Patent Application
20080095882
An embodiment of the invention includes using a composition of the present invention to further protect a food product from the growth of gram-positive pathogenic bacteria, including but not limited to L. monocytogenes.
Another embodiment of the invention includes a method for treating a food product to give a predictable storage life. As used herein, predictable storage life refers to a known period in which the food product remains acceptable for human consumption. For example, predictable storage life includes applying Carnobacterium piscicola NCIMB 702852 or UAL26 to frankfurters to achieve a minimum storage life of 10 weeks at refrigeration temperatures.
An embodiment of the invention includes a method of treating fresh food by applying a microorganism, its pasteurized or unpasteurized fermentate, or combinations thereof to the food. In these embodiments of the invention the microorganism and its pasteurized or unpasteurized fermentate produce a predictable or controlled storage life.
Another embodiment of the invention is the use of selected natural bacteria to give a predictable storage life by treating the food with an amount of a natural bacterium that exceeds the level of natural contamination of the bacterium in the food.
In another embodiment of the invention, the composition applied to the food comprises one or more natural bacterial cultures, pasteurized or unpasteurized fermentate produced by the selected natural bacteria, or combinations thereof. In preferred embodiments of the invention, the food is treated with the combination of the natural bacteria and its pasteurized or unpasteurized fermentate. In the most preferred embodiment of the invention, the food is treated with the combination of selected natural bacteria and the bacterial pasteurized or unpasteurized fermentate of a selected natural bacterial culture.
In another embodiment of the invention, a method of predicting spoilage of a food product wherein a known spoilage bacterium is added to the food product to provide a bactericidal or bacteriostatic effect against L. monocytogenes.
As used herein, gram-positive pathogenic bacteria refer to, but are not limited to, Staphylococcus aureus, Enterococcus spp. and L. monocytogenes.
The procedures provided herein are applicable to all 13 known serotypes of L. monocytogenes. The compositions of the present invention are effective against strains of L. monocytogenes serotypes 1/2a, 1/2b, 3a and 4b. The spoilage bacteria used herein may be psychrotrophic. Specific spoilage bacteria that may be used include Carnobacterium piscicola NCIMB 702852 or UAL26, and Lactobacillus sakei UAL185. Combinations of two or more species may be used which are synergistic in action against the growth of L. monocytogenes.
The compositions of the present invention include one or more selected spoilage bacteria to achieve a predictable storage life and/or protect against the growth of pathogenic bacteria. The selected spoilage bacteria of the present invention include, but are not limited to, Carnobacterium piscicola NCIMB 702852, UAL26, CB1, CB2 and CB3, and Lactobacillus sakei UAL185. The method of the present invention includes the use of one or more natural bacterial cultures, homologous pasteurized or unpasteurized fermentate, heterologous pasteurized or unpasteurized fermentate, or combinations thereof. The natural bacterial cultures of the present invention are described above. A homologous fermentate refers to the culture supernatant of a single bacterial culture processed according to standard culture preparation techniques. A heterologous fermentate refers to the culture supernatant derived from a different bacterial culture processed according to standard culture preparation techniques. The homologous or heterologous fermentates may be pasteurized or unpasteurized, lyophilized or freeze dried. Two or more bacterial cultures may be mixed or added separately. Two or more bacterial fermentates may be mixed or added separately. A bacterial culture combined with one or more fermentates may be mixed, or added sequentially.
Combinations of two bacteria or pasteurized fermentate bacterial cultures may be used including combination of Carnobacterium piscicola NCIMB 702852 and/or Carnobacterium piscicola UAL26 and/or a heat resistant strain of Carnobacterium sp. and/or Carnobacterium divergens and/or Leuconostoc gelidum UAL187 and/or Lactobacillus sakei UAL185.
The compositions and methods of the present invention may also include additional additives or metabolites, which are bacteriocins or which are not bacteriocins, and not lactic acid or hydrogen peroxide.
Specific bacteriocins which may be used include bacteriocins of specific structure and molecular weight isolated from Carnobacterium piscicola NCIMB 702852 or UAL26, a heat resistant strain of Carnobacterium piscicola, Carnobacterium divergens, Leuconostoc gelidum UAL187 and Lactobacillus sakei UAL185. The bacteriocin may be produced by genetic engineering wherein the gene(s) encoding the bacteriocin is expressed from a microorganism. The spoilage bacteria used herein may be initially screened for high activity towards virulent L. monocytogenes. The spoilage bacteria are preferably selected such that the inhibitory activity of the bacteriocin is not affected by glutathione.
The packaged food product may be introduced with specific spoilage bacteria and stored under refrigeration conditions. During storage, the specific spoilage bacteria do not form carbon dioxide nor do they cause discoloration of meat. The procedure extends the “bloom” or red color of fresh meat products for a longer time than is observed with the resident spoilage bacteria. However, when color is used to determine storage life, the present invention extends the storage life of the product beyond that produced by the resident spoilage bacteria.
In another exemplary embodiment of the invention, the invention includes a method of preserving foods or beverages comprising the steps of adding to the food or beverage an effective amount of a bacterial culture of the present invention, alone or in combination with a fermentate, to the food or beverage. The inventors have found that an amount of 102 CFU/g or lower is not typically sufficient to compete with the existing adventitious microbial population. The inventors have found that greater than about 103 CFU/g or higher are sufficient or overcome the growth of the existing adventitious microbial population. One skilled in the art will recognize that the amount of adventitious bacteria in a food product is variable; in accordance with the present invention, the amount of the composition should be about ten times or more higher than the amount of adventitious spoilage or pathogenic bacteria. The known spoilage bacteria bacterium usually is added to the food product in an amount greater than the natural levels of spoilage bacteria in the food product, generally about 103 CFU to about 104 CFU per gram of food product. As used herein, pre-determined storage refers to the capability of controlling spoilage for a discrete period, at which point spoilage becomes evident. For example, bacteria can be applied to a food product to attain a spoilage period of 10 weeks, at which point off-odors or off-flavors, such as sourness, may occur. Within the 10-week period, the composition of the present invention controls spoilage by one or more of the following: by applying bacteria having a known spoilage period; by applying bacteria that produces one or more bacteriocins that kill or control spoilage bacteria.
In accordance with the present invention, the composition has the added benefit of controlling or killing pathogenic bacteria, including but not limited to L. monocytogenes.
The known spoilage bacterium may be introduced to the food product in any convenient manner. In one procedure, the surface of the food product may be introduced with the known spoilage bacterium in the form of a live bacterial culture, a pasteurized or unpasteurized fermentate or with a combination of live bacterial culture and pasteurized or unpasteurized fermentate, which may be from the same or a different bacterium.
Alternatively, the protective live bacterial culture, protective pasteurized or unpasteurized fermentate of the bacterial culture, mixture of protective live bacterial culture and protective pasteurized or unpasteurized fermentate of the bacterial culture, bacteriocin or mixture with live bacterial culture, may be introduced to the food product by mixing with the food product.
Any other desired method of application of a liquid or powder to a substrate may be used, including dipping, spraying, mixing, injecting, tumbling and incorporation into a plastic film.
The food product to which the present invention is applied may vary widely and may be a cooked or uncooked food product. For example, the food product may be a cooked or cured ready-to-eat meat product, including tissues from poultry, beef, pork, lamb, goat and seafood.
The food product also may be a whole cooked vegetable or plant or a chopped or comminuted cooked vegetable or plant. The food product may be a fresh whole uncooked vegetable or plant or a chopped or comminuted cooked vegetable or plant. The food product also may be a non-pasteurized or pasteurized cheese, a batter, a grain or an egg or liquid egg.
The food product may be in packaged form. For meat products, modified-atmosphere packaging (MAP) with elevated levels of carbon dioxide, including vacuum packaging, has been employed. Such packaging may be used herein. The modified atmosphere may have gases in the range of nitrogen ≦70%, oxygen close to 0%, carbon dioxide ≧20%, preferably nitrogen 60%, oxygen 0% and carbon dioxide 40%. Another modified atmosphere that would be preferred would be 100% carbon dioxide. Alternatively, a modified atmosphere of 220% carbon dioxide and ≧50% oxygen would also be applicable for this application, preferably 20% carbon dioxide and 80% oxygen.
The known spoilage bacteria and related materials discussed above also may be used as a sanitizing agent and/or aerosol to provide an environment hostile to L. monocytogenes. For example, the known spoilage bacteria may be applied to brine chiller water used to cool hot dogs from the smoke house of a hot-dog factory. Another possible application would be to apply the spoilage bacteria and related materials discussed above to a drain to inhibit the growth of L. monocytogenes.
This Example illustrates controlled spoilage by selected bacteria of regular BBQ frankfurters.
Freshly manufactured Regular BBQ frankfurters were inoculated by dipping in cultures of either Carnobacterium piscicola NCIMB 702852 or Leuconostoc gelidum UAL187. For the preparation of inocula, bacteria from frozen culture were subcultured twice in APT broth (Difco; Becton Dickinson) and incubated for 24 hours at 25° C. The cultures of Carnobacterium piscicola NCIMB 702852 or Leuconostoc gelidum UAL187 were standardized with sterile distilled water such that the dipped frankfurters were inoculated with preferably ≦103 per cm2. In the control samples, sterile water was substituted for the cultures. The inoculated frankfurters were dried on a sterile rack and vacuum packaged (2 per pack). The frankfurters were stored at 4° C. At the times indicated in
Samples were prepared for microbial analysis by excising a piece of frankfurter with flame sterilized scalpels. A ten-gram sample of frankfurter was placed in a sterile Stomacher bag, homogenized for 2 minutes with 90 ml of sterile 0.1% peptone water using a Colworth Stomacher 400 or similar. Bacterial numbers were enumerated by standard dilution and plating techniques. Total aerobic plate count was determined on Plate Count agar (PCA, Difco) incubated aerobically at 25° C. for 48 hours. Lactic acid bacteria counts were determined on Bacto APT agar (Difco) incubated anaerobically (BBL Anaerobic System with 5 to 10% CO2) for 48 hours. Carnobacterium piscicola numbers were determined by difference on Lactobacilli MRS agar (Difco) for 48 hours. Acetate inhibits or retards the growth of Carnobacterium. Numbers of Leuconostoc gelidum were determined by difference on APT with sucrose added.
Bacterial numbers were enumerated by standard dilution and plating techniques. Total aerobic plate count was determined on Plate Count agar (PCA, Difco) incubated aerobically at 25° C. for 48 hours. Lactic acid bacteria counts were determined on Bacto APT agar (Difco) incubated anaerobically (BBL Anaerobic System with 5 to 10% CO2) for 48 hours. Carnobacterium piscicola numbers were determined by difference on Lactobacilli MRS agar (Difco) for 48 hours. Acetate inhibits or retards the growth of Carnobacterium.
Bacterial numbers are reported as Colony Forming Units per gram (“CFU/g”).
Samples for sensory analysis were evaluated unheated at room temperature.
A trained panel was used to evaluate the sensory quality of the frankfurters throughout the storage period. The flavor of the inoculated frankfurters was compared with the control on a five point scale, 1=acceptable, 3=marginal, 5=unacceptable.
The results obtained are shown graphically in
This Example illustrates antimicrobial activity of two strains of Carnobacterium piscicola.
Three compatible strains of L. monocytogenes (List4, HPB65 and HPB642) were grown separately, centrifuged and washed three times with sterile 0.85% saline and resuspended in sterile 0.85% saline for use as the “Listeria” inoculum “cocktail”. L. monocytogenes CDC 7662 was grown separately and inoculated as a single bacterial culture. The lactic acid bacteria (Carnobacterium piscicola UAL26 or UAL26/8A) were grown separately, centrifuged and washed three times with sterile 0.85% saline and resuspended separately in sterile 0.85% saline for use as the “lactic” inocula.
Freshly prepared, regular frankfurters were obtained from a meat processor in Edmonton and they were inoculated by immersion in the inoculum containing either the washed Listeria cocktail or the single L. monocytogenes culture, and the lactic acid bacterium. The inoculated frankfurters were dried on a sterile rack and vacuum packaged. The following treatments were prepared:
Un-inoculated control. Dipped in sterile 0.85% saline.
Inoculated control. Dipped in 0.85% saline containing the L. monocytogenes CDC 7662 or the L. monocytogenes cocktail to give ˜1000 CFU of Listeria per cm2.
L. monocytogenes CDC 7662 or L. monocytogenes cocktail+Carnobacterium piscicola UAL26 to give 1000 CFU of Listeria and 10,000 CFU of Carnobacterium piscicola UAL26 per cm2.
L. monocytogenes CDC 7662 or L. monocytogenes cocktail+Carnobacterium piscicola UAL26/8A to give 1000 CFU of Listeria and 10,000 CFU of Carnobacterium piscicola UAL26/8A per cm2.
Two frankfurters from each treatment were aseptically transferred to a high barrier film plastic bag and vacuum packaged. The frankfurters were stored at 4° C. and sampled once per week (days 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70).
Samples were prepared for microbial analysis by excising a piece of frankfurter with flame-sterilized scalpels to give a sample with 10 cm2 surface area. The sample of the frankfurter was placed in a sterile Stomacher bag, homogenized for 2 minutes with 90 ml of sterile 0.1% peptone water using a Colworth Stomacher 400 or similar.
Bacterial numbers were enumerated by standard dilution and plating techniques. Total aerobic plate count was determined on Plate Count agar (PCA, Difco) incubated aerobically at 25° C. for 48 hours. Lactic acid bacterial numbers were determined on Bacto APT agar (Difco) incubated anaerobically (BBL Anaerobic System with 5 to 10% CO2) at 25° C. for 48 hours. Listeria monocytogenes counts were determined on PALCAM Agar Base (Oxoid, Unipath Ltd., England) supplemented with selective supplement (SR150E, Oxoid). Plates were incubated at 37° C. for 24 h and enumerated.
Bacterial numbers are reported as logarithms (“log10”) Colony Forming Units per gram (“LOG CFU/g”). The results obtained are shown graphically in
This example illustrates the impact of the growth of Carnobacterium piscicola and Leuconostoc gelidum on the sensory characteristics of vacuum packaged frankfurters.
The following laboratory-scale study was designed to investigate the sensory characteristics of Carnobacterium piscicola and Leuconostoc gelidum on vacuum packaged frankfurters inoculated under controlled conditions.
Two strains of Carnobacterium piscicola NCIMB 702852 and UAL26 and Leuconostoc gelidum UAL187 were investigated.
For the preparation of inocula, bacteria from frozen culture were subcultured twice in APT broth (Difco) over 24 hours at room temperature. Cultures were centrifuged (10,000 rpm for 10 min, at 7° C.) and pelleted cells were resuspended with 0.85% sterile saline and washed three times by centrifugation. The final cell pellet was resuspended in 10 mL of sterile 0.85% saline to a final concentration of 1×109 CFU per mL. Prior to dipping, a 10 mL aliquot of washed bacterial cells was added to 4 L of sterile 0.85% saline to provide an inoculum solution of 2.5×106 CFU per mL. Groups of 5 frankfurters were dipped into the inoculum suspension for 1 minute, drain dried, and vacuum packaged (high barrier, low O2 permeability bags). For control samples, frankfurters were dipped in 0.85% sterile saline without bacterial inoculum. Reference samples were frankfurters that had been dipped in a 0.85% saline solution and stored as described for treated samples.
Treated and control samples were placed into refrigerated 4° C. storage (monitored with a Delphi temperature recorder) for up to 12 weeks. Sampling of frankfurters for sensory evaluation was performed on day 0 and after 2, 4, 6, 7, 8, 10 and 12 weeks of storage.
Prior to sensory evaluation by a trained panel, frankfurters (4-5) were heated by placing in a saucepan containing 2 L of boiling tap water, which was immediately removed from the heat element and allowed to stand for 5 min (internal temperature approx. 83° C.). frankfurters were cut into 1.27 cm (0.5 inch) pieces and placed in coded foil-covered jars, and just prior to evaluation, heated for 15 min in a 200° F. (94° C.) oven (internal temperature approx. 66° C.).
Sensory evaluation was conducted by a group of 9 panelists trained over a three-month period, and was performed in sensory booths under dim red lighting using data collection software. Samples were evaluated for overall aroma intensity, meat flavor intensity, seasoned flavor, smoke intensity, sourness/acidity, off flavor and overall acceptability using a 15 cm unstructured line scale with 0=none (or very bland) and 15=extreme (or very strong). Between samples, palates were cleansed with crackers and a 1:1 dilution of 7-up.
All collected data were analyzed using the GLM of SAS version 6.12 (SAS Institute, 1996) and the Student Newman Keul's multiple range test was used to test for significant differences among treatments and storage times.
After 12 weeks of cold storage, frankfurters inoculated with Carnobacterium piscicola NCIMB 702852 and UAL26 were similar to control and reference samples for all characteristics. The results obtained are shown in
Based on sensory evaluations using a trained nine-member panel over the 12-week storage period, there were no significant adverse effects on aroma, off-flavors, sour intensity, or overall acceptability resulting from inoculation of frankfurters with Carnobacterium piscicola.
Although a few preferred embodiments have been shown and described, it will be appreciated by those skilled in the art that various changes and modifications might be made without departing from the scope of the invention. The terms and expressions in the preceding specification have been used herein as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding equivalents of the features shown and described or portions thereof, it being recognized that the scope of the invention is defined and limited only be the claims that follow.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA03/00986 | 6/27/2003 | WO | 00 | 3/14/2006 |
| Number | Date | Country | |
|---|---|---|---|
| 60391645 | Jun 2002 | US |
