CN101155910A - Lactic acid bacteria for processing food - Google Patents

Abstract

The methods and compositions of the present invention involve the use of Carnobacterium maltaromaticum strains (previously known as C. piscicola) or associated fermentate or bacteriocin compositions to treat foods, such as fresh or processed meats, against bacterial contamination.

Description

Be used to handle the lactic-acid-bacterium of food

The present invention is the U.S. serial 10/870 that is filed on June 18th, 2004,032 and as being filed in the Canadian Patent 2 on June 20th, 2003, part continuation application 432,907 continuation application, that be filed in the international application no PCT/CA2004/000909 on June 18th, 2004; Part continuation application with the international application no PCT/CA03/00986 that is filed on June 27th, 2003.

I. invention field

The present invention relates to produce the new bacterial strain of the Carnobacteriummaltaromaticum of bacteriocin molecule with antimicrobial acivity.Bacterium of the present invention and can be used to handle food and as food antiseptics by described bacterium or other bacteriogenic bacteriocin.In concrete application of the present invention, the bacterial isolates of bacteriocin and the described bacteriocin of generation is used to control pathogenic bacteria, and the shelf-life that can not damage meat, described pathogenic bacteria includes but not limited to the monocyte hyperplasia listeria spp (Listeria monocytogenes) (" L.monocytogenes. ") in the meat product.

II. background of invention

Carnobacterium maltaromaticum is a kind in the various bacteria, and it is classified as lactic-acid-bacterium (LAB).Since several centuries, LAB has been used in the milk preparation industry of food and generation fermented foodstuff.Importantly they produce ability (Stiles and Holzapfel, 1997 of the compound that improves aromatising flavour and local flavor in this production; Carr etc., 2002).LAB is characterized from the ability that fermentable carbohydrates produces the multiple isomer of lactic acid by them.Atypical meat bacillus is unique, although because can produce in fact pure L (+)-lactic acid from glucose, they can not be grown on the acetate agar when pH 5.6, and because they can ferment glycerin and N.F,USP MANNITOL, character (Holzapfel and Gerber, 1983 of uniqueness in lactic-acid-bacterium; Shaw and Harding, 1984).

A kind of method that C.maltaromaticum can suppress possible pathogenic bacteria is by producing bacteriocin.Bacteriocin is the material (Klaenhammer, 1993) that can kill the low-molecular-weight antimicrobial protein of the bacterium that is closely related by the rrna synthetic.Bacteriocin is from beef, putrid ham, and from the soft cheese of French mould slaking, separate (Jack etc., 1996; Herbin etc., 1997).Because bacteriocin separates from food such as the meat that comprises LAB usually and milk preparation, the two has all been consumed LAB and bacteriocin several centuries.The bacteriocin that produces from C.maltaromaticum has shown for proteolytic ferment it is responsive., boil in 30 minutes processes and be stable after 15 minutes 62 ℃ of thermal treatments from the bacteriocin of C.maltaromaticum LV17 at 121 ℃ of autoclavings.Trypsinase, I, IV, VIII, XIV type proteolytic enzyme, alpha-chymotrypsin, β-Quimotrase and papoid make the bacteriocin inactivation, but not proteolytic ferment then not can (Ahn and Stiles, 1990b).Piscicolin 126, and a kind of bacteriocin that is produced by C.maltaromaticum JG 126 is by α-and β-Quimotrase, proteolytic enzyme (I, XIV, XXIII type and trypsinase) inactivation, but catalase, lipase or N,O-Diacetylmuramidase do not have this effect (Jack etc., 1996).Similarly, by the bacteriocin heat resistanceheat resistant (100 ℃, 20 minutes) of C.maltaromaticum LV61 generation, but by alpha-chymotrypsin, trypsinase, stomach en-, papoid and the passivation of Proteinase K institute.

Use catalase, α-Dian Fenmei, lipase, Phospholipase C, DNase I and N,O-Diacetylmuramidase are handled does not influence anti-microbial activity (Schillinger etc., 1993).This evidence shows that the absorption of bacteriocin can not impact useful enteric microorganism.Shown that trypsinase makes bacteriocin, nisin inactivation (Hara etc., 1962).

For having the new needs that continue with new food antiseptics existence useful property.In addition, for using effectively " natural " sanitas, there is more and more keen interest in those replacements " chemistry " food antiseptics commonly used that especially suppresses pathogenic micro-organism.In this respect, the bacterioprotein that is known as bacteriocin has been carried out considerable research, it has thermostability usually and has antimicrobial acivity.

In recent years, have at the corruption relevant and pathogenic microbes in exploitation and obtained remarkable progress aspect the microbe metabolite of antagonistic activity with food.There are many bacteriocins at present, but only have minority fully to be characterized and assess at foodstuff applications.In addition, the human consumer emphasizes the food that minimum level is handled at present, and it is natural and preservative-free.Thus, there is sizable resistance for the applied chemistry additive as food antiseptics.At present studying and to be used in the food by other biostats of microorganisms.Interested especially is that antimicrobial substance is such as the bacteriocin that is produced by lactic-acid-bacterium (" LAB ").

Bacteriocin is antibacterial peptide and the protein that is produced as their metabolic normal by products by LAB, and it may be very attractive natural antiseptic agent.Many LAB are fully understood, on industrial important bacterium, it comprises lactococcus (Lactococcus), streptococcus (Streptococcus), Pediococcus (Pediococcus), leuconos toc (Leuconostoc), lactobacillus (Lactobacillus) and meat Bacillaceae (Carnobacterium).They have been used in the period of thousands of by the production of the fermented foodstuff of safe consumption.Because they have reached the state as " safety " microorganism, they are natural antimicrobial materials, such as the particularly suitable source of bacteriocin, and are used for food.Bacteriocin can have wide in range or narrow anti-microbial activity spectrum, and their cell of generation that can not cause death.Bacterium protects them to avoid the lethal effect of their bacteriocins own by producing immunoprotein.

C.maltaromaticum is a Gram-positive, non-mobility, and the rod-shaped bacteria of non-formation spore redefines the meat Bacillaceae with it by lactobacillus recently.Pointed out that C.maltaromaticum is the wherein a kind of of big and various lactic acid producing bacteria group, its metabolizable glucose lactic acid producing and suppress other acid of some pathogenic bacterias growths.C.maltaromaticum at first finds on salmon, is still finding to the various food of fruits and vegetables from meat and fish since then, to surpass 1 * 10 ⁷The level of cfu/g is by present agricultural practice production and storage.Lactic-acid-bacterium is used for the fermentation of food (for example, sour milk, sausage, vegetables, bread, wine, cheese and milk) and has anticorrosionly reached several centuries.In France with the part of C.maltaromaticum as the initial bacterial cultures in the sausage fermentation.

Although used above-mentioned natural antiseptic agent, still there are needs to the lactic-acid-bacterium that can in certain food, control pathogenicity bo and spoilage organism and their bacteriocin.

III. summary of the invention

The present invention relates to the new bacterial strain of the Carnobacterium maltaromaticum (" C.maltaromaticum ") of bacteriocinogeny, it was known as the flesh of fish bacillus (Carnobacteriumpiscicola) (" C.piscicola ") that dwells in the past, had superior antimicrobial acivity.New bacterial strain CB1 of the present invention, CB2 and CB3 produce the various bacteria element, comprise meat bacillin (Carnobacteriocin) BM1 and piscicolin 126.These bacteriocins have the activity profile that wide in range anti-listeria belongs to bacterium, and produce strain growth in freezing temp and can not cause and other similar relevant spoilage microorganisms food spoilage relevant or in the typical shelf-life of food.

Embodiment of the present invention are included in instant (RTE) and fresh cold charge is in the meat product of processing, preferably with 1 * 10 ⁴The maximum inoculum density of colony-forming unit (cfu)/g is as Carnobacterium maltaromaticum bacterial strain CB1, CB2, CB3, LV17, UAL26, ATCC 35586 and the ATCC 43225 of sanitas.

Embodiment of the present invention comprise by with C.maltaromaticum, and it is through pasteurization or without the fermented product of pasteurization, or its applied in any combination is handled the method for fresh food in the food.In these embodiments of the present invention, bacterium and its produce predictable or in check storage life through pasteurization or without the fermented product of pasteurization.

In embodiment preferred of the present invention, with natural bacteria and its through pasteurization or without the fermented product of pasteurization, or by the combined treatment food of different bacteriogenic one or more bacteriocin fermented products.In the most preferred embodiment of the present invention, handle food through pasteurization or without the combination of the fermented product of pasteurization with selected natural bacteria and selected natural bacteria culture.

Embodiment of the present invention comprise uses composition of the present invention further to protect the food Gram-positive pathogenic bacteria that can not grow, and it includes but not limited to, the monocyte hyperplasia listeria spp.Composition of the present invention is for opposing monocyte hyperplasia listeria spp serotype 1/2a, and 1/2b, the bacterial strain of 3a and 4b are effective.

Method of the present invention comprises uses one or more natural bacteria cultures, and homologous is through pasteurization or without the fermented product of pasteurization, allogenic through pasteurization or without the fermented product of pasteurization, or its combination.Natural bacteria culture of the present invention is described in the above.The homology fermented product refers to typically the culture supernatants according to the single bacterial cultures of standard fabrication technique preparation.The allos fermented product refers to from typically according to the culture supernatants of the different bacterial cultures of standard fabrication technique preparation.Homology or allos fermented product can be i) through pasteurization or do not pass through pasteurization; Ii) freeze dried; Or iii) exsiccant otherwise.Two or more bacterial culturess can be blended or add separately.Two or more fermented products can be blended or add separately.Can be blended or add in proper order with the bacterial cultures of one or more fermented product combinations.

In another exemplary embodiment, the present invention includes the culture of bacterial isolates CB1.CB1 is preserved in American type culture collection (10801UniversityBoulevard, Manassas, Virginia USA 20118) on July 9th, 2003, and the registration number of receiving is PTA-5313.

In another exemplary embodiment, the present invention includes the culture of bacterial isolates CB2.CB2 is preserved in American type culture collection (10801UniversityBoulevard, Manassas, Virginia USA 20118) on July 9th, 2003, and receive be numbered PTA-5314.

In another exemplary embodiment, the present invention includes the culture of bacterial isolates CB3.CB3 is preserved in American type culture collection (10801UniversityBoulevard, Manassas, Virginia USA 20118) on July 9th, 2003, and receive be numbered PTA-5315.

In another exemplary embodiment, the present invention includes and use CB1, CB2, and/or CB3, or it makes up and handles food, handles the spoilage organism on the food, handles the pathogenic bacteria on the food, and/or determines the storage life of predictable food or food.Bacterial strain CB1, CB2, and/or CB3 can be used alone or in combination; Can with or do not use with their bacteriocins separately; Can with or do not use with the fermented product that comprises their bacteriocins separately; Can use with bacteria combination including, but not limited to one or more bacteriocinogeny of lactic-acid-bacterium; And/or can with use together by different bacteriogenic one or more bacteriocins; And/or can with or do not use with comprising the fermented product of generation from one or more bacteriocins of different bacteriocins.

In another exemplary embodiment, the present invention includes the method for preserving food or beverage, described method comprises the bacterial cultures of the present invention with significant quantity, makes up separately or with fermented product to add food or beverage.The inventor has been found that every gram or every cm ²In 10 ²Or the amount of colony-forming unit still less (" cfu ") typically is not enough to and the external microbial population competition that exists.The inventor has been found that bigger than 10 times of the background micro-floras of beginning, typically every gram or every cm ²About 10 ³Cfu or the bigger growth that is enough to overcome existing alien bacteria (for example, background micro-flora) population.The amount that those skilled in the art will recognize that the alien bacteria in food is variable.According to the present invention, the amount of described composition should be about 10 times or bigger of amount of external spoilage organism.

In embodiment preferred of the present invention, described method comprises handles fresh meat.In the most preferred embodiment of the present invention, described method comprises handles or preserves fresh sausage or vacuum-packed wiener.

The invention still further relates to use bacteria composition and/or usually handle listeria spp and belong to (Listeria spp.) to suppress the growth that listeria spp belongs in the meat by the bacterium that described composition produces.

The invention still further relates to and comprise by bacterial strain CB1 CB2, and/or the fermented product of one or more bacteriocins of CB3 generation.In embodiment preferred of the present invention, fermented product comprises piscicolin 126, but meat bacillin BM1 and can identify the protein compound that characterizes not yet with anti-microbial activity.

In comprising embodiment of the present invention of bacteriocin, bacteriocin can separate from natural origin, can be produced by one or more bacterial strains of the present invention, can be produced by another bacterial isolates, maybe can pass through genetic modification, for example use recombinant expression vector to produce.

An advantage of the invention is from the activity of undiscovered resisting-listeria spp.Wide in range so anti--listeria spp spectrum is outstanding.Another advantage of the present invention is sterilization and antibacterial potential.To be these bacteriums grow being low to moderate under 0 ℃ the temperature another advantage of the present invention, and this illustrates that they grows and effective under the anticorrosion necessary freezing temp for meat.Another advantage of the present invention is that these bacterial strains itself do not cause meat significantly corrupt.

Accompanying drawing shows exemplary embodiment of the present invention, by them, and these and other objects, new feature and advantage will become apparent easily.

IV. accompanying drawing summary

Fig. 1 is the active graphic representation of anti-listeria spp of composition of the present invention, illustrates inoculating 10 with every gram pork sausage sample ³With 10 ⁴Under the situation of the C.maltaromaticum CB1 of cfu, the minimizing of bacterial number and to the inhibition of the mixture of four strain bacterial strains of monocyte hyperplasia listeria spp, described sample surpasses 15 days refrigerated storage time limits of the prediction of sausage 5 ℃ of storages.

Fig. 2 is first a graphic representation of three revision tests, illustrates on the surface of vacuum-packed wiener, at every em ²Have 10 ⁴Under the C.maltaromaticum CB1 of cfu or the situation of CB3, the minimizing of bacterial number and to every cm ²10 ²To 10 ³The inhibition of the mixture of four strain bacterial strains of the monocyte hyperplasia listeria spp of cfu inoculation, described wiener surpass the refrigerated storage time limit in 12 weeks of product 5 ℃ of storages.

Fig. 3 is second a graphic representation of three revision tests, illustrates on the surface of vacuum-packed wiener, at every cm ²Have 10 ⁴Under the C.maltaromaticum CB1 of cfu or the situation of CB3, the minimizing of bacterial number and to every cm ²10 ²To 10 ³The inhibition of the mixture of four strain bacterial strains of the monocyte hyperplasia listeria spp of cfu inoculation, described wiener surpass the refrigerated storage time limit in 12 weeks of product 5 ℃ of storages.

Fig. 4 is the 3rd a graphic representation of three revision tests, illustrates on the surface of vacuum-packed wiener, at every cm ²Have 10 ⁴Under the C.maltaromaticum CB1 of cfu or the situation of CB3, the minimizing of bacterial number and to every cm ²10 ²To 10 ³The inhibition of the mixture of four strain bacterial strains of the monocyte hyperplasia listeria spp of cfu inoculation, described wiener surpass the refrigerated storage time limit in 12 weeks of product 5 ℃ of storages.

V. specific descriptions of the present invention

Composition of the present invention comprises the bacterial strain of Carnobacterium maltaromaticum, and each generation is at least a, three kinds of bacteriocins typically, C.maltaromaticum CB1 produces bacteriocin piscicolin 126, the bacteriocin of meat bacillin BM1 and another kind of unidentified demonstration antibacterial activity. C.maltaromaticum CB2 produces piscicolin126, meat bacillin BM1, and can produce one or more other unidentified bacteriocins. C.maltaromaticum CB3 produces piscicolin 126, meat bacillin BM1 and can produce one or more other unidentified bacteriocins.

The compositions and methods of the invention comprise one or more natural bacteria cultures of use, process fermentate pasteurization or that do not pass through pasteurization of homology, process fermentate pasteurization or that do not pass through pasteurization or its combination of allos. Natural bacteria culture of the present invention has been described above. The fermentate of homology refers to the culture supernatants according to the single bacterial cultures of standard fabrication technique preparation. The allos fermentate refers to the culture supernatants that is derived from different bacterial cultures according to the standard fabrication technique preparation. Fermentate homology or allos can be i) through pasteurization or do not pass through pasteurization; Ii) freeze-drying; Or iii) otherwise dry. Two or more bacterial cultures can mix or add respectively. Two or more fermentates can mix or add respectively. The bacterial cultures that combines one or more fermentates can mix, or continuous adding.

An importance of the present invention comprises the bacterial fermentation thing at the anticorrosion of fresh meat and the purposes in processing. According to instruction of the present invention, by bacterial strain CB1, CB2, thereby or the bacteriocin that produces of CB3 as if synergy protection and the effectiveness bigger than the single bacteriocin of independent use is provided.

The meat (under freezing conditions storage) that green meat product used herein refers to raw meat or do not boil, it can comprise or not comprise other flavouring mixture, and comprises meat complete or that ground. Finished meat product refers to such meat, it is i) modulate and boiled; Ii) through pickling; Or iii) but without pickling to produce the commercially available prod. Hope is used " fresh " and " finished " with common meaning well known by persons skilled in the art. Typical meat includes, but not limited to wienerwurst, sausage, fish, and poultry.

The compositions and methods of the invention can also be used to process other food, include, but not limited to the vegetables of controlled atmosphere (modified atmosphere) packing, the product of vacuum-packed pasta and fresh packing.

The storage period of expectation used herein is accused the corrupt ability that continues one independent period of system, and corruption becomes obvious till that time. For example, bacterium can be applied to food to reach about 10 week or longer storage periods, corruption can detect till that time. In storage period in 10-week, composition of the present invention is controlled corruption by in the following approach one or more: the bacterium that i) has the known corrupt time by application; Ii) by using the bacterium that produces one or more protein or bacteriocin, described protein or bacteriocin kill or control spoilage organisms; Or iii) by their combination.

The security of raising used herein refers to the inhibition of latent pathogen growth and/or the minimizing of number, and scope is from sterilization to antibacterial.

Hue preserving used herein refers to that food keeps its required painted time lengthening. This concept is well known to a person skilled in the art.

Embodiment

Embodiment 1.

Collins etc. (1987) report L.piscicola, and L.divergens and L.carnis are synthetic mainly as oleic C18:1 isomer (Δ 9,10), show different unsaturated fatty acids acid enzyme approach.Genetic homogeny classification and chemistry and physical property are also with L.piscicola, and L.carnis places identical dna homology group with L.divergens.In addition, biochemical and chemical data shows that L.piscicola and L.carnis should be simplified to same genus L.piscicola by (and by).L.piscicola, and L.ivergens was subsequently reclassified to new a genus, and (L.gen.N.carnis belongs to meat to Carnobacterium; Gr.dim.n.bakterion, little bar-shaped; M.L.neut.N.Carnobacterium, the meat rod), see (1987) such as Collins.When 16S rRNA sequential analysis shows that the meat Bacillaceae constitutes phylogenetic evolution branch 4 unique in the lactic-acid-bacterium, this point is further confirmed, and comprise lakebed meat bacillus (C.funditum), class lakebed meat bacillus (C.alterfunditum), chicken bacillus (C.gallinarum) and movable meat bacillus (C.mobile) (table 1), Lactobacillusmaltaromaticus is further defined as objective synonym (Miller etc., 1974 of the flesh of fish bacillus that dwells; Collins etc., 1991; Lai and Manchester, 2000; Lai etc., 2004).In addition, although the primary classification of meat Bacillaceae becomes lactobacillus, on phyletic evolution, this symbolic animal of the birth year is for enterococcus spp (Enterococcus) and roaming Coccus (Vagococcus) sibship nearlyer (Hiu etc., 1984)

Subsequently, Lactobacillus maltaromicus (Lactobacillus maltaromicus) strain DSM 20342T, the phenotype of DSM 20344 and JCM1154 and heredity characterize has determined that these bacterial strains also belong to the meat Bacillaceae.Further should be considered to identical with flesh of fish bacillus this two genus of relatively reaching a conclusion of dwelling.As a result of, dwell the flesh of fish bacillus reclassified into Carnobacterium maltaromaticum comb.nov. (Collins etc., 1991; Mora etc., 2003).So, will use the popular name of Carnobacterium maltaromaticum about genus of the present invention.

Table 1. meat Bacillaceae, relation and habitat (Collins etc., 1987 of they and previously described bacterium; Collins etc., 1991; Mora etc., 2003).

Current name	Name in the past	The habitat
			C.divergens C.gallinarum C.mobile C.maltaromaticum * C.funditum C.alterfunditum	L.divergens L.piscicola L.carnis L.maltaromicus	Meat, poultry, slaking mould cheese surface poultry poultry meat, poultry or utmost point lake, Hunan, the salmon South Pole

^*Propose to be C.maltaromicus (Collins etc., 1991) and C.maltaromaticum (Mora etc., 20C.=meat bacillus; The L.=Bacterium lacticum.

Embodiment 2.

Naturally occurring C.maltaromaticum belongs to LAB group in history, its heterofermentation metabolizable glucose is so that produced the lactic acid of equimolar amount by sugar, carbonic acid gas and ethanol or acetate, it before had been included in (Stanier etc., 1957 in the lactobacillus (Lactobacillus); Hiu etc., 1984).Although it is homo-fermentative [generation acetate, formate and CO for the L-lactic acid salt that some researchs have shown the meat Bacillaceae ₂End product (Hiu etc., 1984 as some secondary decarboxylation/catabolic reactions of pyruvate salt; De Bruyn etc., 1988)], the nearest description of C.maltaromaticum and sign are pointed out L (+)-lactic acid, ethanol and acetate are that heterofermentation produces (Mora etc., 2003).So for present embodiment, C.maltaromaticum has been characterized as being has the heterofermentation feature.Suffer through being everlasting to find C.maltaromaticum in the fish of coercing of certain form, described coerce as occur in when laying eggs or be accompanied by transportation and take place coerce (Hiu etc., 1984; Baya etc., 1991).Ringo etc. (2000) find that also C.maltaromaticum is associated with the digestive tube of atlantic salmon (Salmo salar L.).The meat bacillus separates from the vacuum-packed fish of refrigerated and crude beef and lamb meat, and it belongs to dominant LAB (Ahn and Stiles, 1990a on the meat in these habitats; Baya etc., 1991; Barakat etc., 2000; Carr etc., 2002; Paludan-Muller etc., 1998; Sakala etc., 2002; Yamazaki etc., 2003).Not enrichment or select any concrete bacterium classification or genus of the method for in these researchs, using.

Be compared in (table 2) and provide blazoning biochemistry between meat bacillus (C.divergens) and the C.maltaromaticum and physiology.C.maltaromaticum bacterial strain B270T have following properties (Hiu etc., 1984 have been described; Collins etc., 1987):

. Gram-positive, non-sports type, non-sporulation type is shaft-like, occurs separately or occurs with short chain;

. well-grown on many standard laboratory substratum, described substratum comprise TSA (tryptone beans peptone agarose substratum) and brain heart infusion agar sugar culture-medium and at deMan, in Rogosa and Sharpe (MRS) meat soup and the thioglycollate medium;

. when when 25 ℃ are grown in TSA and go up 24h, the clone is tiny, projection, white, circular and non-pigmented;

. the temperature range of growth is 6 ℃-40 ℃; Optimum temps is about 30 ℃;

. best pH scope is from 6.0 to 7.0;

. amphimicrobian.Produce D by homofermentation, the L-lactic acid salt, but this genus can present the heterofermentation characteristic under certain conditions; Lactic acid-producing is increased under the anaerobic growth condition;

. growth needs folic acid, riboflavin, pantothenate and nicotinic acid; Vitamin B12, vitamin H, VITMAIN B1 and pyridoxal are also nonessential;

. do not produce catalase and oxydase;

. nitrate is not reduced into nitrite;

. gas produces indefinite (depending on substrate) and often is negative; Produce gas by the glucose in arginine-MRS meat soup;

. by glycerine, ribose, semi-lactosi, gluconate, glucose, fructose, seminose, N.F,USP MANNITOL, the N-acetyl glucosamine, amygdaloside, arbutin, saligenin, cellobiose, sucrose and trehalose produce acid; By pectinose, wood sugar, sorbose, rhamnosyl, melampyrum, inositol, methyl D-mannoside, inulin or melizitose do not produce acid;

. arginine and esculin are hydrolyzed;

. at TSI Triple Sugar Iron agarose) do not detect H2S in the inclined-plane;

. tolerance 0.4 and 0.6% teepol (Teepol);

. whole cell peptidoglycan comprises diaminopimelic acid;

.DNA G+C content is 33.7-36.4mol%;

. main cell fatty acid is the saturated and single unsaturated type of straight chain, tetradecanoic acid wherein, and palmitinic acid, Zoomeric acid and Δ 9,10-oleic acid is preponderated;

. typical strain is B270T (ATCC 35586), separates from the adult mountain trout of being coerced in 1970, and it raises the Bandon trout hatching institute in Oregon Coos county.

The biochemistry of table 2. meat Bacillaceae and physiology are relatively ¹

Feature	Blazon the meat bacillus	C.maltaromaticum 2
			Produce sour 3 Amidon amygdaloside semi-lactosi β-gentiobiose gluconate inulin N.F,USP MANNITOL melibiose melizitose Alpha-Methyl-D-glucoside Alpha-Methyl-D-mannoside D-tagatose D-turanose D-wood sugar Voges-Proskauer by following material 5Mobility Δ 9,10-methylene radical octadecanoic acid 6	- + - + +(-) 4---ten (-)-----+-+	- + + + + + + + +(-) + + +(-) - - + - -

¹Rewrite from (Collins etc., 1987). ²Called after occupied fish Bacterium lacticum (Lactobacilluspiscicola) and dwelt flesh of fish bacillus in the past; ³That carried out at seven days reads. ⁴+ (-)=accidental bacterial strain feminine gender; ⁵The glucose metabolism test of in API 10E system, carrying out; Two bacterial strains all produce arginine dihydrolase and beta-galactosidase enzymes; Two bacterial strains are for lysine decarboxylase, tryptophane deamidase, urase, ornithine decarboxylase, indoles and H ₂S is negative; ⁶Total cell fatty acid greater than 15%.

Alkaline pH (up to pH 9.5) promotes the growth of meat bacillus bacterium colony, suppresses other lactobacillus simultaneously.Can realize C.maltaromaticum and other bacterium are made a distinction by changing growth substrate.C.maltaromaticum and enterococcal differentiation are included in microscopically and distinguish bar-shapedly and spherical, and are grown in and comprise 2% inulin but not on Cresol Red Thallous Acetate Sucrose (CTAS) substratum of sucrose.The unfermentable inulin of faecalis, and when C.maltaromaticum fermentation inulin, it is light yellow to peach bacterium colony that formation has the metallic copper glossy, the yellow of substratum changes and precipitation is removed.When in the CTAS agarose during with the inosine substituting saccharose, C.maltaromaticum forms bacterium colony projection or the β type.Faecalis also produces the yellow and the sedimentary removing of substratum, but does not have metalluster (Carr etc., 2002).The different strains of C.maltaromaticum shows that generation suppresses lactobacillus, bacteriocin (the rrna synthetic that listeria spp belongs to and other meat Bacillaceae is grown, lower molecular weight, the germ resistance proteinaceous substances, it can suppress to be close to the growth of bacterium or kill them) (McMullen and Stiles, 1996; Duffes etc., 1999c; Schillinger etc., 1993).

Embodiment 3.

The bacterial strain of enumerating (for example, Carnobacteriummaltaromaticum bacterial strain CB1, CB2, CB3, LV17, UAL26, ATCC 35586 and ATCC43225) is tested it for 27 kinds of antibiotic resistance (tables 3 in this GRAS document; Griffiths Labs, 2004).Generally, the C.maltaromaticum bacterial strain of test is to amoxycillin+clavulanic acid, paraxin, Ciprofloxacin, erythromycin, gentamicin, imipenum, Ethylsisomicin, Rifampin, tsiklomitsin and tobramycin sensitivity.With reference to antibiotics resistance pattern (table 3), the meat bacillus strain for the Gram-positive fungal component in common those the related main microbiotic of transferable genetic elements; Particularly, erythromycin, paraxin and sensitive tetracycline.Borriello etc. (2003) propose that selected bacterial strain should be to two or more main microbiotic susceptibles when as probiotic agent.By (1991) such as Baya, Duffes etc., (1999b) or the used microbiotic of Euzeby (2004) comparison shows that C.maltaromaticum bacterial strain (CB1, CB2, CB3, LV17, UAL26, ATCC 35586 and ATCC43225) to various antibiotic susceptibility with in that to separate the antibiotics resistance of finding in the C.maltaromaticum bacterial strain in natural fish source fully consistent, such as in the table 3 notes.The antibiotics resistance pattern of the C.maltaromaticum bacterial strain of in these GRAS archives, enumerating with add in the food or in food the antibiotics resistance pattern of the Bacterium lacticum Pseudomonas of natural discovery fully consistent.This shows these C.maltaromaticum bacterial strains are added in the food and will can not add any new or significant antibiotics resistance determiner that described determiner is under normal circumstances found in symbiosis or probiotic agent lactic-acid-bacterium.

Microbiotic	?CB1	CB2	CB3	LV17	UAL26	ATCC 35586	ATCC 43225	Baya etc. 1991	Euzby， 2004	1999b such as Duffes
											Amikacin amoxycillin+clavulanic acid aztreonam cefepime Cefotazim ceftazime Cefuracetime paraxin Ciprofloxacin clindamycin Polymyxin E erythromycin erythromycin imipenum kantlex Minocycline HCl Moxolactam Nalidixic Acid netilmicin piperacillin Rifampin Streptomycin sulphate tsiklomitsin ticarcillin tobramycin vancomycin	R S R R R R R I I R R I I S R P P R I R I R S R S R	R S R R R R R I S R R I R S R P P R S R I R I R S S	R S R R R R R S S R R S S S I P P R S R S R S R S R	I S R R R R R S S R R I I S R P P R S R S R I R S S	R R R R R R R I I R R I R S R P P R I R I R I R S R	R R R R R R R S S R R I S S I P P R S R S R S R S R	R S R R R R R S S R R I I S R P P R S R S R S R S R	R S R R R S	S R R S	R S R R R R R S S R R S R S R R R S S R S S R S

Existence and the use of embodiment 4. lactic-acid-bacteriums in food

Utilize the method for direct inoculation to identify from meat and meat product, milk and milk preparation, vegetables, the LAB strain isolated of the bacteriocinogeny of fruit and sea-food, to (suckling and milk preparation for 32 parts from 72 parts of foodstuff samples, 40 portions of meat) 663,533 bacterium colonies have altogether been checked bacteriocin production (Coventry etc., 1997).Many being predicated of these foodstuff samples surpassed acceptable preservation period.15% meat and meat product produce the meat Bacillaceae of bacteriocinogeny altogether.In investigated 72 parts of foodstuff samples, 44% produces the bacterium of bacteriocinogeny.From the bacterium colony of 663,533 tests altogether, find that 80,992 bacterium colonies (12.2%) are the meat Bacillaceae, wherein 0.15% bacteriocinogeny.The culture supernatants fluidic antibacterial activity of filter degerming of selected bacterial strain of producing bacterium from bacteriocin is not by catalase, lipase or N,O-Diacetylmuramidase influence, but but by the deactivation wholly or in part of at least a proteolytic ferment, the prompting antibacterial activity is associated with proteinaceous substances.This research also shows the human bacterium that always contact the generation bacteriocin that meat Bacillaceae and other food grows.

Amezquita and Brashears (2002) have reported from commercially available instant (RTE) meat product and have isolated 49 kinds of LAB bacterial strains.In the agarose spot test, these are screened it in 5 ℃ of abilities that suppress the growth of monocyte hyperplasia listeria spp.Pediococcus acidilactici, Lactobacillus casei and L.paracasei are accredited as three kinds and have the active kind of the highest inhibition.When adding Pediococcus acidilactici to the RTE meat product, during the selected bacterial strain of Lactobacillus casei and L.paracasei three kinds, in all evaluated RTE meat products (five parts be purchased boil ham sample and five parts of frankfurter samples that are purchased), pair remarkable inhibition (P＜0.05) of monocyte hyperplasia listeria spp growth is arranged all.This studies show that the selected bacterial strain of LAB can separate from RTE meat product and these bacterial strains in 5 ℃ of growths that effectively suppressed frankfurter and boiled monocyte hyperplasia listeria spp in the ham in the storage at 28 days.At duration of storage, the number of LAB only increases about 1 logarithm period and the corrupt sign of no obvious visible is (for example on product surface, relate to outward appearance, as colour-change, what undesirable fragrance and hardness or quality changed influences the disadvantageous effect of sense organ for some).By the research that implement (2002) such as Sakala, investigated in the refrigerator storage, having a liking on the vacuum-packed beef cold (can under freezing temp, grow, but on the best bacterium of growth more than 20 ℃) spoilage microorganisms fauna.This studies in 7 ℃ heated culture temperature and utilizes low optionally glucose-blood-liver agarose and tryptone beans peptone agarose culture medium inoculated method (allowing bacterial growth the most widely) down.

Various on the vacuum-packed beef of identifying in the time in six weeks and having quantized to preserve under freezing temp are had a liking for cold genus, and bacterium belongs to or the variation of bacterial number to measure.Utilize type and the quantity of five parts of fresh beef cutting samples (butchering about 48 hours of back acquisition and vacuum packaging) to be determined at the various bacteriums of finding in the vacuum-packed beef.1493 bacterial isolateses are accredited as altogether: heat kill microbacterium (Brochothrix thermosphacta) (64), Carnobacterium maltaromaticum (27), blazon meat bacillus (79), Lactobacillus algidus (637), lactobacillus (4), fish galactococcus (Lactococcus piscium) (270), cold living leukonid (Leuconostoc gelidum) (375), acinetobacter calcoaceticus (Acinetobacter) (3), Aeromonas (Aeromonas) (1), genus bacillus (Bacillus) (10), rod bacillus (Corynebacterium) (3), enterobacteria (Enterobacteriaceae) (1), pseudomonas (Pseudomonas) (13) and Psychrobacter (Psychrobacter) (6).Cold living leukonid, fish galactococcus and L.algidus the storage the junior three cycle between from about 5 * 10 ³Cfu/g rises to about 1 * 10 ⁸Cfu/g, and in the remaining time of six weeks research, keep stable.C.maltaromaticum is detected inconsistently, but when it exists, is increased to about 5 * 10 between the junior three cycle of storage ⁷Cfu/g, and remaining on this level in last three weeks in research.Vacuum or controlled atmosphere (CO ₂) packing (CO ₂-MAP) influence separates the bacterium genus (Labadie, 1999) from meat.Under the low temperature and the oxygen of limiting the quantity of, LAB comprises about 1 * 10 ⁷Cfu/cm ²The predominant bacteria population (Gill and Newton, 1978) of CO2-MAP packing meat.Still research is not directly compared at CO ₂The concrete quantity of not generic of lactobacillus, leuconos toc (Leuconostoc) and Carnobaterium on the fresh packing meat under-the MAP condition.Nilsson etc. (1999) isolate 2 * 10 after 32 days respectively from the cold smoking salmon when buying and at incubation ⁴With 5 * 10 ⁷Cfu/g LAB.

Embodiment 5.Carnobacterium maltaromaticum is at meat, the natural existence on fish and the cheese product

As summing up in the table 4,, isolate meat Bacillaceae (Ahn and Stiles, 1990a in fish and the French soft junket from vacuum-packed meat; Buchanan and Klawitter, 1992b; Stoffels etc., 1992; Pilet etc., 1995; Milliere and Lefebvre, 1994a; Milliere etc., 1994b).The research of Lewus etc. (1991) has been identified the C.maltaromaticum bacterial strain of two kinds of bacteriocinogeny from the meat from the different piece of retail meat product.Other C.maltaromaticum strains separation is from fish, meat and cheese (Milliere etc., 1994b; Nissen etc., 1994; Pilet etc., 1995; Schillinger etc., 1993; Shaw and Harding, 1984).Leisner etc. (1994) find initial separation from vacuum-packed halibut, and 18 strains in 80 bacterial isolateses of salmon or mackerel are lactic-acid-bacteriums.Wherein, 28% be accredited as C.maltaromaticum.Sakala etc. (2002) have carried out a kind of cold spoilage microorganisms fauna of investigating on the vacuum-packed beef of refrigeration of having a liking for of studying, and determine separating in 1493 bacterial strains altogether of five parts of fresh beef cutting samples (every part all from different butcher's), 27 strains are accredited as C.maltaromaticum.The 0th, 1,3,5 and 6 weeks detected described bacterium for two parts of C.maltaromaticum positive what preserve, average number is respectively 2 * 10 ³, 2 * 10 ⁴, 2.5 * 10 ⁶, 1 * 10 ⁷With 2.5 * 10 ⁷Cfu/g, and during last three weeks of six storage periods in week, maintain about 5 * 10 ⁷The level of/cfu/g.

C.maltaromaticum was put down in writing by Montel (1999) in the growth in the meat of fermentation, and " when yeast phase finished, lactic-acid-bacterium generally was the predominant bacteria fauna in his record.Following Pseudomonas: lactobacillus curvatus (Lactobacillus curvatus), lactobacillus sake pure mellow wine subspecies (L.sakei), plant lactobacillus (L.plantarum), lactobacillus viridescens (L.viridescens), blazon Bacterium lacticum, C.maltaromaticum and leuconos toc are naturally occurring, but the sheet coccus is only found when inoculating as starting culture.Their counting generally surpasses 10 ⁶Cfu/g and remaining on this level between the whole maturation period.Carnobacterium is present between yeast phase, but disappears thereafter.″

Table 4. is separation of C .maltaromaticum. from food

The food classification	Food	The C.maltaromaticum bacterial strain *	Reference
				Fish	The striped perch of cultivating, tabby squama and bullhead catfish be cold-smoke cured fresh-water fishes salmon is cold-the vacuum-packed halibut of smoke cured salmon, and salmon or mackerel be cold-smoke cured salmon fish	A9a，A9b，A9c， A9J，A10a，A10b， A10f，A10J，S1， S2，S3，S4 V1	(Baya etc., 1991) (Gonzalez-Rodriguez etc., 2002) (Hiu etc., 1984) (Leroi etc., 1998) (Leroi etc., 1994) (Paludan-Muller etc., 1998) (Pilet etc., 1995)

Beef lamb meat chicken milk preparation

The chicken leg poultry softer cheese of the lamb meat modified atmosphere packaging of the vacuum-packed beef meat of the beef meat modified atmosphere packaging of the grinding that vacuum-packed beef is given birth to

GN，DX LV?61 CP5

(Ahn and Stiles, 1990a) (Buchanan and Klawitter, 1992A) (Lewus etc., 1991) (Sakala etc., 2002) (Shaw and Harding, 1984) (Nissen etc., 1994) (Bakarat etc., 2000) (Collins etc., 1987) (Milliere etc., 1994B)

*=and given bacterial strain, if known.

Cold smoking salmon (CSS) is as easy as rolling off a log septic food and the pollution that is subject to the monocyte hyperplasia listeria spp especially.The CSS corruption mainly be since microorganism active during chilled storage cause (Duffes, 1999a).For CSS, according to estimates after packing at once, bacterial count is from 1 * 10 ³To 1 * 10 ⁴Cfu/g, gram negative bacterium is preponderated (64%), such as corrupt Shiva Salmonella (Shewanella putrefaciens) and Aeromonas (Aeromonas spp).There is LAB (32%) in discovery, and majority is meat Bacillaceae (Donald and Gibson, 1992; Huss etc., 1995).Under 8 ℃, the level of bacterial flora is increased to 1 * 10 in three weeks ⁷-1 * 10 ⁸Cfu/g, the Related Bacteria population changes to some extent, thereby makes LAB preponderate (60%), is mainly meat Bacillaceae (47%) and lactobacillus (Lactobacillus spp.) (13%).Paludan-Muller etc. (1998) have reported a series of researchs, have assessed C.maltaromaticum in the effect of preserving in the cold smoking salmon corruption of 5 ℃ of following vacuum and modified atmosphere packaging.Usually on corrupt CSS, find the mixture of LAB and gram negative bacterium.

The initial number of bacterium is low, and total psychrophilic bacteria counting is less than 5 * 10 ³Cfu/g, particularly, the LAB counting is 10-1 * 10 ²Cfu/g.(to pass through the sensory evaluation) in addition, according to surveying and determination vacuum-packed, and the cold smoking salmon preserves aspire to around being up under 5 ℃.Micro-flora in the time of all around is by LAB (1 * 10 ⁶-1 * 10 ⁷Cfu/g) with the gram-negative micro-organism fauna (1 * 10 of different levels ⁵-1 * 10 ⁷Cfu/g) form.

Modified atmosphere packaging reduces growth and the specificity of gram negative bacterium and selects LAB, although the duration of storage that was grown in for five weeks of LAB is lower than 3 * 10 ⁵Cfu/g (Paludan-Muller etc., 1998).The LAB micro-flora is dominant to be C.maltaromaticum, accounts for 87% in 255 LAB strain isolateds identifying.Inoculate about 1 * 10 by every gram in the CSS that preserves under 5 ℃ ⁶Cfu C.maltaromaticum further studies the corrupt potentiality (Paludan-Muller etc., 1998) of C.maltaromaticum.In the vacuum-packed salmon that has inoculated the C.maltaromaticum bacterial strain, only the LAB counting reaches 1 * 10 after the storage in a week ⁷Cfu/g, and this level is higher than 1 * 10 in the storage period of remainder ⁸Cfu/g.But, after storage all around, salmon is not repelled by the sensory experience group, and vacuum-packed contrast then is ostracised after four to five weeks.In the salmon of the modified atmosphere packaging of inoculating, two week back LAB countings reach terminal level 1 * 10 ⁶-1 * 10 ⁷Cfu/g, but salmon is not repelled by sense organ after four to five weeks of storage.Even according to inferring at high number (1 * 10 ⁷-1 * 10 ⁸Cfu/g) under, the grow corrupt process of the cold smoking salmon that several weeks also can not quicken to pack of C.maltaromaticum.

The cold-peace of having a liking for for the dried soft junket of the French surface mould slaking of being made by raw milk is had a liking for warm microbiotic bacteriological study and is found, C.maltaromaticum be when slaking finishes on five kinds of Brie cheese samples predominant bacteria (Milliere and Lefebvre, 1994a).The C.maltaromaticum bacterium also separates from Coulommiers, Camember, Pon-I`Eveue and Munster cheese.In various cheese samples, separation is 5 * 10 from the number range of the meat bacillus bacterium colony of these cheese ⁵-8 * 10 ⁸Cfu/g.Milliere etc. (1994b) continue to identify that separation is from the C.maltaromaticum of five kinds of Brie cheese samples bacterial strain.The pH value of cheese is between 6.8 and 7.6 and does not record peculiar smell (off-odors) or sense organ defective.The meat Bacillaceae is preponderated in described cheese samples, 1 * 10 ⁸With 1 * 10 ⁹Between the cfu/g.The result of DNA-DNA hybridization shows that 33 kinds in 36 kinds of strain isolateds is the C.maltaromaticum Pseudomonas, and remaining three kinds (all pickings from identical sample) are for blazoning the meat bacillus.

In a word, the meat Bacillaceae is at vacuum-packed meat, poultry, and microbiotic common component on fish and the cheese product, in some cases, they can representative products such as smoked fish, the advantage on chicken, beef and the cheese is formed population, reaches 1 * 10 ⁸Cfu/g or higher level, and can not cause can detected corruption.

The production of embodiment 6.Carnobacterium maltaromaticum culture

The C.maltaromaticum bacterial strain is kept with lyophilized form under 4 ℃, or kept as the frozen cultures in 20% (v/v) glycerine down at-80 ℃.Carry out API  strip analysis (a kind of test kit of identifying bacterium to the genus level) to guarantee viability and to confirm bacterial strain purity by no bacteriology pollution and/or by randomly amplified polymorphic DNA (RAPD) and microbiological analysis.For seven freeze dried bottles of each bacterial strain preparation (main kind).By single bottle main kind, 15 freeze-drying bottles of preparation under vacuum, and preserve in 4 ℃ (secondary kinds).By every bottle of secondary kind, prepare the production needs that enough cryovials are used for a year, and preserve in-80 ℃.Get one bottle in per 10 bottles and carry out microorganism testing to confirm bacterial strain purity and not have bacteriology and pollute.

Inoculum and starter culture

By being transferred among the 10ml APT (general Tween), freezing main kind of a full ring or the freeze dried culture of a bottle prepare inoculum.Then, seed growth is spent the night.Prepare the starter culture by inoculum (grow overnight) being transferred in the 6L APT substratum and once more it being incubated overnight from inoculum.

Fermentation and concentrated

The starter culture is transferred under aseptic condition in the production fermentation container, and described fermentation container comprises growth medium and maintains 25 ℃.Reach about 10 by spectrophotometer (650nm) and by being inoculated on the APT agar up to cell density ⁹Cfu monitors fermentation.Then collect the growth medium (comprising C.maltaromaticum) of fermentation and with its freeze-drying.

Freeze-drying

Freeze dried material scraped from dish get, grind also and pulverize, be placed in the polyethylene bag before and in the double-ply bag of packing in refrigeration (4-8 ℃).

Embodiment 7 microbiological analysiss

Freeze dried material is carried out total lactic-acid-bacterium, non-lactic-acid-bacterium, yeast, mould, total coliform group, streptococcus aureus (Staphylococcus aureus), intestinal bacteria (Escherichia coli), and the microbiological analysis (table 5) of salmonella (Salmonella spp).

The description of the freeze dried bacterial powder of table 5Carnobacterium maltaromaticum

Activeconstituents vehicle shelf-life storage requirement physics aspect	Carnobacterium maltaromaticum Star Dri 5＞1 year room temperature (22 ℃) explanation	Method
			Outward appearance concentration residual moisture microorganism is described the non-lactic-acid-bacterium yeast of lactic-acid-bacterium mould anaerobism spore forming bacteria Clostridium botulinum total coliform staphylococcus aureus e coli salmonella	By between 3.2*10 6/ g and 3.2*10 7Between viable cell/g＜5% between 3.2*10 6/ g and 3.2*10 7Between the cfu/g or between 1.3*10 9And 1.4*10 10Between the cfu/ packing＜the every 50g shortages＜10/g of 100/g＜100/g＜100/g＜10/g＜every 25g of 100/g lacks every g shortage	Visual observation APT is dull and stereotyped and compare A.P.H.A./USP O ' Haus A.P.H.A./USP A.P.H.A./USP A.P.H.A./USP A.P.H.A./USP A.P.H.A./USP A.P.H.A./USP A.P.H.A./USP A.P.H.A./USP A.P.H.A/USP A.P.H.A/USP with the photo and the description of the flat board of standard

A.P.H.A=American Public Health Association (APHA); The USP=American Pharmacopeia

The reconstruct of embodiment 8 starting cultures

Standardized viable cell adulterant is packaged in the plastic foil film packaging, thereby makes every gram the finished product reach 10 with nitrogen wash and by the requirement regulation packed weight that the finished product are used ³With 10 ⁴Between C.maltaromaticum cell alive.Then described packing material is stored in room temperature.

Stdn viable cell adulterant to packing carries out total lactic-acid-bacterium, non-lactic-acid-bacterium, and yeast, mould, total coliform group, streptococcus aureus, (table 5) identified in the microbiological analysis of intestinal bacteria and salmonella.

Composition mix and further grind and the packing material of packing into before, can also be with doses comprise bacterial strain CB1, CB2, CB3, LV17, UAL26, ATCC 35586 or ATCC 43225 (about 1 * 10 ³To 1 * 10 ⁴The reconstruct C.maltaromaticum of one or more the cfu/g the finished product) directly adds in the meat of grinding.Then, with these sausages-50 ℃ of quick freezing up to freezing at the center.Then, pack these sausages in plastic wraps and remain on freezing state and carry out retail sales up to thawing.

The growth characteristics of Carnobacterium maltaromaticum on the embodiment 9. vacuum-packed wieners

The research of Design Laboratory scale come can with the growth characteristics of the C.maltaromaticum of research in vacuum packaging under the commercial production condition relatively, described wiener is inoculated with C.maltaromaticum.In addition, research is to the influence of organoleptics property such as fragrance and local flavor pattern.The method and the result of this research are as follows:

Select the two strain bacterial strains of C.maltaromaticum to study: LV17 (synonym of UAL 8) begins to separate from vacuum-packed, the refrigerated fresh pork is also described by Shaw and Harding (1984), with bacterial strain UAL 26, it separates from vacuum-packed beef (Stiles and Holzapfel, 1997).Bacterial cell by will washing adds 0.85% salt solution of sterilization to provide 2.5 * 10 ⁶The inoculum level of cfu/ml prepares inoculum.Wiener that will be independent immerses in the inoculum suspension and reaches one minute, dehydrates and carries out vacuum packaging (high barrier, low O with every bag 5 one group wiener ₂Transmit the VP bag).In contrast, wiener is not being had under the situation of bacterial inoculum, in the sterile saline of immersion 0.85%.Then, place refrigeration (4 ℃) storage to reach for 12 weeks with what handle with control sample.At 0 day and back to wiener sampling carrying out microbiological analysis and sensory evaluation in 2,4,6,7,8,10 and 12 weeks of storage.

(be equivalent to 10cm by downcutting the long wiener piece of 1.8cm ²Surface-area), be placed in the tissue homogenate bag of sterilization and carry out homogenate and prepare sample and carry out microbiological analysis.Bacterial count is undertaken by standard dilution and inoculation technique and is comprised 1) at 25 ℃, 48 hours the total aerobic plate count on plate count agar of aerobic incubation; 2) at 25 ℃, 48 hours the lactic-acid-bacterium on APT agar of anaerobism incubation; 3) adding of 35 ℃ of incubations 18 hours the intestinal bacteria on the Violet Red BileAgar of 1% glucose.The concentration of bacterium is reported as every cm ²Cfu (the cfu/cm of product ²).

Will carry out wiener boiling in the water of " boiling just " that organoleptics property is estimated, and make its static 5 minutes (temperature of inner wiener is approximately 83 ℃).Wiener is cut into piece, and places the container with the paper tinsel covering of coding, heating is 15 minutes in 94 ℃ baking oven, assesses immediately.The sensory evaluation is undertaken by one group of 9 people's who has carried out training in three months periods panel.Use the informal line grade of 15cm, sample has been carried out total aromatising flavour intensity, meat flavor intensity, seasoning (seasoned) local flavor, flue dust intensity, tart flavour/acidity, stink (off-flavor) but and the assessment of total acceptance, wherein 0=as mild as a dove, 15=is very strong.Between sample, tongue purifies with the 7-Up  of cracker and dilution in 1: 1.

This research has reported that the sample wiener of having inoculated with C.maltaromaticum bacterial strain LV17 or UAL 26 reaches 2.75 * 10 respectively after through the refrigeration of 7 or 8 weeks ⁶With 1.2 * 10 ⁵Cfu/cm ²Maximum anaerobism lactic-acid-bacterium (LAB) quantity.At duration of storage, C.maltaromaticum grows on vacuum-packed wiener and grows with speed slowly and is accompanied by the quite little minimizing of surface p H.LV17 changes to the pH6.1 in the 10th week from the pH 6.2 in 0 week, and UAL 26 changes to about 5.9 from the pH6.2 that begins during 6-8 week and 12 weeks.

Reach a conclusion for: with other lactic-acid-bacterium such as cold living leukonid relatively, when being inoculated into refrigeration (4 ℃), in the time of on the vacuum-packed wiener, C.maltaromaticum is the kind of slowly growing.The level of C.maltaromaticum reaches maximum 5 * 10 after the refrigeration in 12 weeks ⁷Cfu/cm ²Based on the sensory evaluation that the panel by 9 members of training carries out at the duration of storage in 12 weeks, the inoculation of carrying out with C.maltaromaticum does not cause aromatising flavour, stink, but the tangible disadvantageous effect of acidity or total acceptance.

Embodiment 10. is in being inoculated into sausage the time, the growth characteristics of Carnobacterium maltaromaticum

At the production period of sausage, with C.maltaromaticum CB1 as inoculum in three tests, add pork.Handle the odor intensity and the freshness quality of sausage (estimating) as unprocessed the two with boiling with 21 dotted line grades.The inoculation horizontal extent is from 1 * 10 ³To 1 * 10 ⁵Cfu/g meat.The 0th, 5, carry out bacteriological analysis over 10,15 and 20 days with the growth of assessment C.maltaromaticum and the generation of bacteriocin.

Refrigerated pork shoulder and pork fat are weighed, slightly grind and be divided into 4 batches, water with 2.76% and 1.8% condiment add wherein.With reaching 10 ⁵The C.maltaromaticum of cfu/g inoculates test products.The meat and the composition that grind are mixed, lappingout and fill out Collagent casing for sausages (casing) (UniPac, Edmonton) in.The casing of filling is cut into the ring of 3.5-3.75 inch, to obtain being about the sausage of 20.4g/ sausage.At-50 ℃ with about 35 minutes of independent sausage ring quick freezing.The refrigerated sausage is packaged in (each packs about 10oz) on the Styrofoam dish, with its sealed envelope and be sealed in the plastic wrapping.Before bacterial sampling, sample is thawed and preserve in 4 ℃.

The general work program of meat comprises the program of " chilling " meat to transport, and afterwards for selling or further process thawing of carrying out.

Thawing and preserving, carrying out the bacteriology sampling on 10,15 and 20 days the sample in 4 ℃ 0,5.Bipartite 10g sample is placed the bacterium separator bag (VWR International) of sterilization and mix with 0.1% peptone water of 90ml sterilization.The serial dilution degree that is fit to that will be in 0.1% peptone water on APT agar that pours in advance and MRS agar plate, rule and with it 30 ℃ of incubations 48 hours.After date when incubation writes down the duplicate counting (cfu/g meat) of each sample.

In 20 day period, the total anaerobic bacterium counting on MRS agar is from 10 ³-10 ⁵The cfu/g product is increased to 10 ⁹The cfu/g product.The microbiotic growth of background in test products is not different from the background microbiotic growth relevant with nonvaccinated sample, as by as shown in the growth on APT agar.This explanation does not increase total incidence of bacterial growth in the sausage with C.maltaromaticum inoculation sausage meat.The sum that the bacterial growth on APT and MRS agar is pointed out in microbioassay is similar on nonvaccinated contrast and test products.So the C.maltaromaticum culture of adding is not increased in the number of bacteria of finding on the test products, do not make described meat comparison according to corrupt sooner yet.

The 0th, 5, the bacteriocin of testing in the sausage sample by direct and indirect measurement in 10,15 and 20 days produces, and it is detected, and indication is produced by the bacteriocin that the C.maltaromaticum that adds carries out.To the 10th day by indirect measurement [part of sausage is by thermal treatment (kill produce biological)] and directly be embedded in monocyte hyperplasia listeria spp CDC 7762 (serotype 4b)] observe inhibition in the APT agar inoculated to indicator organism monocyte hyperplasia listeria spp, wherein this inhibition is maintained to the 20th day of described mensuration.The direct mensuration that in the sausage of C.maltaromaticum inoculation bacteriocin is produced (supernatant liquor of heat treated homogenate sausage is directly added in the APT agar plate that covers with monocyte hyperplasia listeria spp indicator organism) is indicated to sausage in the generation of the 15th day generation bacteriocin of 4 ℃ of storages and last till the 20th day of storage.

Embodiment 11.Carnobacterium maltaromaticum be added on instant (RTE) and fresh cold charge, processing meat product in application

RTE meat and fresh cold charge, the meat product of processing need suppress the aseptic technic that possible pathogenic bacteria grows.Fatal monocyte hyperplasia listeria spp outburst has swept across Northeastern United States recently, cause FDA and USDA ' s food safety inspector-general's department (FSIS) to issue healthy alarm (Morbidity and Mortality weekly Report, 2003) in September, 2003.

Suggestion is as alleviating the means of the influence of the pollution that is caused by people pathogenic bacteria such as monocyte hyperplasia listeria spp, C.maltaromaticum added instant (RTE) meat product of modified atmosphere packaging and FF, in the meat product of processing.During the packing of RTE meat product such as wiener, advise every 454g (1 pound) packaging application doses (about 1.5ml, or 5 * 10 ⁶Cfu) reconstruct C.maltaromaticum.

Before composition mixes and further grinds and be filled in the casing, the aliquot of reconstruct C.maltaromaticum (is sent about 1 * 10 ³To 1 * 10 ⁴Cfu/g) adding is FF, and is FF to produce in the meat product of processing, the meat product of processing.FF, the meat product of processing will be rapidly frozen up to the center at-50 ℃ and be frozen, then sealed parcel and chilled storage in plastic wrapping.

For RTE meat product and fresh food frozen, the inoculation scope of the meat product of processing is about 1 * 10 ³To 1 * 10 ⁴C.maltaromaticum cell (the cfu)/g product of living.

Embodiment 12

By (2000) such as Duffes carry out to separating from commercial, vacuum-packed cold-the bacteriocinogeny bacterial strain C.maltaromaticum (bacterial strain SF668) in the smoke cured salmon (CSS) is suppressed at the inspection of the potential of the monocyte hyperplasia listeria spp growth on the CSS, find that C.maltaromaticumSF668 is being housed on vacuum-packed cold-smoked salmon of 4 ℃, can be in 21 days from 1 * 10 ⁵Be increased to 3 * 10 ⁷Cfu/ml (table 8).Back from 1 * 10 with the monocyte hyperplasia listeria spp that C.maltaromaticum cultivates altogether 4 ℃ of three week ³Cfu/ml is increased to 3.5 * 10 ³Cfu/ml.The common cultivation of this C.maltaromaticum and monocyte hyperplasia listeria spp has caused that (when lacking C.maltaromaticum, the growth of monocyte hyperplasia listeria spp reaches about 5 * 10 to the tangible bacteriostatic effect in the monocyte hyperplasia listeria spp on cold-smoke cured salmon growth ⁴Cfu/ml).

When belonging at the listeria spp of 21 strains when screening, the inhibition circle of the uniqueness that forms by C.maltaromaticum LK5 for 17 strain bacterial strains be tangible (Buchanan and Klawitter, 1992a).Find that C.maltaromaticum LK5 does not form hydrogen peroxide, but produce bacteriocin.The ability that C.maltaromaticum LK5 suppresses the listeria spp genus is temperature-dependent form (5 ℃ and 19 ℃ of mensuration), with 19 ℃ of comparisons, at 5 ℃ significantly bigger inhibition is taken place in the monocyte hyperplasia listeria spp of cultivating altogether with C.maltaromaticum LK5.C.maltaromaticum LK5 is presented at and can grows obviously sooner than monocyte hyperplasia listeria spp on the freezing temp, and approximately is identical 19 ℃ of speeds of growth.At 19 ℃, (inoculum density remains on 1 * 10 to the monocyte hyperplasia listeria spp ³Cfu/ml is constant) inhibition depend on the inoculation ratio, wherein only 〉=1: 1 LK5: monocyte hyperplasia listeria spp ratio produces significant inhibition degree.At 5 ℃, during the commitment of incubation,, observe anti--listeria spp activity of increase level for higher inoculation ratio, still by about 300 hours of incubation, LK5 inoculation size was to suppressing the active influence that do not have; (be respectively 10: 1 * 10 for scope from 0.01: 1 to 1000: 1 ³With 1 * 10 ⁶: 1 * 10 ³Cfu/ml) ratio), the degree of inhibition is identical.

In freezing temp, meat bacillus strain isolated has competitiveness very much, illustrates even inoculum in a small amount can be used to control the monocyte hyperplasia listeria spp in frozen product.This studies confirm that the report of Schillinger and Holzapfel (1990), it has been reported in 13 strain C.maltaromaticum bacterial strains, there are 10 strains significantly to suppress the growth of monocyte hyperplasia listeria spp DSM 20600, test determined as passing through the agar spot.

Embodiment 13.7.2. exposes the background of Carnobacterium maltaromaticum

Research has shown before the deadline finds lactic-acid-bacterium usually in retail food, particularly C.maltaromaticum (modified atmosphere packaging and freezing preferential selection anaerobism meat Bacillaceae) (Milliere and Lefebvre, 1994a; Kelly etc., 1996; Schobitz etc., 1999; Amezquita and Brashears, 2002; Sakala etc., 2002).So, the quantity of the C.maltaromaticum that can at utmost consume for accurate assessment, must consideration the C.maltaromaticum of any theoretical amount on the target food Already in.

Document has been carried out broad research, found that the reference of two pieces of concrete analyses in the amount that is purchased the C.maltaromaticum that finds on the food.Sakala etc. (2002) have measured two beef samples that comprise C.maltaromaticum.The 0th, 1,3,5 and 6 all detected average quantitys in storage (vacuum-packed and preserve in 2 ℃) are respectively 2 * 10 ³, 2 * 10 ⁴, 2.5 * 10 ⁶, 1 * 10 ⁷And 2.5 * 10 ⁷Cfu/g meat.Montel (2000) found in the latter stage in fermentation period of sausage, the micro-flora that lactic-acid-bacterium is normally main, wherein C.maltaromaticum under native state, fermentation period with about 5 * 10 ⁷The level of cfu/g sausage exists, but disappears subsequently., find after week at 6 ℃ of storage 2-3 with 10 ⁴-10 ⁵The aseptic cold smoking salmon of cfu/g salmon inoculation has scope between 5 * 10 ⁷To 10 ⁹The final counting of cfu/g (Stohr etc., 2001).Nadon etc. (2001) are presented at initial 6 weeks of storage, at vacuum-packed or carbonic acid gas controlled atmosphere (CO ₂-CAP) packing in the pork of handling, LAB (it comprises the meat bacillus) is from initial 100cfu/cm ²Be increased to 1 * 10 ⁶The mean level (ML) of cfu/cm2, and in the time of the remainders of 13 week researchs, keep the level of LAB.At CO ₂In-CAP pork the sample, LAB just has tangible increase up to the 11st week of storage, and wherein the maximum horizontal of LAB is 3.2 * 10 ⁵Cfu/cm ²Under the tangible anoxybiotic situation of undocumented of service of being undertaken by (2001) such as Nadon, at-1.5 ℃ of duration of storage, the meat bacillus occupies superiority in the LAB micro-flora.

Embodiment 14

The bacterial strain of C.maltaromaticum produces several different meat bacillins (Quadri etc., 1994), and it has been accredited as heat-stable peptide, is stable in wide in range pH scope and can work as sterilant (Jack etc., 1996).Thereby the bacteriocin to C.maltaromaticum L103 has carried out measuring this bacteriocin is controlled the growth of monocyte hyperplasia listeria spp in vacuum-packed meat ability (Schobitz etc., 1999) in a research recently.Partially purified bacteriocin is inoculated ox semitendinosus strip of muscle with the concentration of 100AU/ml (the active arbitrary unit of AU/ml=).The monocyte hyperplasia listeria spp is added in the incarnation as indicator strain, and ultimate density is 1 * 10 ³Cfu/cm ²Guaranteeing with after meat better contacts, described cutlet is carried out vacuum packaging and preserve in 4 ℃ 21 days.Each is taken a sample day, all comprises nonvaccinated contrast and only comprises the meat of indicator strain.The growth and the LAB growth of bipartite cutlet being taken a sample and observing the monocyte hyperplasia listeria spp the 0th day and per 7 days.After 7 days, observe the obvious minimizing of monocyte hyperplasia listeria spp counting 4 ℃ of storages, counting is from 2 * 10 of beginning ³Cfu/cm ²To 4cfu/cm ², suppressed pathogenic agent (＜1cfu/cm fully at the 14th day that preserves ²).LAB is multiplied on vacuum-packed meat, after 14 days, reaches 1 * 10 ⁷Cfu/cm ²Counting, wherein original level is 1.6 * 10 ²Cfu/cm ²The color of meat and smell still are acceptable at 14 days duration of storage.The presentation of results of this research can be suppressed at monocyte hyperplasia listeria spp on the vacuum-packed meat from the bacteriocin of C.maltaromaticum in time of 14 days nearly, and still keeps the edibility characteristics (Schobitz etc., 1999) of meat.C.maltaromaticum LV61 produces such bacteriocin, it is to C.maltaromaticum2762 and monocyte hyperplasia listeria spp (bacterial strain R2, Lud 1033, Br1246, Lud 905 and T) have an activity, but by pronase e, Proteinase K and trypsinase institute deactivation (Pilet etc., 1995).Other research has illustrated that the bacteriocin from the purifying of C.maltaromaticum LV61 suppresses meat bacillus and enterococcal several bacterial strains, but does not suppress several bacterial strains (Holck etc., 1994) of listeria spp.So inference, C.maltaromaticum LV61 is except producing piscicolin 61, and generation relates to the active another kind of factor of anti-listeria spp.

Embodiment 15

The meat Bacillaceae is had a liking for cold, growth and fermentation inulin on the high pH value of 8-9.Exist under the culture condition of inulin, C.maltaromaticum forms has metallic copper glossy yellow to peach bacterium colony, and the yellow of substratum changes and sedimentary disappearance.Various C.maltaromaticum bacterial strains have shown the generation bacteriocin, have other meat bacillus of inhibition, and Bacterium lacticum and listeria spp belong to the protein compound of the ability of growth.

Recommendation with C.maltaromaticum with 1 * 10 ³To 1 * 10 ⁴The scope of cfu/g is inoculated into various instant and fresh food frozens, thereby improves anticorrosion in the meat product of processing and the growth of minimizing pathogenic bacteria.Based on these inoculation scopes, and bacterium will be at the theory vision of the prolonging period growth of preserving, and average everyone consumption of C.maltaromaticum of adding in the selected RTE food is estimated as for 60kg people's 4.3 * 10 ⁹Cfu/ days or 7.2 * 10 ⁷Cfu/kg/ days.

When salmon, chicken, pork, when assessing in beef and other meat product that is purchased, C.maltaromaticum is to pathogenic agent, and the inhibition of monocyte hyperplasia listeria spp takes place.The common cultivation of C.maltaromaticum and monocyte hyperplasia listeria spp causes the logarithm of monocyte hyperplasia listeria spp growth to reduce.At low temperature, with monocyte hyperplasia listeria spp growth fraction than the time, C.maltaromaticum is improved at low temperature to the restraining effect of monocyte hyperplasia listeria spp growth.The monocyte hyperplasia listeria spp suppresses can be by the generation of lactic acid, and the generation of nutraceutical competition and bacteriocin is regulated.The generation of bacteriocin is relevant with the inhibition of the increase of monocyte hyperplasia listeria spp growth.When being subjected to simulating hydrochloric acid in gastric juice or followed by action of proteolytic enzymes, the active of bacteriocin that is produced by C.maltaromaticum reduces fast, is designated as non-toxicity and anallergic protein.

C.maltaromaticum increases the storage time of the meat product of RTE and vacuum-packing, reduces growth of pathogenic bacteria simultaneously.The growth that has been found that C.maltaromaticum is from limit, and wherein at RTE meat product and vacuum-packed, the horizontal stable of the C.maltaromaticum on the salmon of cold smoking is about 1 * 10 ⁹Cfu/g.With C.maltaromaticum with between 1 * 10 ³With 1 * 10 ⁴Level between the cfu/g is added target RTE and fresh food frozen to, will can obviously not increase in the meat product of processing from total mankind's consumption of the LAB of these food (to determine that theoretic consumption naturally is 4.3 * 10 ⁹Cfu/ days).The growth that has shown C.maltaromaticum itself is from limit; The C.maltaromaticum growth will be about 1 * 10 ⁸With 1 * 10 ⁹Stop between the cfu/g meat.Infer that this is because the release of the specificity bacteriocin of the higher bacterial density of restriction causes.

Embodiment 16 is from separation and the screening of the lactic-acid-bacterium (LAB) of meat product

Refrigeration or refrigerated, the sample with meat instant processing that give birth to is:

A) purchase is from the sample of retail market; Take the laboratory to and carry out microbiological analysis

B) the freezing sample of producing from the pilot plant of raw pork sausage thaws and carries out microbiological analysis

C) purchase is from the sample of the meat of the instant processing of retail market, and it is stored in 4 ℃ of " the best date finish " up to them in the laboratory, carry out microbiological analysis.

In triplicate 10g sample is downcut from each packing under aseptic condition, be diluted in 0.1% peptone water of sterilization of 90ml and bacterium separator Lab-Blender 400 (Seward, England) in homogenate 2 minutes.The serial dilution thing of homogenate is prepared and is layered on pre-dabbling APT (general tween in 0.1% peptone water; Difco) on agar (1.5%) flat board.With flat board 25 ℃ (A, B) or 15 ℃ of (C) anaerobism (A, B) and aerobic (C) incubation 48 hours.From the dull and stereotyped single bacterium colony selected at random with the toothpick picking of sterilizing of APT, and it is scoring on the pre-dabbling APT plate pack of requirement (for one group of the every kind of indicator strain that is used to screen).With flat board in 25 ℃ of anaerobism (A) with aerobic (B, C) incubation is 24 hours.Being seeded in the lawn that monocyte hyperplasia listeria spp indicator strain on the soft APT agar (0.75%) or general indicator strain blazon meat bacillus LV13 in order to 1% is coated on every group of flat board.With the flat board that was coated with 37 ℃ of incubations 24 hours.The inhibition zone that will be viewed as transparent circle in coating layer is recorded as the biology that produces antimicrobial substance.Screen this active biology of demonstration at the susceptibility of pronase e (Sigma) with for the susceptibility of heat.Selection for the PRONASE A sensitivity and stablize those of 30 minutes at 60 ℃ and further identify.

Embodiment 17.

Many bacteriums produce antibacterium peptides or protein (for example, bacteriocin), and it has activity at other bacterium, the bacterium that typically is closely related usually.The exemplary list of bacterium and their bacteriocin is presented in the table 6.

Table 6

Bacterial strain	Bacteriocin
		The 3.C.maltaromaticum CB3 4.C.maltaromaticum UAL26 5.C.maltaromaticum LV 17 6.C.maltaromaticum UAL26/8A 7. of the 2.C.maltaromaticum CB2 of LAB 1.Carnobacterium maltaromaticum CB1 the unknown that collect in our laboratory+the unknown blazon meat bacillus LV13 8. cold living leukonid UAL 187 9. Lactobacillus saki pure mellow wine subspecies UAL 185 10. leukonids and belong to non-LAB 11. sarson rope silk bacterium (Brochothrix campestris) ATCC43754 that UAL280 suppresses Listeria	Meat bacillin BM1, piscicolin 126+ meat bacillin BMl, piscicolin 126 meat bacillin BM1, piscicolin 126 piscicolin 126 meat bacillin A, BM1 and B2 piscicolin 126, the brochocin C of the unknown of meat bacillin A divergicin A leucocin A the unknown

12.A53 13. ( Brevibacterium linens ) ATCC9175 14.OC2 15.NCFB1454 LAB 16.C.maltaromaticum LV61 17.C.maltaromaticum V1 18.C.maltaromaticum CP5 19.C.maltaromaticum JG 126 20.377 21.C.maltaromaticum U 149 22.750 23. ( P.acidilactici ) PAC 1.0 24.E 25.F 26.H 27.JD1-23 28.M 29. ( P.pentosaceus ) Z102 30.WHE92 31.ALC01 32.Lb706 33.CTC494 34. ( L.curvatus ) LTH1174 35.LTH673 36.674 37. ( Lactobacillus bavaricus ) M1401 38.MN 39.CTC492 40.T136 41.WHE81 42.BFE900 43.L50

The linenscin OC2 bifidocin B meat bacillin A meat bacillin BM1 of duomycin (aureocin) A53 the unknown, piscicolin 126 meat bacillin BM1 and B2 piscicolin 126 carnocin H carnocin U149 divergicin 750 pediocin PA-1 pediocin PA-1 pediocin PA-1 pediocin PA-1 pediocin PA-1 pediocin PA-1 pediocin PA-1 pediocin PA-1 pediocin PA-1 sakacin A sakacin A sakacin A sakacin P sakacin P sakacin P bavaricin MN enterocin A and B enterocin A and B enterocin A and B enterocin A and B enterocin L50A and L50B, P, Q

44.DPC1146 45.EK13 46.P13 47.AA13 48.G16 49.JCM5804T 50. ( Enterococcus casseliflavus ) IM416K1 51. ( Leuconostoc carnosum ) 4010 52.UG1 53.CRL35 54. ( Lactobacillus casei ) CRL705 55.CTC494 56. 57. 58. ( Lactobacillus brevis ) VB286 59.CTC305 60.CTC306 61.CTC372 LAB 62.C.maltaromaticum CS526 63. ( Streptococcus thermophilus ) Sfi13 64. ( E.faecalis ) EJ97 65.BFE1071 66.FAIR-E309 67.Y1717 68.LMG2333 69.DPC5280 70.S-48 71.INIA4 72.ALC01 73.Lb.Sake 2512 74.423

Enterocin A enterocin A and P enterocin P enterocin P enterocin P enterocin A; B, the thermophyllin 13 enterocin EJ97 enterocin 1071 enterocin 1071 bacteriocins 31 enterolysin A enterolysin A enterocin AS-48 enterocin AS-48 pediocin PA-1 sakacin G plantaricin 423 of the unknown of the unknown of the unknown of P enterocin 416K1 leucocin and AC plantaricin UG1 enterocin CRL35 lactocin CRL705 sakacin K leucocin F10 leucocin B-Tal1a brevicin 286 the unknowns

75. ( Enterococcus mundtii ) ATO6 76.NFR17393 77. ( Lactobacillus buchneri ) 78. ( L.lactis ) MMFII 79.UL720 80. ( Enterococcus gallinarum ) 012 81. 82. ( Leuconostoc mesenteroides ) FR52 83.Y105 84. 85. 86.61-14 87.DPC3147 88. 89.LMG280 90.IPLA972 91.DPC5552 92.BGMN1-5 93. ( Lactobacillus johnsonii ) VPI 11088 94. ( Lactobacillus acidophilus ) M46 95.N2 96.LA39 97.UCC118 98.C11 99.NC8 100. ( Propionibacterium jensenii ) DF1 101. ( Escherichia coli ) 102

Mundticin mundticin KS buchnericin-LB lactococcin MMFII diacetin B enterocin 012 plantaricin NA mesenterocin 52A mesentericin Y105 nisin nisin Z nisin Q lacticin 3147 lactococcin A, B, M lactococcin G lactococcin 972 lacticins 481 LsbA, LsbB lactucin (lactacin) F acidocin B lactucin B gassericin A ABP-118 plantaricn E/F, J/K plantaricin NC8 propionin SM1 colicin V colicin Y101

103. it is Microbial anticorrosive additive by the gramnegative bacterium generation that the dried bacterium SA6 112. Lactobacillus saki pure mellow wine subspecies L45 of Escherichia coli 104. MRSEs (Staphylococcus epidermis) 105. bacillus subtilis bacterium (Bacillus subtilis) 168 106. Lactobacillus gasseris, 107. Friedlanders bacillus (Klebsiella pneumoniae) 108. junket clostridium butyricums (Clostridium tyrobutyricum) ADRIAT932 109. Bai Shi shuttle bacterium (Clostridium beijerinckii) ATCC25752 110. food starch milk bacillus (Lactobacillus amylovorus) DCE471 111. plants breast will following biology be called usually: 1. Friedlanders bacillus RYC492 2. Escherichia coli 3. Escherichia coli 4. Escherichia coli 5. Escherichia coli 6. Escherichia coli

The plain E492 Closticin of the plain H47 epidermin of microorganism subtilosin A gassericin K7B microorganism 574 circularin A exocellular polysaccharide L471 plantaricin SA6 lactocin S swash the plain V of vitamin H E492 (identical with 107) microorganism (with 101 identical, being once called as colicin) the plain L microorganism of the plain H47 microorganism of plain Y101 (identical with the 102) microorganism of microorganism element 24

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Claims (18)

1. handle the method that food is resisted listeria spp for one kind, described method comprises described food is contacted with composition, described composition comprise being selected from the flesh of fish bacillus strain CB1 (ATCC number PTA-5313) of dwelling, the flesh of fish bacillus strain CB2 (ATCC numbers PTA-5314) and dwell and oppress one or more bacterial culturess of bacillus strain CB3 (ATCC numbers PTA-5315) of dwelling.

2. method of handling food, described method comprises:

Determine that at least a harmful microbe exists in the food;

Select specificity control or eliminate one or more beneficial microorganisms that described harmful microbe influences; Preparation comprises the composition of at least a beneficial microorganism or at least a bacteriocin or their combination; With

With the described food of described compositions-treated.

3. the method for claim 2, wherein said harmful microorganism comprises spoilage organism or pathogenic bacteria.

4. the method for claim 3, wherein said pathogenic bacteria is one or more bacteriums that are selected from the group of being made up of listeria spp.

5. the method for claim 3, wherein said spoilage organism is to one or more bacteriocin sensitivities that is produced by lactic-acid-bacterium.

6. the method for claim 2, wherein said beneficial microorganism comprises one or more lactic-acid-bacteriums.

7. the method for claim 6, wherein said beneficial microorganism comprise being selected from dwell the flesh of fish bacillus strain CB1 (ATCC numbers PTA-5313), dwell and oppress bacillus strain CB2 (ATCC numbers PTA-5314) and at least a meat bacillus of the flesh of fish bacillus strain CB3 (ATCC numbers PTA-5315) of dwelling.

8. the method for claim 5, wherein said lactic-acid-bacterium has known corrupt speed.

9. the method for claim 8, wherein known corrupt speed is used to predict the shelf-life of described food.

10. the method for claim 7 comprises that also the fermented product that will comprise bacteriocin is applied to described food.

11. comprising, a method for preparing the meat of processing, described method use the flesh of fish bacillus strain of dwelling that being selected from of significant quantity dwelt and oppressed bacillus strain CB1 (ATCC numbers PTA-5313), dwell the flesh of fish bacillus strain CB2 (ATCC numbers PTA-5314) and the flesh of fish bacillus strain CB3 (ATCC numbers PTA-5315) of dwelling.

12. the bacteriocin composition that is produced by the flesh of fish bacillus strain CB1 (ATCC numbers PTA-5313) of dwelling that a method of preserving food or beverage, described method comprise significant quantity adds in food or the beverage.

13. the bacteriocin composition that is produced by the flesh of fish bacillus strain CB2 (ATCC numbers PTA-5314) of dwelling that a method of preserving food or beverage, described method comprise significant quantity adds in food or the beverage.

14. the bacteriocin composition that is produced by the flesh of fish bacillus strain CB3 (ATCC numbers PTA-5315) of dwelling that a method of preserving food or beverage, described method comprise significant quantity adds in food or the beverage.

15. the method for claim 1 or claim 2 also comprises and the described bacterial strain of fermented product combined administration.

The flesh of fish bacillus strain CB1 (ATCC numbers PTA-5313) 16. dwell.

The flesh of fish bacillus strain CB2 (ATCC numbers PTA-5314) 17. dwell.

The flesh of fish bacillus strain CB3 (ATCC numbers PTA-5315) 18. dwell.

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