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WO2003045172A1 - Conservation au moyen de bacteries lactiques - Google Patents

Conservation au moyen de bacteries lactiques Download PDF

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Publication number
WO2003045172A1
WO2003045172A1 PCT/AU2001/001549 AU0101549W WO03045172A1 WO 2003045172 A1 WO2003045172 A1 WO 2003045172A1 AU 0101549 W AU0101549 W AU 0101549W WO 03045172 A1 WO03045172 A1 WO 03045172A1
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Prior art keywords
admixture
bacteria
food product
producing
botulinum
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PCT/AU2001/001549
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English (en)
Inventor
Svetlana Rybka-Rodgers
Paul Peiris
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University Of Western Sydney
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Priority to AU2002223305A priority Critical patent/AU2002223305A1/en
Priority to PCT/AU2001/001549 priority patent/WO2003045172A1/fr
Publication of WO2003045172A1 publication Critical patent/WO2003045172A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B2/00—Preservation of foods or foodstuffs, in general
    • A23B2/70—Preservation of foods or foodstuffs, in general by treatment with chemicals
    • A23B2/725—Preservation of foods or foodstuffs, in general by treatment with chemicals in the form of liquids or solids
    • A23B2/729—Organic compounds; Microorganisms; Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B4/00—Preservation of meat, sausages, fish or fish products
    • A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
    • A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
    • A23B4/20—Organic compounds; Microorganisms; Enzymes
    • A23B4/22—Microorganisms; Enzymes; Antibiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B7/00—Preservation of fruit or vegetables; Chemical ripening of fruit or vegetables
    • A23B7/14—Preserving or ripening with chemicals not covered by group A23B7/08 or A23B7/10
    • A23B7/153—Preserving or ripening with chemicals not covered by group A23B7/08 or A23B7/10 in the form of liquids or solids
    • A23B7/154—Organic compounds; Microorganisms; Enzymes
    • A23B7/155—Microorganisms; Enzymes ; Antibiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/157—Lactis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/41—Pediococcus
    • A23V2400/427—Pentosaceus

Definitions

  • the invention relates to the use of bacteriocin-producing lactic acid bacteria for limiting C. botulinum growth and toxin production in .food products.
  • These food products are distinguished from other products by their preparation, and are typically prepared by vaccuum packing fresh or freshly prepared foods, pasteurising in water autoclaves, and rapidly cooling to chilled temperatures. Typical of these products is their enhanced sensory and nutritional quality. The sensory quality of these products is due to the minimal processing of raw materials, combined with mild to moderate pasteurisation and limited use of salt and other preservatives .
  • the microbiological quality and safety of these food products relies mainly upon refrigerated storage.
  • the pasteurisation temperatures which are applied are typically 65-95°C. These temperatures are sufficient for destroying most vegetative bacterial cells, however, they are too low to destroy bacterial spores such as C. botulinuir? spores. Further, C. jbotulinum has the capacity to form toxin at refrigerated temperatures and has been reported to grow and produce toxin at temperatures as low as 3°C.
  • a problem with the above described food products is that often they are exposed to abuse temperatures, i.e. temperatures greater than 4°C, and usually, 10°C or higher.
  • a further problem is that it is expensive and inconvenient to provide the refrigeration environment of 4°C or lower for storing these foods, particularly when even at these temperatures, safety from botulinum toxin cannot be warranted.
  • jbotulinum spores when cultured simultaneously with those spores.
  • the minimum numbers of the lactic acid bacteria required for direct antagonism of C. botulinum spores is described as ranging from 10 s -10 7 CFU/ml . As acknowledged in the document, these numbers are specific for the growth medium and initial load of C. botulinum spores used in the study. This study did not consider the growth or toxin production of non-proteolytic C. botulinum in food products, or growth or toxin production of non-proteolytic C. botulinum at refrigeration temperatures.
  • Jotulinum spores at higher incubation temperatures for example, at 15 and 30°C
  • lower incubation temperatures for example 4 and 10°C
  • the document states that the reason for this increased inhibition with increasing incubation temperature is that the lactic acid bacteria are under less stress and grow optimally (more dense and larger colonies were observed) at the higher temperatures, which consequently led to the production of more bacteriocins .
  • the study shows that complete inhibition of non-proteolytic C. botulinum spores can be obtained using as little as 10 2 CFU of lactic acid bacteria, contrary to an earlier study. This study did not consider the growth or toxin production of non- proteolytic C. jbotulinum in food products, or growth or toxin production of non-proteolytic C. botulinum at refrigeration temperatures.
  • Protection 56: 458-488 discloses a study of the capacity of lactic acid bacteria, specifically, P. pentosaceus 43200, L . plantarum Lb75, BN, and Lb592, and Lc . lactis 11454 to inhibit non-proteolytic C. botulinum spores in a model gravy system, by bacteriocin production and acidification.
  • the study was performed at 15, 25 or 35°C.
  • the document states that at 15°C, inhibition of C. botulinum spores was caused solely by acidification of the model gravy system caused by the lactic acid bacteria. Further in the only circumstance where bacteriocin production appeared to inhibit C. botulinum spores, the inhibitive effect could not be conclusively attributed to bacteriocin production.
  • the invention provides a method for determining whether a bacteriocin-producing bacteria would be capable of limiting toxin production of a non proteolytic C. botulinum strain in a food product at about 10°C.
  • the method comprises forming a culture of the bacteria and a non-proteolytic C. botulinum strain in the food product at t about 10°C, measuring the amount of bacteriocin in the culture at pre-determined intervals, and determining whether the measured amount of bacteriocin is at least about 15 AU of pediocin A per mL of culture, or 50 IU of nisin per mL of culture, within about 5 days of forming the culture.
  • non-proteolytic C. botulinum growth is increasingly inhibited with decreasing temperature.
  • the inventors believe that the reason for this increased inhibition is that at temperatures in the range of 10°C, non-proteolytic C. botulinum strains have increased sensitivity to bacteriocin. This is in contrast to earlier studies in which it was observed that at higher temperatures, for example, in the order of 30°C, there is increased inhibition of non-proteolytic C. botulinum growth.
  • These studies teach that inhibition of growth increases with higher temperatures because the bacteriocin producing lactic acid strain are capable of producing more bacteriocin at these higher temperatures.
  • bacteriocin in amounts of about 10 8 CFU/ml of culture of nisin producing lactic acid bacteria, and 10 9 CFU/ml of culture of pediocin A producing lactic acid bacteria, are necessary to produce a concentration of nisin and pediocin A of about 50-100 IU/ml and 20-35 AU/ml of culture, respectively, at 3-5 days after forming the culture.
  • concentration of nisin and pediocin A producing bacteria are significantly higher than those suggested for use in earlier studies.
  • concentrations of at least about 15 AU of pediocin A per mL of culture, or 50 IU of nisin per mL of culture, within about 5 days of forming the culture, are required for inhibition of growth and toxin production of a non- proteolytic C. jbotulirzum strain at 10°C, in a food product such as those described above, particularly, a REPFEP food product having a pH not less than 5.0.
  • bacteriocin producing lactic acid bacteria capable of producing concentrations of at least about 15 AU of pediocin A/ mL culture, or 50 IU of nisin/ mL culture, within about 5 days of forming a culture with non- proteolytic C. botulinum at 10°C, are capable of limiting toxin production of the strain, and accordingly, capable of providing a food product which can be stored at about 10°C, and that is adapted for reduced botulinum toxin production.
  • the invention provides a method for producing a food product adapted for limiting the production of a toxin by a non-proteolytic strain of C. botulinum.
  • the method comprises forming an admixture of a food product and bacteria for producing nisin for limiting production of the toxin, the bacteria in an amount sufficient for permitting the bacteria to produce at least 100 IU of nisin per ml of the admixture within about 5 days after forming the admixture, and storing the admixture at a temperature greater than 5°C but less than 15°C, to produce the adapted food product.
  • the admixture is typically stored at 10°C, to produce the adapted food product .
  • the invention provides a method for producing a food product adapted for limiting the production of a toxin by a non-proteolytic strain of C. jbotulinum.
  • the method comprises forming an admixture of a food product and bacteria for producing pediocin A for limiting production of the toxin, the bacteria in an amount sufficient for permitting the bacteria to produce at least 15 AU of pediocin A per ml of the admixture within about 5 days after forming the admixture, and storing the admixture at a temperature greater than 5°C but less than 15°C, to produce the adapted food product.
  • the admixture is typically stored at 10°C, to produce the adapted food product .
  • the food product is of the class of food products described above, including REPFEP food products.
  • the food product has a pH of about 5.0 of more, as these products are capable of supporting non- proteolytic C. botulinum growth and toxin production. At lower pH ranges, for example, those ranges observed in fermented products, non-proteolytic C. botulinum growth is inhibited by the acidified environment of the product.
  • the bacteria for producing nisin is admixed in an amount for producing a concentration of the bacteria in the admixture of at least about 10 7 bacteria/ ml admixture.
  • the bacteria for producing pediocin A is admixed in an amount for producing a concentration of the bacteria in the admixture of at least 10 8 bacteria/mL.
  • non-proteolytic C. botulinum growth and toxin production is reduced when the bacteria for producing pediocin A is admixed in an amount for producing a concentration of the bacteria in the admixture of 10 9 bacteria/ml admixture.
  • the C. botulinum spores and vegetative cells were present in an amount of 10 3 CFU/ml of food product, which is typical of the spore concentrations observed in C. botulinum contaminations.
  • the food product may be heated to pasteurise the food product, before forming the admixture.
  • Typical temperatures range between 60 and 95°C, for about 2 to 15 minutes . Examples of suitable methods are described in "Reference Code For An Extended Shelf-Life Cook-Chill System", NSW Health Pept . Capital and Infrastructure Services Branch, Sydney, 1998.
  • the admixture may be heated to pasteurise the food product in the admixture, before storing the admixture. Where the admixture is heated to pasteurise the admixture, it is important that the bacteria for producing pediocin A or nisin are protected from pasteurisation.
  • the bacteria for producing nisin and/or pediocin A may be encapsulated in protecting means for protecting these bacteria from pasteurisation, when the admixture is heated.
  • protecting means capable of protecting the bacteria is suitable, and in one example, the protecting means comprises a lipid, for example, a liposome, for encapsulating the bacteria.
  • a protecting means is a pouch for encapsulating the bacteria. The pouch comprises material for permitting the pouch to degrade during pasteurisation sufficient to release the bacteria from the pouch into the product after pasteurisation.
  • the pasteurised food product may be cooled, for example, to refrigerate the pasteurised food product, before forming the admixture .
  • Typical temperatures range between 1 and 50°C. Examples of suitable methods are described in "Reference Code For An Extended Shelf-Life Cook-Chill System", NSW Health Pept . Capital and Infrastructure Services Branch, Sydney, 1998.
  • the admixture is typically formed at a temperature greater than 5°C but less than 15°C, for example 10°C.
  • the temperature at which the admixture is formed may be higher.
  • the temperature may be as high as 50°C.
  • the temperature at which the admixture is formed may be as low as 1°C.
  • the bacteria for producing nisin and/or pediocin A may be freeze dried before forming the admixture. Methods of preparing freeze dried bacteria and for application of freeze dried bacteria are described herein.
  • any lactic acid bacteria capable of producing pediocin A or nisin at the amounts, and in the conditions described above, could be used in the invention.
  • these bacteria include P. pentosaceus 43200 and Lc. lactis 146. Other Lc .
  • lactis nisin-producing strains include 6F3 , NIZO 22186, NIZO R5, NDCO 497, NPCO 933, BB 24, N8 , SIK-83, NCFB 894, NCFB 497, NCK 400, LJH 80, CECT 539, INIA 415, PPC 496, 354, MG 1363, TAB50, TAB 50-M5 (mutant of TAB 50), A 164, ATCC 19649, IFO 12007, BFE 1500, F15876, ITAL 383, CNRZ 150, SBT 1212, CNRZ 150, 140, G 35, IO-l, ESI 515, R5 , 725, 1605, LL 34-1, IL 1403 and biovar diacetylactis UL 719.
  • pediocin producing strains include P. acidilacti PAC-1.0, E, F, H, M, CFR, K7 , NPPL, JPJ-23, SJ-1, L-7230, N5p.
  • a bacterial strain genetically modified for producing nisin and/or pediocin A could also be used, such as that described in Horn et al . 1999 Appl . Environ. Microbiol. 65 (10), 4443-4450 which is a nisin producing Lc . lactis F15786 which comprises a pediocin A producing gene from P. acidilactici 347.
  • the invention provides a food product adapted for limiting the production of a toxin by a non- proteolytic strain of C. botulinum, the food product being produced by the method of the second or third aspects of the invention.
  • the invention provides a method for preserving a food product from non proteolytic C. botulinum growth and toxin production comprising forming an admixture of a food product and bacteria for producing nisin for limiting production of the toxin, the bacteria in an amount sufficient for permitting the bacteria to produce at least 100 IU of nisin per ml of the admixture within about 5 days after forming the admixture, and storing the admixture at a temperature greater than 5°C but less than 15°C, to preserve the food product.
  • the invention provides a method for reducing the production of toxin by a non proteolytic strain of C. botulinum in a food product.
  • the method comprises forming an admixture of a food product and bacteria for producing nisin for limiting production of the toxin, the bacteria in an amount sufficient for permitting the bacteria to produce at least 100 IU of nisin per ml of the admixture within about 5 days after forming the admixture, and storing the admixture at a temperature greater than 5°C but less than 15°C, to reduce the production of the toxin in the food product.
  • the invention provides a method for preserving a food product from non proteolytic C. botulinum growth and toxin production.
  • the method comprises forming an admixture of a food product and bacteria for producing pediocin A for limiting production of the toxin, the bacteria in an amount sufficient for permitting the bacteria to produce at least 15 AU of pediocin A per ml of the admixture within about 5 days after forming the admixture, and storing the admixture at a temperature greater than 5°C but less than 15°C, to preserve the food product .
  • the invention provides a method for reducing the production of toxin by a non proteolytic strain of C. botulinum in a food product.
  • the method comprises forming an admixture of a food product and bacteria for producing pediocin A for limiting production of the toxin, the bacteria in an amount sufficient for permitting the bacteria to produce at least 15 AU of pediocin A per ml of the admixture within about 5 days after forming the admixture, and storing the admixture at a temperature greater than 5°C but less than 15°C, to reduce the production of the toxin in the food product.
  • the methods of the above described aspects of the invention comprise the further step of adding a bacteria for producing a further bacteriocin to the admixture.
  • the bacteria for producing a further bacteriocin may produce, for example, lactostrepcin, lacticin, dricin, lactococcin, diplococcin, acidophilin, acidolin, lactacin, acidophilucin, bavaricin, lactobrevin, caseicin, curvacin, bulgarican, lactobacillin, lacticin, gassericin, helveticin, lactolin, rutericin, sakacin, carnibacteriocin, piscicolin, carnocin, leuconocin, mesentrerocin and thermophilin. Examples of such bacteria are described in Table 6.
  • FIGURES Figure 1 Growth and bacteriocin production by fresh and freeze-dried Lc . lactis in TPGY broth at 10°C.
  • Figure 2. Growth and bacteriocin production by fresh and freeze-dried P. pentosaceus in TPGY broth at 10°C.
  • Figure 3. Inhibition of C. botulinum 17B (A) , C. botulinum 17B with Lc . lactis (B) , and C. botulinum type 17B with P. pentosaceus (C) at 5°C in TPGY broth.
  • Figure 4. Incubation of C. botulinum 17B (A), C. botulinum 17B with Lc . lactis (B) and C. botulinum 17B with P. pentosaceus (C) at 10°C in TPGY broth "-" negative for C. botulinum type B toxin, "+" positive for C. botulinum type B toxin.
  • FIG. 5 Incubation of C. botulinum 17B (A), C. botulinum 17B with Lc . lactis (B) and C. botulinum 17B with P. pentosaceus (C) at 15°C in TPGY broth.
  • Figure 6. C. botulinum 17B Sensitivity to pediocin A at 10°C (A), 30°C (B) , and nisin at 10°C (C) , 30°C (D) .
  • FIG 8. Survival of C. botulinum type 17B in Roasted Vegetable soup (initial pH 5.2, final pH 4.9) (A), co- incubated with Lc . lactis (final pH 4.3) (B) and P pentaceous (final pH 4.3 (C) at 10°C.
  • Figure 9. Survival of C. botulinum type 17B in Seafood Chowder soup (initial pH 6.2, final pH 5.3) (A), co- incubated with Lc . lactis (final pH 5.5) (B) and P pentaceous (final pH 5.5 (C) at 10°C.
  • FIG. 10 Survival of C. botulinum type 17B in Seafood Chowder soup (initial pH 6.4, final pH 5.2) (A), co- incubated with Lc . lactis (final pH 4.6) (B) and P pentaceous (final pH 5) (C) , and a mixture of Lc . lactis and P. pentaceous (final pH 4.5) (P) at 10°C.
  • the aim of this study was to demonstrate the ability of Lc . lactis and P. pentosaceus to grow and produce bacteriocins at 10°C, to observe the effect of inoculation level and freeze-drying.
  • Lc . lactis was supplied by Food Science Australia's Melbourne Laboratory, P. pentosaceus 43200 - by the ATTC. Lactic cultures were maintained at -40°C in sterilised 11% non-fat dry milk supplemented with 1% glucose and 0.3% yeast extract. Fresh cultures of P. pentosaceus and Lc . lactis were prepared by inoculating 20 ml of MRS and M17 agar respectively, growing overnight at 37°C, adding it to 500 ml of sterile broth and incubating at 37°C for 24h with gentle shaking. The cultures were centrifuged at 5000G for 10 min, washed in sterile water and centrifuged again. Pried cultures were prepared by resuspending in 5 ml of 1% glycerol and 12% skim milk solids and freeze-drying at - 40°C for 18 hours.
  • Bacteriocin sensitivity and detection Bacteriocin preparations of known concentrations were: nisin (Sigma Chemicals - 2.5% in milk solids and sodium chloride, equivalent to 10 6 IU/ml) ; pediocin A partially purified preparation with activity of 3.083 AU/mg was supplied by University of Bologna, Italy.
  • Standard solutions were prepared by dissolving pediocin A in sterile water and filter-sterilising; dissolving nisin in TPGY broth (g/1 : Pancreatic digest of soybean meal 3.0, Pancreatic digest of casein 17.0, Sodium chloride 5.0, Pi- potassium phosphate 2.5, Glucose 2.5, Yeast Extract 2.5 adjusted to pH2 with HC1) and autoclaving at 121°C for 15 min.
  • Well diffusion assay was used to detect bacteriocin production.
  • a second decimal dilution (1 ml) of the overnight indicator strain Micrococcus luteus in TPGY broth was mixed with 75 ml of TPGY agar at 45°C for seeding and pouring the plates (20 ml each) . Seeded agar was overlayed with unseeded agar to prevent surface colonies spreading.
  • Wells of 0.5 cm diameter were made in the agar and filled with 50 ⁇ l of the filter sterilised samples. Sealing of the bottoms of the wells with agar was omitted. The plates were kept under refrigeration overnight to allow the samples' diffusion through the agar and were then incubated for at 30°C for 24 h aerobically.
  • the samples were 1:2 diluted and MID (minimum inhibitory dilution) was established.
  • MID minimum inhibitory dilution
  • a standard solution of the bacteriocins 100 IU nisin and 35 AU pediocin A were used to monitor the sensitivity of the detection.
  • TPGY adjusted to pH 5 was tested to ensure the absence of inhibition by low pH.
  • Enzyme sensitivity of the bacteriocins was demonstrated by making additional wells (3 mm in diameter) at the distance of 3 mm from the wells with the standard solutions and samples. These wells were filled with 30 ⁇ l of 10 mg/ml of pronase E (Sigma - Aldrich) , protease enzyme powder (Novo Nordisk A/S) and trypsin (TPCK treated from Bovine
  • lactis (10 8 , 10 7 , 10 6 and 10 5 cfu/ml) and P. pentosaceus (10 8 and 10 7 cfu/ml) were inoculated into 100 ml bottles of freshly autoclaved and rapidly chilled to 10°C TPGY broth. Inoculation and hermetic sealing of the bottles was carried out in the COY Laboratories anaerobic chamber (85% N 2 , 5% H 2 , 10% C0 2 ) in order to create an anaerobic headspace atmosphere. The bottles were incubated at 10°C for eight days.
  • the state of the culture did not affect the bacteriocin-producing ability at higher inoculums.
  • nisin produced by 10 s cfu/ml of freeze-dried Lc was detected on the eighth day instead of the fifth day by the fresh culture. It should be noted that these levels were close to the sensitivity limit of the MIC method.
  • the actual titre can range between 0.75 X and 1.5 X, where X is the obtained titre.
  • Lc . lactis and P. pentosaceus were able to grow and produce bacteriocins at 10°C. Bacteriocin production was associated with the culture growth reaching high population levels - 10 8 and 10 9 cfu/ml correspondingly. We showed that a higher cell density produced higher levels of bacteriocins.
  • Pediocin A production by P. pentosaceus depended on the initial inoculum.
  • Lc . lactis populations and nisin titre were similar by the fifth day of storage amongst the inoculums tested. Freeze-drying of the cultures did not affect their bacteriocin - producing abilities .
  • the aim of the study was to enumerate C. botulinum and lactic acid bacteria populations and detect toxin and bacteriocin production at the same time. Materials And Methods. a. Microbial cultures.
  • Microbial cultures were obtained from the sources listed in Table 2. The cultures were activated by inoculating in the medium based on reconstituted skim milk (RSM: 9.5% skim milk powder, 0.5% yeast extract, and 2% glucose) . The cultures were maintained on MRS (OXOID) plates. For long term storage, the original cultures were diluted 1/10 with 12.5% RSM and 1% glycerine and dispensed into sterile o
  • TPGY tryptose peptone yeast extract glucose broth
  • OXOID Tryptone Soy Broth
  • the degree of sporulation was estimated by microscopic observation of the suspension stained with crystal violet.
  • the spores were centrifuged (2160 x g, 20 min) and washed eight times with sterile distilled water to minimise residual toxin and was then re-suspended in 1 ml of sterile distilled water. Cell lysis was induced by frequent ultrasonic oscillation between the washes. The pellet was resuspended in 1 ml of sterile distilled water and stored at - 40°C. Prior to use the spores were heat- shocked at 60°C for 30 min.
  • each sample was placed on ice whilst in the chamber and used immediately for microbiological analysis. A portion of each sample (about 10 ml) was centrifuged (2160 x g, 10 min, 5°C) , filter sterilised (0.20 microns pore size, Ministart single use filter unit, Sartorius) and stored at -20°C for toxin and bacteriocin detection. All trials were repeated three times.
  • C. botulinum could not be differentiated from Lc . lactis and P. pentosaceus on TPGY or RCPB agar on the basis of their colony morphology.
  • Salicin Tryptone Soy Agar STSA was devised for elective enumeration of C. botulinum type B, which is capable of digesting salicin with production of gas and acid.
  • Lc . lactis and P. pentosaceus cannot ferment salicin and did not grow on STSA.
  • STSA was prepared by adding 10% w/w salicin (Sigma) to Bacto Tryptic Soy Broth without dextrose (DIFCO) .
  • a well diffusion assay was used (sealing of the bottoms of the wells with agar was omitted) with Micrococcus luteus as an indicator strain.
  • TPGY agar 75 ml, 45°C was seeded with M. luteus incubated overnight in TPGY broth at 30°C (1 ml of 10 "2 dilution) .
  • Plates with cooled seeded agar (20 ml) were overlaid with sterile agar (7 ml) to prevent the spreading of colonies.
  • Wells (0.5 cm in diameter) made with a core borer were filled with 50 ⁇ l of the filter- sterilised samples.
  • the plates were kept under refrigeration overnight to allow diffusion of the sample through the agar, then incubated aerobically at 30°C for 24 h. In consecutive trials the samples that produced a clearing zone were serially diluted (1:2) and the MID (minimum inhibitory dilution) was established. At each determination, standard solutions of nisin (100 U/ml, Sigma) and pediocin A (35 AU/ml) were used to monitor the sensitivity of detection. TPGY adjusted to pH 5 and all control treatments inoculated with C. botulinum alone were tested to ensure the absence of inhibition by other factors .
  • Toxicity of 'the samples (2 ml) was tested using a C. botulinum BoNT/B in vitro bioassay (Rhone-Diagnostics Technologies Ltd) according to manufacturer's instructions. Samples with a net absorbance of 0.3 or higher (430 nm against a 630 nm reference) were considered positive for botulinum toxin. As recommended for non- proteolytic strains, trypsin activation was tested.
  • TPCK treated trypsin (0.4 mg) from bovine pancreas (Sigma T- 1426) was dissolved in 1 ml sterile distilled water. This solution (0.4 mg/ml) was added to the samples (2 ml) and incubated for 30 min at 37°C. Trypsin inhibitor type I-S from soybean (Sigma 9003) was added at ten times the concentration of the trypsin. Results and Discussion.
  • the growth rate of interacting cultures and the rate of diffusion of inhibitory substances through the agar have an impact on the inhibition pattern.
  • the inhibition zones reported by Okereke and Montville (1991a) were smaller than the ones observed in this study. If the other strain in the cocktail was more resistant to inhibition than 17B and/or had a different growth pattern, it could determine the size of the clearing zones.
  • Lc . lactis 146 and P. pentosaceus 4300 were co-incubated with C. botulinum 17B in TPGY broth at 5, 10 and 15°C for 31, 14 and 10 days respectively ( Figures 3, 4, 5 B,C) .
  • Lc . lactis 146 is a nisin producing strain
  • P. pentosaceus 4300 produces pediocin A. The sensitivity of M. luteus, the indicator strain, to nisin was confirmed as 100 IU/ml and coincided with the minimum inhibitory concentration for C. botulinum 17B (unpublished data) .
  • lactis 146 is required to produce >100 IU/ml of nisin and 10 9 CFU/ml of P. pentosaceus 4300 - 35 AU/ml of pediocin A at 10°C (unpublished data) .
  • the inoculums of lactic the acid bacteria strains were chosen in order to achieve these population levels during incubation.
  • lactis 146 grew from 5 xlO 5 to" above 10 8 CFU/ml within 10 days, which coincided with the production of a detectable level of nisin (>100 IU/ml) . It did not reach 10 8 CFU/ml at 5°C and nisin was not detected at this temperature.
  • P. pentosaceus 4300 grew from 10 7 to above 10 9 CFU/ml within 6 days at 15°C and within 10 days at 10°C. It did not grow at 5°C. M. flavus was not sensitive to the levels of produced pediocin A in all trials. Okereke and Montville (1991a) also could not detect pediocin A production by P. pentosaceus 4300 at 4 and 10°C using L . sake 15521 as an indicator strain.
  • pentosaceus 4300 indicated the bacteriocin - mediated nature of inhibition and low likelihood of an impact on sensorial qualities of foods.
  • a number of strategies can be employed to accelerate inhibition: the addition of substances inducing bacteriocin production; synergetic combination of different bacteriocins by the combination of species; or production of different bacteriocins by a genetically engineered species.
  • STSA agar was found suitable for elective enumeration of C. Jbotulinum type B in co-incubation trails.
  • C. botulinum colonies on STSA were cream in colour, approx. 1.5 mm in diameter and gas production was often observed.
  • Comparison of C. botulinum counts on STSA and TPGY agar from 32 enumerations of control trials (without lactic acid bacteria) were not significantly different (one factor ANOVA, 1% significance level, data not shown) .
  • the population of C. botulinum 17B incubated without lactic acid bacteria as a control treatment was static at 5°C, increased to 2xl0 4 CFU/ml at 10°C and 9x10 s CFU/ml at 15°C ( Figure 3, 4, 5 A) .
  • the C. Jbotulinuin 17B growth rate was lower than predicted by Gomperts and Baranyi models or Food MicroModel (FMM) - the increase in populations to 10 6 -10 7 CFU/ml within the first five days at 10-15°C and within 25 days at 5°C. Furthermore, the rise in temperature from 5 to 10°C resulted in only a 1.5 log increase in C. botulinum population during 10 days of incubation, unlike the six log increase predicted by the FFM model .
  • the FMM used a cocktail of spores, in which the 17B and 202F strains were included. Different non-proteolytic strains of C. jbotulinum have a different growth pattern. Type F grew more frequently from spores than type B. We observed that this type had a higher growth rate than Beluga and type 17B (data not shown) . This could account for the higher growth rates predicted by the model .
  • Inhibition zones produced by simultaneous antagonism between non-proteolytic C. botulinum (10 4 CFU/ml) and lactic acid bacteria on buffered TPGY agar C. botulinum C. botulinum C. botulinum Beluga 17B 202F
  • Pediococcus >20 3 ⁇ 2 >20 0.5 ⁇ 2 >20 1 ⁇ 2 pentosaceus
  • Lactic acid bacteria bacteriocin-producing cultures in commercial refrigerated soups was tested and for the first time successful inhibition of psychrotrophic C. botulinum was demonstrated.
  • the strain C. botulinum 17B was chosen as the most resistant to inhibition amongst non-proteolytic type E (Beluga) and 202F.
  • P. pentosaceus 43200 was supplied by ATCC, Lc. lactis 146 - by Food Science Australia (Melbourne Laboratory) , non- proteolytic C. botulinum strains 17B, Beluga (type E) and 292F - by Food Science Australia (Sydney Laboratory) , Micrococcus luteus - by University of Western Sydney. Lactic acid bacteria cultures were freeze-dried and C. botulinum spore were prepared as described herein.
  • Bacteriocin sensitivity of C. botulinum Bacteriocin sensitivity of vegetative cells and spores of C. botulinum 17B was tested by seeding TPGY agar supplemented with 0.01% cycteine - HCl (Sigma) and different concentration of bacteriocins. A 50% decrease in the number of colonies in comparison to the control, which had no nisin, was considered as a sensitivity threshold after incubation anaerobically at 10 and 30°C for 20 days and 48 h correspondingly.
  • the spore suspension used for seeding (1 ml of 1/10 dilution per plate) was prepared by dissolving 100 ⁇ L of the spore stock in 10 ml of sterile water and heating it (60°C, 30 min) .
  • Vegetative C. botulinum cells were prepared by inoculating 100 ⁇ L of the spore stock in 200 ml of freshly prepared TPGY broth, heating (60°C, 30 min) and incubating for 24 h at 37°C in COY Laboratories anaerobic chamber (N 2 85%, H 2 10% and C0 2 5%) .
  • the culture was examined microscopically to ensure the vegetative state of the cells. This preparation (1 ml of 10-3 dilution per plate) was used for seeding. All manipulations were carried out in an anaerobic chamber.
  • Standard bacteriocin solutions were prepared by the following method: pediocin A preparation was dissolved in distilled water and filter-sterilised; nisin (Sigma Chemicals, 2.5% in milk solids and sodium chloride, 10 6 IU/g) was dissolved in TPGY broth adjusted to pH 2 with HCl and autoclaved at 121°C for 15 min. The trials were repeated three times.
  • STSA Salicin Tryptone Soy Agar
  • DIFCO Bacto Tryptic Soy Broth without dextrose
  • P. pentosaceus and Lc . lactis were enumerated on MRS agar and M17 agar (OXOID) respectively and incubated at 30°C for 48 hours aerobically. A pour plates technique was used. In the trials with mixed cultures these two species were enumerated on Tryptone Soy agar without dextrose (DIFCO) supplemented with 1% w/w of filter sterilised maltose (Sigma) . Both P. pentosaceus and Lc . lactis ferment maltose. The identities of the colonies were confirmed by microscopic observation of Gram-stained smears .
  • each sample (about lOg) was centrifuged (2160 x g, 10 min, 5°C) , the supernatant was filter sterilised (0.20 microns pore size, Ministart single use filter unit, Sartorius) and stored at -20°C for toxin and bacteriocin detection.
  • a sample (10 g) of each non-inoculated commercial product was enumerated on all media described above as a control and on Plate Count Agar (OXOID) to enumerate background microflora. All trials were repeated three times.
  • a well diffusion assay was used (sealing of the bottoms of the wells with agar was omitted) with Micrococcus luteus as an indicator strain.
  • TPGY agar 75 ml, 45°C was seeded with M. luteus incubated overnight in TPGY broth at 30°C (1 ml of 10 "2 dilution) .
  • Plates with cooled seeded agar (20 ml) were overlaid with sterile agar (7 ml) to prevent the spreading of colonies.
  • Wells (0.5 cm in diameter) made with a core borer were filled with 50 ⁇ l of the filter- sterilised samples.
  • TPGY adjusted to pH 5 and all control treatments inoculated with C. botulinum alone were tested to ensure the absence of inhibition by other factors.
  • Toxicity of the samples (2 ml) was tested using a C. botulinum BoNT/B in vitro bioassay (Rhone-Diagnostics Technologies Ltd) according to manufacturer's instructions. Samples with a net absorbance of 0.3 or higher (430 nm against a 630 nm reference) were considered positive for botulinum toxin. Trypsin activation was tested.
  • TPCK treated trypsin (0.4 mg) from bovine pancreas (Sigma T- 1426) was dissolved in 1 ml sterile distilled water. This solution (0.4 mg/ml) was added to the samples (2 ml) and incubated for 30 min at 37°C. Trypsin inhibitor type I-S from soybean (Sigma 9003) was added at ten times the concentration of the trypsin. Results and Discussion.
  • Pediocin A - producing P. pentosaceus and nisin - producing Lc . lactis were added to the soups in order to inhibit the growth of C. botulinum 17B (10 3 cfu/ml) and prevent toxogenesis.
  • Three soups representing different food categories - seafood (seafood chowder) , vegetables (roasted vegetable soup) and meat (chicken gumbo) - were selected for the co-incubation trials.
  • C. botulinum population remained static in the chicken gumbo ( Figure 7A) and roasted vegetable (Figure 8A) soups. It increased to 10 8 cfu/g in seafood chowder ( Figure 9A) . No background microflora were detected in these products.
  • P. pentosaceus (10 7 cfu/g) and Lc . lactis (10 6 CFU/g) populations increased slightly during incubation in all products except P. pentosaceus in the roasted vegetable soup. The ability of these cultures to produce bacteriocins at 10°C is demonstrated herein. However, in all trials P. pentosaceus populations were not high enough ( ⁇ 10 9 cfu/ml) to produce 35 AU/ml of pediocin A, the minimum sensitivity level for M. luteus . Consequently, pediocin A was not be detected in this study. However, it did not exclude the possibility of its production at lower concentrations .
  • Co-incubation did not result in reduction of C. botulinum in chicken gumbo and roasted vegetable soups during 15 days at 10°C ( Figure 7, 8) . These products had a low pH range close to pH 5. Most likely the spores did not germinate in the controls and co-incubated samples: no toxin was detected after 10 days of storage; the populations of C. botulinum was static during incubation; spore counts were not significantly different (data not shown) to total C. botulinum populations (vegetative cells and spores) .
  • botulinum were reduced to undetectable levels by the ninth day in all co-incubation trails. Neither of bacteriocins reached detectable level in mixed culture trials. Gas formation in the control and sample showed the absence of any visual signs of spoilage in the sample preserved with the protective cultures .
  • Toxicity of the samples is the indication of the efficiency of inhibition.
  • Non-proteolytic C. botulinum can grow without toxin production in sous vide beef.
  • the control was negative for toxin type B on the fifth day and became positive on the eighth ( Figure 10) .
  • the speed of toxin production was similar to that in observed in TPGY broth.
  • All co-incubated samples were negative on the ninth day of incubation at 10°C.
  • prevention of toxogenesis through the application of protective cultures was demonstrated. High inoculums were needed, this explained the inability of 10 4 CFU/g P. pentosaceus to prevent toxogenesis in sous vide beef with gravy at 10°C (Crandall et al . , 1994).
  • Example 3 showed successful inhibition of non-proteolytic C. botulinum 17B in seafood chowder by nisin-producing Lc . lactis (10 7 CFU/ml) and pediocin A - producing P. pentosaceus (10 8 cfu/ml) after 10 days of incubation at 10°C.
  • lactis (10 7 CFU/ml)
  • pediocin A - producing P. pentosaceus (10 8 cfu/ml) after 10 days of incubation at 10°C.
  • the aim of this study was to demonstrate the effect of these protective cultures in a wide range of commercial sous vide products and develop a protocol for practical application.
  • lactis produced 300-400 IU/g of nisin in singular culture and 100 IU/g or below in mixed cultures trials.
  • Pediocin A production by P. pentosaceus did not reach detectable level of 35 AU/g. This did not exclude it reaching 15 AU/g - the inhibition level for C. botulinum.
  • the final pH levels of the samples with protective cultures were lower than the control samples.
  • C. botulinum Active growth of C. botulinum is a good indicator of a successful outcome. This preservation technique was effective in less preserved catering products, which contributed to the significant improvement of their safety. It also provides an opportunity to increase storage temperature and save on energy costs.
  • the performance of mixed cultures was similar to that of singular cultures.
  • Other ways of enhancing inhibition are incorporating into recipes the ingredients with antibacterial properties, which can act in a synergetic relationship with the protective cultures, or the ingredients enhancing bacteriocin production such as glucose, yeast extract, biotin and others.

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Abstract

L'invention concerne l'utilisation d'une bactérie produisant une bactériocine destinée à limiter ou à réduire la production d'une toxine par une souche non protéolytique de C. botulinum.
PCT/AU2001/001549 2001-11-29 2001-11-29 Conservation au moyen de bacteries lactiques WO2003045172A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008036033A1 (fr) * 2006-09-19 2008-03-27 Scania Cv Ab (Publ) Moyeu pour frein à disque, frein à disque et véhicule
US9277763B2 (en) 2013-06-27 2016-03-08 Starbucks Corporation Biopreservation methods for beverages and other foods
CN108606049A (zh) * 2018-05-09 2018-10-02 北京农学院 一种薄荷map包装的保鲜方法
US10538818B2 (en) * 2013-03-13 2020-01-21 Dsm Ip Assets B.V. Methods and kits to determine the sensitivity of strains of Lactococcus lactis bacteria to phage infection

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4584199A (en) * 1983-06-10 1986-04-22 Aplin & Barrett, Ltd. Antibotulinal agents for high moisture process cheese products
US4597972A (en) * 1983-06-10 1986-07-01 Aplin & Barrett, Ltd. Nisin as an antibotulinal agent for food products
US5573800A (en) * 1989-02-21 1996-11-12 Viskase Corporation Antimicrobial composition for surface treatment of foodstuffs
WO2001005254A2 (fr) * 1999-07-14 2001-01-25 Rhodia Inc. Composition antibacterienne contre les bacteries gram positives dans les applications alimentaires

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4584199A (en) * 1983-06-10 1986-04-22 Aplin & Barrett, Ltd. Antibotulinal agents for high moisture process cheese products
US4597972A (en) * 1983-06-10 1986-07-01 Aplin & Barrett, Ltd. Nisin as an antibotulinal agent for food products
US5573800A (en) * 1989-02-21 1996-11-12 Viskase Corporation Antimicrobial composition for surface treatment of foodstuffs
WO2001005254A2 (fr) * 1999-07-14 2001-01-25 Rhodia Inc. Composition antibacterienne contre les bacteries gram positives dans les applications alimentaires

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
MAZZOTTA ET AL.: "Nisin-resistant (NisT) listeria monocytogenes and NisT clostridium botulinum are not resistant to common food preservatives", JOURNAL OF FOOD SCIENCE, vol. 65, 2000, pages 888 - 890 *
SCOTT ET AL.: "Effect of nisin on the outgrowth of clostridium botulinum spores", JOURNAL OF FOOD SCIENCE, vol. 46, pages 117 - 120, 126 *
SCOTT ET AL.: "Temperature, pH and spore load effects on the ability of nison to prevent the outgrowth of clostridium botulinum spores", JOURNAL OF FOOD SCIENCE, vol. 46, pages 121 - 126 *
SOMERS ET AL.: "Further studies on the antibotulinal effectiveness of nisin in acidic media", JOURNAL OF FOOD SCIENCE, vol. 46, 1981, pages 1972 - 1973 *
TAYLOR ET AL.: "Antibotulinal properties of nisin in fish packaged in an atmosphere of carbon dioxide", JOURNAL OF FOOD PROTECTION, vol. 53, 1990, pages 953 - 957 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008036033A1 (fr) * 2006-09-19 2008-03-27 Scania Cv Ab (Publ) Moyeu pour frein à disque, frein à disque et véhicule
US10538818B2 (en) * 2013-03-13 2020-01-21 Dsm Ip Assets B.V. Methods and kits to determine the sensitivity of strains of Lactococcus lactis bacteria to phage infection
US9277763B2 (en) 2013-06-27 2016-03-08 Starbucks Corporation Biopreservation methods for beverages and other foods
CN108606049A (zh) * 2018-05-09 2018-10-02 北京农学院 一种薄荷map包装的保鲜方法

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