate, tripolyline Sequestrants containing acid salts, other polyphosphates, or substituted or unsubstituted amino groups such as EDTA.
[0010]
European Patent Publication 0 453 860 proposes a combination of nisin and a phosphate buffer effective between pH 5.5 and 6.5 to eradicate gram-negative bacteria on the surface.
[0011]
International Patent Publication No. WO 97/23136 involves treatment with an alkaline metal salt solution of low concentration of orthophosphate combined with either osmotic shock and / or lysozyme in solution and / or nisin in solution. We propose a method for removing bacterial contamination. For this reference, a combination of low concentrations of trisodium orthophosphate and lysozyme for certain bacteria on lettuce leaves or chicken skin and low concentrations of trisodium orthophosphate for certain bacteria on chicken skin A combination of nisin was tested.
[0012]
Australian Patent Publication No. AU-A-18604 / 88 discloses the use of a product of N-acetylmuramidase, a bacterial lytic enzyme containing lysozyme, for example, with a non-enzymatic preservative for food preservation There is. The nonenzymatic preservatives described in this publication are: citric acid and EDTA; amino acids, in particular amino acids such as cysteine, alanine, tyrosine and glycine; and 5'-inosine monophosphate of inosine, or pyrroline Complexing agents such as nucleosides and nucleotides such as acid tetrasodium (diphosphate), sodium tripolyphosphate (triphosphate), and polyphosphates; or reding agents such as alkali metal salts of nitric acid.
[0013]
U.S. Pat. No. 5,286,506 has a bacteriostatic action against Listeria Monocytogenes in food, so that 6 to 50 ppm of fat-soluble hop beta acid extract per weight of foodstuff can be used Have been described. In addition, US Pat. Nos. 5,370,863 and 5,455,038 show that certain hop acid derivatives that are chemically hydrogenated may have antimicrobial activity against Listeria species. Suggest. However, these extracts are not for food use (GRAS) and can not be used for food except for brewing.
[0014]
Finally, Johnson et al. In International Journal of Food Microbiology 33 (1996) 195-207, presumably hop acids and hop acid derivatives are due to beta acids being transferred or confined into fat emulsions and then Discloses that it has limited efficacy against Listeria species in fat-containing foods such as cheese, meat, sauces, and dressings by not being available to control bacterial growth in the aqueous part of food emulsions There is. The problem of the absence of activity of beta-hop acids in fat-containing foods has made them unusable commercially as natural antimicrobials for control of Listeria or other Gram-positive pathogens.
[0015]
All of the references cited above are specifically incorporated by reference to the extent necessary to complete the present patent application.
[0016]
In the light of the above teachings, there is still a need in the art for a method of treating food with a bactericidal composition having activity at a reasonable dosage in common food applications at risk of gram positive pathogens. There is. More specifically, there is a need for effective processing of these products that can be conveniently incorporated into existing manufacturing design schemes. Finally, to more fully and effectively reduce or even eliminate harmful Gram-positive pathogens by using safe, adequate and economical amounts of food grade antimicrobials such as nisin, lysozyme and hop acids There is a need for
[0017]
Summary of the invention
Quite surprisingly, one or more of the following types of materials: bacteriocins of the lantibiotics, pediocin and / or lacticin class, or one or more gram-positive bacteriostatic agents from degrading enzymes Or a composition having a first component comprising a bactericidal compound and a second component comprising one or more naturally occurring hop acids or hop resins or derivatives thereof is an individual component or a composition of previously published compositions It has now been discovered that the present invention provides superior antimicrobial properties against potentially harmful bacteria, particularly of the Listeria by dramatically surpassing any antimicrobial effect.
[0018]
One embodiment of the present invention is obtained from the following types of materials as the first component (a) bacteriocins of the lantibiotics, pediocin and lacticin class and / or lysozyme ie natural enzymes from egg white Antimicrobial composition comprising one or more gram-positive bacteriostatic or bactericidal compounds, and as a second component (b) one or more hop acids or hop acid derivatives, or hop resins or hop resin derivatives Including things. Particularly preferred are compositions comprising lantibiotic bacteriocin, lysozyme and beta hop acid extract.
[0019]
In another embodiment, the present invention relates to (a) one or more of the following types of materials: lantibiotics, pediocins, and bacteriocins of the class bacteriocins of the class of lacticin, or one or more gram positives obtained from degrading enzymes An effective amount of a composition comprising a bacteriostatic or bactericidal compound as a first component, and (b) one or more hop acids or hop acid derivatives, or a hop resin or hop resin derivative as a second component Treating the surface of the food, slowing the growth in the food, reduce the viable count, or provide a method of completely removing Gram-positive bacteria, in particular Listeria Monocytogenes.
[0020]
The object of the present invention is to provide a method of treating food such that it is protected against harmful bacteria and so that the antimicrobial activity of the compound is maintained on or even in fat-containing foods. is there.
[0021]
Another object of the present invention is to provide novel compositions having antimicrobial properties far superior to those previously observed for the individual components of the composition.
[0022]
These and other objects should be readily apparent to those skilled in the art upon reference to the detailed description of the preferred embodiment.
[0023]
Detailed Description of the Preferred Embodiment
Several terms are used in the description of the preferred embodiments to facilitate understanding. Such terms are intended to encompass the re-cited embodiments as well as all technical equivalents, and are operated in substantially the same way to achieve approximately the same results for substantially the same purpose. Ru.
[0024]
The present invention provides novel antimicrobial compositions and methods of use thereof in methods of reducing, preventing or completely removing harmful bacteria from the surface of food, even in fat-containing foods.
[0025]
The novel antimicrobial composition comprises (a) one or more of the following types of materials: lantibiotics, pediocins, and bacteriocins of the class of bacteriocins of the class of lacticin, or one or more gram-positive enzymes obtained from degrading enzymes A fungal or bactericidal compound, and (b) one or more hop acids or hop acid derivatives, or hop resins or hop resin derivatives.
[0026]
The first component of the composition of the present invention is one or more compounds having gram positive bacteriostatic or bactericidal activity. Such compounds preferably include, but are not limited to, bacteriocins and / or egg whites of the lantibiotics, pediocin and lacticin classes and / or lysozyme from egg white, shellfish, or other natural sources. A combination of two or more compounds having gram-positive bacteriostatic or bactericidal activity (eg, nisin and lysozyme) is considered to fall particularly within the preferred range of the first component of the present invention.
[0027]
The first class of gram positive bacteriostatic compounds comprises lantibiotics. The term "lantibiotics" was coined by Schnell et al. (Nature 333: 276-278, 1988) to describe a bacteriocin group that includes nisin containing amino acid lanthionine and other "non-protein" amino acids. The common properties of these bacteriocins are investigated in detail by Kellner et al. (Eur. J. Biochem. 177: 53-59, 1988), among which "... Polycyclic polypeptide antibiotics have a high content of non- It has saturated amino acids (dehydroalanine, dehydrobutrin) and thioether amino acids (meso-lanthionine, (2S, 3S.6R) -3-methyllanthionine) In addition, lisinoalanine, 3-hydroxyaspartic acid, and S- ( 2-aminovinyl) -D-cystine is found in some members. Members of this group include nisin, subtilin, pep5, epidermin, gallidermin, cinnamycin, Ro09-0198, duramycin, and ancovenin. These ribosomally synthesized peptide-based antibiotics contain 19-34 amino acids and are produced by a variety of microorganisms including Staphococcus species, lactic acid bacteria, Bacillus species, and Streptomyces species. In addition to their unique composition of non-protein amino acids, they can be distinguished from other peptide antibiotics based on their specificity. In general bacteriocins, in particular lantibiotics, are characterized by a very narrow spectrum of action. Thus, only a few species of bacteria are sensitive to specific bacteriocins at practical, safe and appropriate concentrations. Such bacteriocins have at most no bacteriostatic (i.e. growth inhibitory) properties at the usual legally acceptable levels used for foodstuffs (e.g. up to 12.5 ppm pure nisin in some foodstuff systems) It is. It is active against polymyxin B1, which exhibits active bactericidal activity against a wide range of bacteria even at very low doses, as well as against most bacterial, yeast and even mammalian cells, International Patent Publication No. WO 89/00194. In contrast to other broad spectrum peptide based antibiotics such as the "lytic peptides" discussed by Jaynes et al.
[0028]
Nisin is a ribosome-encoded peptide that rarely occurs as a dimer with a molecular weight of about 7000. It contains several unusual amino acids, including β-methyl lanthionine, dehydroalanine, and lanthionine in all its 34 amino acids. There are five unusual thio-ether linkages in the peptide that contributes to its stability in acidic solution. Nisin is one of the most fully characterized bacteriocins and shares remarkable structural and functional homology with other lantibiotics such as subtilin and epidermin (Buchman et al. J. Bio. Chem. 263 (31): 16260-16266 (1988)). For the latest commentary on nisin, its physical properties and usage, see T.K. R. Klaenhammer's Commentary "Bacteriocins of Lactic Acid Bacteria", Biochimie 70: 337-349 (1988), Hurst's comment "Nisin", Avd. Appl. Microbiol. 27: 85-121 (1981), and U.S. Patent No. 4,740,593. Nisin is a collective name that describes several closely related substances that exhibit similar amino acid composition, and a limited range of antimicrobial activity. This phenomenon is described in "Antibiotics and Antibiosis in Agriculture" (M. Woodbine) pp. Of E.C. 103-130 (1988). It is discussed by Lipinska.
[0029]
L. For using nisin to combat monocytogenes, see M. Doyle's paper "Effect of Environmental and Processing Conditions on Listeria monocytogenes", Food Technology, 42 (4): 169-171 (1988). This reference describes the initial suppression of growth of the organism (about 12 hours), L. et al. It has been reported that monocytogenes can grow at pH as low as 5.0 and is also resistant to alkaline pH and has the ability to grow at pH 9.6.
[0030]
Nisin is a standardized 2.5% by weight preparation of Rhodia Inc. Are marketed under the trademark Novasin®. Protein-containing lantibiotics may also be present as minor fermentation by-products in some varieties of Cheddar cheese or American cheese, and also in fermented defatted milk products also known as MICROGARD® MG 300 There is sex. In practice, lantibiotics are preferably used in amounts of about 1 to about 100 ppm (by weight of solution used for treatment) of the active ingredient (nisin), based on the safety and adaptability when used in food, preferably 1 to 12 .5 ppm is added to the food.
[0031]
As an alternative to using lantibiotics in a preferred combination, it is also known that the use of metabolites of Pediococcus spp., In particular pediocin, in place, can yield greater results. Pediocin is not approved for food use, but may still be acceptable for commercial use in the future. In addition, a new class of streptococcal bacteriocin called lacticin, in particular called lacticin 3147 described in Irish patent application 980500, may provide similar activity against gram positive bacteria. Similar to lantibiotics, both pediocin and lacticin are known to have bacteriostatic activity primarily against a limited range of gram positive bacteria.
[0032]
The second class of gram-positive bactericidal proteins comprises degrading enzymes, in particular lysozyme, which are not only most commonly obtained from egg albumin in food extraction processes, but also arctic typhus, human milk, tears, And from other natural sources. When lysozyme is used as a bactericide, an amount of about 50 to about 500 ppm (by weight of solution used for processing), preferably about 50 to about 200 ppm, is mainly to inhibit Clostridium tyrobutyricum in ripened cheese Add to food. Lysozyme is not bactericidal against other gram positive bacteria at this level, but is used at higher levels (above 1000 ppm, generally above 2000 ppm) to remove the cell wall of a wide range of gram positive bacteria. It has
[0033]
Lysozyme (muramidase, mucopeptide N-acetylmucamoyl hydrolase, 1,4-β-N-acetylhexosaminidase E.C. 3.2.1.17) is a mucosal lysis isolated from various sources It is an enzyme and is well characterized. Egg white lysozyme W. in 1922. The first protein first discovered and sequenced by Fleming, a three-dimensional structure was proposed using X-ray crystallography and a detailed mechanism of action was proposed. Its antibacterial activity against Gram-positive bacteria is described, for example, in CRC Crit. In Food Science and Nutrition, 26 (4): 359-395 (1988). Reviews in V. N. A sufficient record is left by the commentary of Procter et al. The molecular weight of egg white lysozyme is about 14,300 to 14,600, and the isoelectric point is pH 10.5 to 10.7. It consists of 129 amino acids linked together by four disulfide bridges. Similar enzymes have been isolated and characterized from different sources, including various producers such as Escherichia coli bacteriophage T4 and human tears. Despite minor differences (eg, human lysozyme has 130 amino acids), the hydrolysability of the acetylhexosamine polymer remains essentially the same. Thus, for the purposes of the present invention, the term lysozyme is intended to include acetylhexosamines and their cell wall or peptidoglycan disrupting enzymes that have the ability to hydrolyze related polymers.
[0034]
Lysozyme is known to attenuate or inhibit bacterial and fungal growth and is used in Europe to control the growth of the spoilage organism Clostridium tyrobutyricum in various cheeses. It is also proposed for use in various other food preservation applications and has been reported to inhibit (and in some cases kill) growth of Listeria monocytogenes (Hughey et al., Appl. Environ. Microbiol. 53: 2165-2170 (1987)). Lysozyme with an activity of about 20,000 Shugar units per mg obtained from egg albumin is commercially available from Rhodia under the trademark NovaGARDTM.
[0035]
In summary, the first component of this novel composition is preferably the combination of lantibiotics and degrading enzymes described above, in particular the combination of nisin and egg white lysozyme is more preferred.
[0036]
The second component of the novel composition is one or more hop acids or hop acid derivatives, or hop resins or hop resin derivatives, or a combination of some or all of these. It has now been found that the bitter taste component of hops used in the preparation of beer, in particular the beta acid, serves in particular as a food germicide, in combination with the above bacteriostatic and / or bactericidal compounds. The most potent groups of bitter acids contained in hops are alpha acids and beta acids, also called humulones and luprons, respectively. Although both contribute to the bitter taste of beer, alpha acids are much more bitter than beta acids and their use is undesirable in most foods. Hop extract producers industrially isolate α-acids and β-acids by various chromatography methods, and recently developed technology to separate two acid fractions using liquid carbonic acid under supercritical conditions It is done. The by-product of this operation is a product containing about 61% by weight of beta acids, with the remainder consisting essentially of hop resin. This by-product can be standardized with maltodextrin or another food grade carrier, spray dried and used as an antimicrobial food grade ingredient. Preferred beta-hop acid compositions are commercially available as natural flavor extracts containing 1% by weight beta-hop acid.
[0037]
The alpha acids contained in hops are generally known as humulones, cohumulones and adhumulones, while the beta acids contained in hops are generally known as lupron, coluplon and adoprone. In the context of the present invention in the context of the present invention hop acids or hop resin chemically modified derivatives which have proven antimicrobial properties such as hexahydrocoluprone and tetrahydroisohumulone as disclosed in US Pat. No. 5,455,038. Especially considered for use. Also, the use of hop acid or acid salt form of hop resin is specifically considered for use in the context of the present invention.
[0038]
In practice the hop acid or resin or derivative thereof is added to the food in an amount of about 0.1 to about 50 ppm (by weight of the solution used for treatment), more preferably about 0.40 to about 20 ppm, of the active ingredient Be done.
[0039]
Other additives that may be present in the compositions of the present invention include, but are not limited to: That is, another antimicrobial and / or chelating agent such as chitosan or its derivatives, natural or synthetic seasonings, essential oils and / or flavors, dyes and / or colorants, vitamins, minerals, nutrients, enzymes, guar gum and Binders such as xanthan gum. In a particularly preferred embodiment, guar gum is present in the composition of the invention to promote the bonding of the antimicrobial component and the food surface to be treated. The addition of these materials is not considered critical to the success of the present invention and should be considered within the skill of the art.
[0040]
The antimicrobial composition of the present invention can be used with any food susceptible to microbial degradation. These include, but are not limited to, fruits and vegetables including cereal products, products obtained from grains and cereals, daily foodstuffs, meat, poultry and seafood. In a particularly preferred embodiment, this composition is used with meat, chicken and / or seafood, and more specifically cooked with fats such as hot dogs, sausages, roast beef, turkey meat, corn beef and deli meats Used with pre-cooked meat.
[0041]
The novel composition is simply applied to the food surface before or after cooking to reduce the amount of bacteria on the food surface. In practice the application of the composition in question to the food surface may be either direct or indirect application. The use of the term "food surface" is defined as including some or all of the inner or outer surface of the food product being treated.
[0042]
The composition according to the invention is applied to the outside of blended foods such as hot dogs or bologna sausages or to the outside of solid foods such as pieces of roast beef to minimize loss of activity in the fat phase of the food. Used most easily by doing. Alternatively, the composition may be contained in an emulsion or raw ingredient of a food product such as sauce or salsa before or after cooking, or may be contained inside a solid product such as ham by injection or tumbling. . In yet another embodiment, the composition may be applied as a marinade, breading, rubbing of seasonings, glazes, mixtures of colorants, etc., the basic condition being that the antimicrobial composition is susceptible to bacterial degradation. It is to be used on the surface. In a preferred embodiment, the composition can be placed in contact with the food surface indirectly by applying the composition to a food packaging material or packaging container and then affixing the packaging material to the food surface. The use of methods of surface treatment, whether direct or indirect, benefits from minimizing the loss of fat in the fat phase of the food. The optimal amount for bacteriostatic or sterilizing used will depend on the composition of the particular food product being treated and the method used to apply the composition to the food surface, but by simple experimentation You can decide. In a preferred embodiment of the invention, the antimicrobial composition comprises at least one bacteriostatic or bactericidal compound selected from the group consisting of lantibiotics, pediocin, bacteriocins of the class of lacticin, and degrading enzymes 61. A second component comprising about 38.5 to 99.8 parts by weight of one component and at least one compound selected from the group consisting of hop acids, hop acid derivatives, hop resins, and hop resin derivatives; It contains 5 to 0.2 parts by weight. All parts by weight are based on the total weight of the first and second components of the composition.
[0043]
In an embodiment of the particularly preferred composition of the present invention wherein the first component comprises two compounds, the composition comprises, as first component, about 1.0 to 2.5 parts by weight of lantibiotics and about 37. 5 to 97.3 parts by weight, and about 61.5 to 0.2 parts by weight of at least one compound selected from the group consisting of hop acid, hop acid derivative, hop resin, and hop resin derivative as the second component It is preferable to include. All parts by weight are based on the total weight of the first and second components of the composition.
[0044]
The following non-limiting examples illustrate a wide range of antimicrobial compositions that constitute the present invention.
[0045]
Example 1
Formulations of nisin To demonstrate the inhibitory effect of NovasinTM, lysozyme, and β-hop acid alone or in combination, two gram-positive strains were used in trypticase soy broth, pH 6.0 as shown in Table 1. At 30 ° C. for 48 hours. The study demonstrates the surprising synergy of NovasinTM, lysozyme, and β-hop acid (BHA). Table 1 shows that the combination of NovasinTM, lysozyme and BHA at the same concentration has a significantly better inhibitory effect than each component alone or the combination of the two components. In the case of any single component treatment or two component treatment, the combination of the three components shows a log 5 reduction in both strains, while only a log 1 to 3 reduction is observed.
[0046]
[Table 1]

[0047]
Example 2
Hot dogs are inoculated with Listeria monocytogenes to demonstrate the in vivo efficacy of the compositions of the invention. The hot dog is immersed in a suspension containing either (1) NovasinTM, lysozyme, and BHA, or (2) NovasinTM and BHA, and Inoculated with monocytogenes. The hot dogs were then packed in sterile bags and kept at 10 ° C. for 13 days. The hot dogs are washed with sterile saline each day of sampling and the resin is spread on top of Listeria selective agar to obtain Listeria numbers. The hot dogs are fortified by transferring 1 ml of rinse solution to BHI broth and incubating for 24 hours, followed by spreading on top of Listeria selective agar. Treated products showing a count of zero on Listeria selective agar after enrichment are considered negative and recorded as a fraction for all treated samples.
[0048]
Table 2 shows that immersion of NovasinTM and BHA significantly reduces the number of first Listeria monocytogenes on the hot dog, while the combination of the three components is virtually detected by either direct plating or enhanced recovery methods It has been shown to reduce the number of Listeria monocytogenes to levels that are not. With this three-component combination, survival of proliferative Listeria monocytogenes, as evidenced by the negative result (3/3) after applying the sensitivity enhancement method used to recover small numbers of damaged cells Was not observed. The finding that all the samples treated with the novel three component combination were negative for Listeria monocytogenes even after enrichment is the most unexpected finding of the present application. Such a finding has not yet been reported at the safe and appropriate use levels described herein using any one of these antimicrobial components.
[0049]
【Table 2】

[0050]
Example 3
As shown in Table 3, two gram-positive spore bacteria of Bacillus genus are shown in trypticase soy broth, pH 6.0 to show the inhibitory effect of Novasin, lysozyme, and β-hop acid alone or in combination. It was tested at 30 ° C. for 48 hours. The study demonstrates the surprising synergistic effect of Novasin, lysozyme, and β-hop acid (BHA) on spore bacteria. Table 3 shows that the combination of Novasin, lysozyme and BHA at the same concentration has a significantly better inhibitory effect than either component alone or only two of the three components alone. The combination of the three-component system completely kills all inoculated spores, but when treated with a single component or two components considerable removal is observed but not complete. Thus, it is now concluded that the compositions of Novasin, lysozyme, and BHA exhibit synergistic bactericidal activity against spores.
[0051]
[Table 3]

[0052]
Example 4
The effectiveness of the composition of the present invention on cooked ham is demonstrated in Table 4. Inoculate Listeria monocytogenes on the surface of a heated ham and then topically by spraying the surface with either NovasinTM, lysozyme, and BHA (treated A), or lysozyme and BHA (treated B) Processing was done. The ham was then shrink wrapped, vacuum packaged in sterile bags and stored for 60 days at 40 ° F. (4 ° C.). On each sampling day, the ham is washed with sterile buffer and the resin is spread on top of Listeria selective agar to determine the number of viable Listeria bacteria. Transfer 1 ml of rinse solution to a broth of brain heart infusion (BHI) and incubate for 24 hours, followed by spreading on top of Listeria selective agar to intensify against a sample of the number of Listeria that can not be detected The law was implemented. Treated products with negative counts (less than 1 per ml) on the enriched Listeria selective agar are considered negative and recorded as a fraction for all treated samples.
[0053]
Table 4 shows that while the control group increased to 10,000-fold (log 4), the combination of the three components reduced the inoculated Listeria monocytogenes to levels not detected either by direct plating or by enhanced recovery. Is shown. The combination of the two-component "lysozyme plus BHA" showed an inhibitory effect, but not as bactericidal. In the three-component combination, the Listeria capable of proliferating, as evidenced by the negative results after applying the procedure used to strengthen, ie very few proliferating cells or damaged cells, No survival of monocytogenes was observed. Thus, the data demonstrate that topical application of the preferred three-component composition can completely remove Listeria monocytogenes in high-fat processed foodstuffs such as heat-treated ham.
[0054]
[Table 4]

[0055]
Example 5
Efficacy of the composition of the invention for protecting Frankfurter sausages from Listeria. The composition of Novasin and BHA was delivered to the surface of the frankfurter sausage during cooking by incorporating the composition into a cellulose packaging container containing the hot dog emulsion. After cooking, the packaging container was peeled off and then Listeria monocytogenes was inoculated on the surface of the finished Frankfurter sausage and vacuum-packed in a sterile bag. The packaged and inoculated Frankfurter sausages were stored at 40 ° F. (4 ° C.) for over 60 days and fresh packaging was opened at each sampling point. Frankfurter was washed together with its packaging container with sterile buffer at each sampling time and then spread on top of Listeria selective agar to determine the number of viable Listeria bacteria.
[0056]
Table 5 shows that the combination of the two components inhibited the growth of Listeria monocytogenes for a period of more than 61 days and resulted in a reduction of Listeria by more than 4 log (10,000-fold) compared to the control at the end of the incubation. Show. This data indicates that a packaging container delivering an application of the preferred two-component composition prevents the growth of Listeria monocytogenes on a Frankfurter sausage and delivers the composition, thereby improving the safety of the Frankfurter Sausage and sausages. Demonstrate that it may be a method. More importantly, this surface application method allows the activity of the ingredients to be fully realized even in the high fat environment of a typical hot dog emulsion. In contrast, when this composition is used as an ingredient in a pre-cooked hot dog emulsion, the antimicrobial activity is dramatically reduced (data not shown), which contains significant amounts of fat. Loss of activity of nisin in diets (Muriana, P. M. and Kanach, L., Use of Nisaplin to Inhibit Spoilage Bacteria in Buttermilk Ranch Dressing, J. Food Protection, Vol. 58, No. 10, This is consistent with observations by previous authors that demonstrated a loss of activity of Hop Acid (1995) and a loss of activity of Hop Acid (Johnson et al., 1996). Thus the disclosed method of surface application reveals a surprising means of maximizing the efficacy of the composition of the invention in foods containing high fat mass (more than 4 w / w%).
[0057]
[Table 5]

[0058]
While the invention has been described in detail and with reference to preferred embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the claims.