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WO1999034679A1 - Methode biologique d'elimination de la salmonelle de processus et de structures - Google Patents

Methode biologique d'elimination de la salmonelle de processus et de structures Download PDF

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Publication number
WO1999034679A1
WO1999034679A1 PCT/FI1998/001034 FI9801034W WO9934679A1 WO 1999034679 A1 WO1999034679 A1 WO 1999034679A1 FI 9801034 W FI9801034 W FI 9801034W WO 9934679 A1 WO9934679 A1 WO 9934679A1
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WIPO (PCT)
Prior art keywords
salmonella
procedure
spaces
pseudomonas
treated
Prior art date
Application number
PCT/FI1998/001034
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English (en)
Inventor
Atte Von Wright
Tiina Mattila-Sandholm
Hanna-Leena Alakomi
Jorma Hirn
Tapio Palmu
Marjaana Hakkinen
Ilkka Helander
Original Assignee
Atria Oyj
Avena Siilot Oy
Broilertalo Oy
Munakunta
Osuusteurastamo Karjaportti
Primalco Oy
Raisio Yhtymä Oyj
Stevena Oy
Suomen Rehu Oy
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Atria Oyj, Avena Siilot Oy, Broilertalo Oy, Munakunta, Osuusteurastamo Karjaportti, Primalco Oy, Raisio Yhtymä Oyj, Stevena Oy, Suomen Rehu Oy filed Critical Atria Oyj
Priority to EP98962467A priority Critical patent/EP1043933A1/fr
Priority to AU17634/99A priority patent/AU1763499A/en
Publication of WO1999034679A1 publication Critical patent/WO1999034679A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K1/00—Housing animals; Equipment therefor
    • A01K1/015—Floor coverings, e.g. bedding-down sheets ; Stable floors
    • A01K1/0152—Litter
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/27—Pseudomonas

Definitions

  • the present invention relates to a procedure as defined in the preamble of claim 1 and 17.
  • the salmonella bacteria are known pathogens causing food poisoning. Being intestinal bacteria, they readily spread in animal spaces, contaminating animal products. A very problematic situation prevails in many Central European countries, where e.g. over 90 % of all broilers may be contaminated by salmonella. Animals bearing salmonella and animal products contaminated by salmonella may spread these bacteria to spaces where foodstuffs are handled, stored or sold and also to homes. In Finland, very small amounts of salmonella have been found in animals used for food production. During 1989-94, less than 5 % of the broiler production batches were salmonella positive.
  • antibiotics are used to curb salmonella infections.
  • widespread use of anti- biotics has certain risks.
  • Microbe populations resistant to antibiotics are readily generated, creating difficulties in the chemotherapy of infections in humans as well.
  • It is possible to reduce the need for antibiotics by improving animals' immunity to salmonella either by vaccination or by a so-called competitive exclusion (CE) method.
  • the CE method is based on the fact that the intestines of a newly born animal are exposed to infections, whereas the varied normal flora of an adult animal prevents pathogenic colonisation.
  • the CE method has been used to prevent the development of salmonella in broilers.
  • To promote the development of a protecting normal flora in the intestines of chickens the chickens have been given a CE preparation containing an adult hen's intestinal flora as early as possible after being hatched.
  • the CE method has proved to be an alternative worth consideration in fighting salmonella in broilers.
  • the sanitation costs of a typical raw material stock amounts to hundreds of thousands of marks (FIM) . Be- sides, even if sanitation is carefully planned, its successful implementation is very uncertain.
  • the object of the present invention is to eliminate the drawbacks described above
  • a specific object of the present invention is to disclose a method for control of salmonella in food industry plants and processes, using protecting microbes and microbial protective substances.
  • substantially dry pro- duction and processing spaces and/or production and processing equipment can be treated with a Pseudomonas bacterial preparation to prevent salmonella contamination. All production and/or processing spaces in food industry can be treated with a preparation of lactic acid bacteria to prevent salmonella contamination.
  • Production and/or processing spaces comprise the surfaces in process spaces, storage spaces, transport spaces, animal breeding spaces and/or other spaces, such as e.g. large cookhouses.
  • Production equipment comprises process materials, media and/or other objects.
  • Substantially dry production and processing spaces and/or production and processing equipment comprise open, non- watery spaces and equipment mentioned above. Substantially dry spaces does not refer e.g.
  • Pseudomonas bacterial preparation refers to an antimicrobial preparation inhibiting growth of salmonella, containing bacteria and/or produced by bacteria, which has a low pro- tease and lipase activity.
  • Lactic acid bacterium preparation refers to an antimicrobial preparation inhibiting growth of salmonella, containing bacteria and/or produced by bacteria, which contains compounds having a molecular weight ⁇ 1000 Da.
  • a Pseudomonas bacterial preparation preferably containing above-mentioned bacteria, which has an inhibiting effect on the growth of salmonella and a low protease and lipase activity, is applied to substantially dry surfaces.
  • Pseudomonas bac- terial preparations are well suited for the prevention of salmonella growth on the surface of various surface materials used in above-mentioned spaces, such as metal (e.g. stainless steel), brick, tile, concrete, asphalt, rock material, plastic, wood and/or other materials.
  • the Pseudomonas bacterial preparation is well suited for the transparent of surfaces in storage spaces. The bacterial preparation is able to survive even in very harsh conditions, forming on the surface a biofilm inhibitive to salmonella.
  • the protective effect of the bacterial preparation is particularly good e.g. on asphalt surfaces, which are favourable to growth of salmonella.
  • a Pseudomonas bacterial preparation having an inhibitive effect on the growth of salmonella is applied to media.
  • the bacterial preparations are well suited for the treatment of desiccants used in broiler breeding plants, such as peat, to fight salmonella.
  • a preparation of lactic acid bacteria containing small -molecule compounds of a mo- lecular weight ⁇ 1000 Da which have an inhibitive effect on the growth of salmonella is applied to the surfaces to be treated.
  • Preparations of lactic acid bacteria are well suited for the prevention of salmonella growth on various surface materials used in above- mentioned spaces, such as metal (e.g. stainless steel), brick, tile, concrete, asphalt, plastic, wood and/or other materials.
  • lactic acid bacteria Especially the use of preparations of lactic acid bacteria is safe because it is generally allowed and approved in food industry. Preparations of lactic acid bacteria are particularly well suited for the treatment of desiccants, such as peat, used in broiler breeding plants.
  • Pseudomonas bacteria are gram-negative, aero- bic, mesophilic or psychophilic organisms. Their natural environments are soil, water and e.g. plant surfaces . Pseudomonas bacteria are not very demanding in respect of nutritives as they are able to synthetise most of their structural elements themselves. They are generally non-pathogenic, although certain Pseudomonas species and strains (e.g. P. aeru ⁇ inosa) may cause e.g. urinary tract infections. Some strains produce heat- resistant proteases and lipases, which may give rise to defects of structure and taste in pasteurised milk products.
  • Pseudomonas bacteria form compounds inhibiting the growth of other microbes.
  • examples of such compounds are phenazine antibiotics and siderophores .
  • Bacterial strains producing phenazine antibiotics are used as biopesticides in plant cultivation.
  • Siderophores iron carriers
  • a mi- crobe that produces siderophores is able to take in iron into its cells in this chelate form, but by binding the iron needed by other microbes it inhibits their growth.
  • Siderophores have been used for control of pathogenic microbes in environments like process spaces containing liquids, e.g. in fermentation vessels or fish breeding environments (EP 360568) .
  • Biofilm means a covering formed in suitable conditions and consisting of microbes and/or exopolysaccharides and proteins secreted by them, which can be used to cover a wide variety of surfaces.
  • a biofilm based on Pseudomonas bacteria providing protection against salmonella is used.
  • the non-pathogenic strain used has a good capacity to produce siderophores and other compounds inhibiting the growth of salmonella. If desirable, the sensitivity of the bacterial strains used in the procedure to the commonest disinfectants used by food industry and commerce can be determined. If necessary, it is possible to define disinfection conditions for the Pseudomonas strain for its elimination, e.g.
  • VTT-E-94557 Pseudomonas aureofaciens , Pseudomonas chlorora-phis (e.g. VTT-E91436 and VTT-E- 95581 ⁇ ) and Pseudomonas fluorescens (e.g. VTT-E-94558 and VTT-E-93443 T ) , preferably Pseudomonas marginalis.
  • the Pseudomonas bacterial preparation may con- sist of a culture solution with or without cells, cells, concentrated or diluted culture solution with or without cells, fractionated culture solution or a mi- crobicidal product completely or partially purified; e.g. in the form of an aqueous suspension.
  • the Pseudomonas bacterial preparation is used in concentrations of e.g. about 10 2 - 10 7 pmy/cm 2 , preferably about 10 4 - 10 s pmy/cm 2 ; if desirable, it is also possible to use considerably lower or higher concentrations, consider- ing the target and conditions of application, in order to fight salmonella.
  • Lactic acid bacteria are gram-positive micro- aerophilic rods or cocci, a typical characteristic of which is fermentative metabolism. Depending on the spe- cies, the main fermentation products besides lactic acid are acetate, carbon dioxide, ethanol, formiate and the like. Lactic acid bacteria are relatively demanding microbes and need a very rich culture medium to be able to grow. These bacteria and the fermentation phenomena caused by them are utilised in many traditional foodstuff processes. For this reason, they have a so-called GRAS (Generally Recognized As Safe) status. In most probiotic preparations, lactic acid bacteria are essential. Lactic acid bacteria often effectively prevent the growth of other microbes. The low pH resulting from the fermentation alone eliminates many rivals.
  • the main fermentation product lactic acid
  • bacterio- sines mi- crobicidal peptides and proteins
  • Most bacteriosines have a very narrow range of action, limiting their usability.
  • An exception is the macromolecular nisin, which is effective against most gram-positive bacteria and which has been approved as a food additive in many countries, Finland among others.
  • Nisin is also used together with a chelating agent, which expands its range of action, to eliminate bacteria from surfaces that come into contact with foodstuffs (WO 96/39842) .
  • Lactic acid bacteria in food and fodder industry is previously known. Lactic acid bacte- ria are a generally used in milk products, meat products, fermentation of vegetables and in bakery products and fodder preservation. Lactic acid bacteria are used in foodstuffs to fight pathogenic microbes, such as Listeria and Salmonella bacteria. However, lactic acid bacteria have not been used to fight Salmonella bacteria on surfaces in spaces and in media.
  • any commonly available lactic acid bacterium can be used which contains and/or produces micromolecular compounds hav- ing a molecular weight ⁇ 1000 Da and inhibiting the growth of salmonella.
  • Usable genera of lactic acid bacteria are Lactobacillus , Pediococcus and Lactococcus , which are described e.g. in the article Kandler, O. and Weiss, N. (1986) Genus Lactobacillus. Section 14. Regu- lar, Nonsporing Gram Positive Rods, un Bergey's Manual of Systematic Bacteriology, Vol 2. Ed. Krieg, N.R. and Holt, J.G., Williams & Wilkins, Baltimore, London, pp.
  • Particularly advantageous strains are the Lactobacillus plantarum or Pediococcus parvulus strains .
  • a lactic acid bacteria preparation may consist of a culture solution with or without cells, cells, a concentrated or diluted culture solution with or without cells, a fractionated culture solution or a completely or partially purified microbicidal product; e.g. in the form of an aqueous suspension.
  • the lactic acid bacteria preparation consists of a cell-free culture solution, a cell-free concentrated culture solution, a fractionated culture solution and/or a completely or partially purified microbicidal product.
  • Active compounds can be fractionated from the culture so- lution using gel filtration and ultrafiltration and different concentrating methods, such as evaporation or cold drying.
  • the treatment is carried out using an ultrafiltered supernatant of lactic acid bacteria which has been concen- trated to a 5-fold level by cold drying and/or using a diluted solution of it.
  • the present invention makes it possible to prevent salmonella contamination in premises and processes used in food industry.
  • the procedure of the in- vention promotes elimination of salmonella from contaminated spaces.
  • Fig. 1 illustrates the effect of the Pseudomonas strain on the growth of salmonella on an asphalt surface
  • Fig. 2 illustrates the effect of the same Pseudomonas strain on the growth of the Salmonella ty- phimurium EELA 144 strain in a peat substrate
  • Fig. 3 illustrates the effect of the ultrafil- tered fraction of a lactic acid bacterial strain on the growth of the Salmonella strain in peat .
  • Fig. 4 illustrates the effect of the same
  • this strain belongs to the collection of bacterial strains at the biological and foodstuff laboratory of VTT, the Technical Research Centre of Finland, was used. In a low-iron sub- strate, this strain has a high siderophoric production activity and an antimicrobial activity on the wild Salmonella strains used. Moreover, the strain has a low lipolytic and proteolytic activity. It tolerates the Alcoran and Salmonkill disinfectants but is sensitive to disinfectants such as e.g. Virkon, Hygicept and Klo- roforte .
  • test strains used were Salmonella infan- tis, Salmonella tvphimurium (EELA 144) and Salmonella enteritidis, isolated and delivered by the Finnish Institute of Foodstuffs and Veterinary Medicine (EELA) . Contamination was effected using a (1:1:1) mixture prepared from the test strains.
  • Aqueous suspension of the Pseudomonas strain was applied in laboratory conditions to asphalt, concrete and Leca brick surfaces in a concentration of 10 4 pmy/cm 2 . Part of the surface material was contaminated with a mixture of the test strains. In the tests, three different initial salmonella levels were used, a) , b) and c) : ⁇ 10 pmy/cm 2 , 10 2 pmy/cm 2 and 10 4 pmy/cm 2 . The fitness of both the Pseudomonas strain and the salmo- nella strains on the surfaces was monitored using plate culture and enrichment methods . By the enrichment method, even a very slight salmonella growth could be discerned.
  • Table 1 presents the results of the surface material tests. Inhibition of salmonella growth was very obvious with all the surfaces used in the test. The inhibiting effect in the case of concrete and Leca bricks was partly due to the high pH of the substrate/material, whereas in the case of asphalt, which is favourable to salmonella growth, the growth inhibition achieved by Pseudomonas inoculum was very distinct.
  • Fig. 1 illustrates the effect of the Pseudomonas marginalis strain on the growth of salmonella on an asphalt surface. On the control surfaces, salmonella grew well, reaching a level of 10 s pmy/cm 2 in a two-day sam- pie.
  • the Pseudomonas marginalis VTT-E- 94557 strain was used.
  • the test strain used was Salmonella typhimurium (EELA-144) .-
  • an autoclaved sterile peat sample was inoculated with both Pseudomonas and Salmonella strains .
  • the pH of the peat substrate was adjusted using ammonia.
  • Fig. 2 illustrates the effect of the Pseudomonas marginalis strain on the growth of the Salmonella typhimurium strain in a peat substrate (pH 7,4) .
  • the addition of Pseudomonas retarded the growth of salmonella during 5 days so that the microbial content of the Salmonella strain in the sample due to the Pseudomonas addition was 100 times lower than in a reference sample to which only salmonella had been added.
  • This experiment was carried out to test the effect of the substances produced by lactic acid bacteria on salmonella in a peat substrate as used in broiler breeding plants.
  • the salmonella inhibiting efficiency of the active compounds produced in the breeding experiments was tested using a culture solution filtrate purified by ultrafiltration (cut-off 1000 D) and concentrated (5x) by cold drying.
  • the reference sample used was lactic acid (10 % stock solution) .
  • an autoclaved sterile peat sample whose pH had been adjusted to the value 6 using ammonia, was inoculated with the Salmonella typhimurium strain and concentrated ultrafiltrate was added to it.
  • the starting levels of the salmonella inoculum were 10 2 and 10 4 pmy/g. Samples were taken after 0, 2, 4 and 7 days from the beginning of the test.
  • Fig. 3 illustrates the effect of a L. Plantarum filtrate on the growth of the S . typhimurium 144 strain.
  • the effect of the filtrate is apparent from the sample taken after 4 days.
  • Salmonella was found in all samples by the enrichment method, but direct plate culture did not reveal salmonella in the sample with a concentrated ultrafiltrate addition.
  • the inhibition of growth was not solely due to the effect of lactic acid. Even at a higher initial contamination level (10 4 pmy/g, Fig. 3a) , the inhibiting effect was visible as a retardation of salmonella growth.
  • the pH of the peat substrate was adjusted to the value 6 by adding CaC0 3 .
  • Some of the test groups were given Broilact" protective culture.
  • the salmonella content of the peat substrate was monitored by the plate culture technique (samples taken on days 1, 4 and 6) .
  • the chickens were killed and the Salmonella content of their caecum was determined quantitatively and by enriching.
  • the effect of the L. piantarum E-98 filtrate addition on the salmonella content in peat substrate is illustrated by Fig. 4a.
  • the salmonella quantity in the peat substrate in the reference group was 10 5 pmy g "1 .
  • Lactic acid bacterium filtrate E-98 reduced salmonella growth to the level of 10 3 pmy g "1 .
  • Ta- ble 2 presents the salmonella quantity in the caeca of the chickens at the end of the test.
  • the salmonella quantity in the reference samples was 10 9 pmy g "1 , 10 7 pmy g "1 in the E-98 filtrate groups and 10 1 pmy g "1 in the Broilact groups. Table 2.
  • Fig. 4b illustrates the effect of the Pseudomonas marginalis VTT-E-94557 addition on salmonella content in peat substrate.
  • the first-day (1 d) sample of the peat substrate of the reference group had a salmonella content of 10 s pmy g "1 and 5xl0 7 pmy g "1 at the end (6 d) of the test.
  • Pseudomonas VTT-E-94557 protective culture reduced salmonella growth in the 1 d sample to the level of 10 4 pmy g "1 and in the 6 d sample to 1 x 1x10 s pmy g "1 .
  • the salmonella quantity in the 1 d sample was lxlO 4 pmy g "1 and lxlO 5 pmy g "1 in the 6 d sample.
  • Table 3 presents the salmonella quantities in the chickens' caeca at the end of the test.
  • the salmonella quantity in the reference samples was 5x10 s pmy g "1 , 10 s pmy g "1 in the Pseudomonas E557 protective culture groups and 10 2 pmy g "1 in the Broilact groups.
  • Broilact protective culture for chickens inhibits the adherence of salmonella to the chickens' intestines even if the peat substrate has a high salmonella content.
  • Lactic acid bacterium filtrate E-98 and Pseudomonas marginalis E-94557 protective culture retard salmonella growth in siccative peat, and the salmonella quantities in caecal samples of the chickens in the corresponding groups were lower than in the reference group. No additive effect was detected between Broilact and the filtrate.
  • EXAMPLE 5 This experiment was carried out to test the mechanisms of action and bioactivity of the Pseudomonas strain toward different Salmonella strains. A cell-free filtrate of the Pseudomonas marginalis VTT-E-94557 bacterium was used in the test.
  • Target organism 0 mM FeS0 4 0.1 mM FeS0 4
  • NPN N-phenyl naphthyl amine
  • the test was carried out using a raw filtrate of Pseudomonas marginalis VTT-E-94557 culture 191, 192 and 198 (with only cells removed) , so in addition to siderophores it also contained all the other metabolic products (e.g. proteins) . Therefore, the results are based on the com- bined effect of a plurality of factors. The tests were performed on three independent samples and their respective substrates.
  • Fig. 5 illustrates the effect of an addition of siderophore filtrates on the permeabilisation of Salmonella. All three siderophore supernatants caused a weakening of the outer membrane of the S . typhimurium ATCC 13311 strain. This appears as a rise in the NPN uptake factor in samples treated with filtrates as compared with mere substrate treatment. The effect of the filtrates on the detergent and lysozy e sensitivity (1 % Triton X-100, 0,1 % SDS, lysozy e) of Salmonella cells was tested using cell lysis measurement.
  • Supernatant 192 renders Salmonella clearly more sensitive to the lysing effect of SDS but not to the effect of neutral detergent TX-100.

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Abstract

L'invention concerne un procédé de lutte contre la salmonelle dans les locaux et les processus de l'industrie alimentaire. Ce procédé consiste à traiter les espaces de production et/ou de transformation, et/ou le matériel de production et/ou de transformation avec une préparation bactérienne d'acide lactique possédant un effet inhibiteur sur la croissance de la salmonelle; et/ou à traiter les espaces de production et/ou de transformation, et/ou le matériel de production et/ou de transformation correspondants, sensiblement secs, avec une préparation bactérienne à base de Pseudomonas possédant un effet inhibiteur sur la croissance de la salmonelle.
PCT/FI1998/001034 1997-12-30 1998-12-30 Methode biologique d'elimination de la salmonelle de processus et de structures WO1999034679A1 (fr)

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Application Number Priority Date Filing Date Title
EP98962467A EP1043933A1 (fr) 1997-12-30 1998-12-30 Methode biologique d'elimination de la salmonelle de processus et de structures
AU17634/99A AU1763499A (en) 1997-12-30 1998-12-30 A biological method for elimination of salmonella from processes and structures

Applications Claiming Priority (2)

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FI974643 1997-12-30
FI974643A FI974643L (fi) 1997-12-30 1997-12-30 Biologinen menetelmä salmonellan eliminoimiseksi prosesseista ja rakenteista

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AU (1) AU1763499A (fr)
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003092382A1 (fr) * 2002-04-30 2003-11-13 Danish Institute For Fisheries Research Composition et procede pour reguler l'adherence microbienne a la formation de biofilms sur des surfaces
WO2012085255A3 (fr) * 2010-12-22 2013-01-24 Research Center Pharmaceutical Engineering Gmbh Composés organiques volatils issus d'antagonistes bactériens pour lutter contre la croissance bactérienne
US10264808B2 (en) 2015-12-29 2019-04-23 The United States Of America, As Represented By The Secretary Of Agriculture Use of phyllosphere associated lactic acid bacteria as biocontrol agents to reduce bacterial growth on fresh produce

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EP0333056A2 (fr) * 1988-03-14 1989-09-20 Microlife Technics, Inc. Procédé d'inhibition de pathogènes humains d'origine alimentaire et de prévention de la contamination microbienne dans des produits alimentaires réfrigérés en utilisant un lactobacillus
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WO2003092382A1 (fr) * 2002-04-30 2003-11-13 Danish Institute For Fisheries Research Composition et procede pour reguler l'adherence microbienne a la formation de biofilms sur des surfaces
WO2012085255A3 (fr) * 2010-12-22 2013-01-24 Research Center Pharmaceutical Engineering Gmbh Composés organiques volatils issus d'antagonistes bactériens pour lutter contre la croissance bactérienne
US10264808B2 (en) 2015-12-29 2019-04-23 The United States Of America, As Represented By The Secretary Of Agriculture Use of phyllosphere associated lactic acid bacteria as biocontrol agents to reduce bacterial growth on fresh produce

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