Technical Field The present invention relates generally to a liquid smoke composition. Liquid smoke has traditionally been used to color and flavor edible food stuffs. More particularly, the present invention relates to application of a liquid smoke composition to meat to affect biological activities including reducing and inhibiting host organism infestation, such as microbial infestation, particularly bacterial, and inhibiting or preventing oxidation.

Background of the Invention

As is well known to those in the meat processing field, contamination and spoilage of meat products by host organisms, especially microorganisms, have been the subject of great concern and attention. Pathogenic microorganisms in particular have been a major problem in food processing. Although contamination of meat such as from animal carcasses during slaughtering procedures is undesirable, it has been viewed as unavoidable in the

conversion of live animals to meat for consumption. While the inner surfaces of animal carcasses are essentially sterile, contamination of meat from the carcasses often occurs during slaughtering of animals and removal of their hides. Initial contamination is sometimes referred to as primary contamination or infestation. Further contamination can occur during processing and transport of meat from the animals.

Recently, foodborne outbreaks of pathogenic microorganisms such as Escherichia coli ( ⁿ E. coli " ) , Salmonella typhimuri m and Listeria monocytogen.es ("L. monocytogenes") have caused great concern to the public and food packers, and such microorganisms are believed to be a major health threat. Consumption of food infested with such bacteria can result in sickness, and death in a worst case scenario. The threat and awareness of pathogenic microorganisms as they relate to meat products for ultimate consumption has focused attention on establishing microbiological guidelines for meat products to minimize or eliminate the level of microbial contamination or infestation.

In response to recent outbreaks of E. coli and L . monocytogenes, the Food Safety and Inspection Service

(FSIS) of the United States Department of Agriculture stated that regardless of the source of the microorganisms present in a finished food product, ready-to-eat products such as fully cooked, uncured meat patties must be free of all pathogenic bacteria. Additionally, the FSIS

identified Salmonella, I*, monocytogenes and E. coli as the organisms to be concerned with regarding the microbiological safety of ready-to-eat products.

Various methods have been developed and utilized in an effort to reduce, and to inhibit microorganisms on meat from animals. To reduce the number of microorganisms present on the carcasses of animals, for instance animals recently slaughtered, it has been common to utilize a washing treatment whereby whole or partial animal carcasses are washed. Chlorinated water, cold and hot water have been utilized as wash treatments for animal carcasses to reduce the number of microorganisms present. Organic acids such as acetic, ascorbic, citric, formic, lactic and propionic acids have also been utilized in the past in wash treatments on animal carcasses as a sanitizer to reduce the number of microorganisms present on the animal carcasses. The use of organic acids, however, is performed at the risk of discoloring the meat and imparting an acidic odor to the meat. Furthermore, some organic acids can promote protein or lipid breakdown and subsequent formation of undesirable end products through acid hydrolysis of myofibrilla or sarcoplasmic proteins.

Acetic acid, in particular, has been the subject of attention regarding its potential effectiveness in reducing the number of microorganisms present on meat surfaces. It has been found, however, that practical limitations exist regarding utilizing acetic acid to reduce the number of microorganisms present on meat

surfaces in that the surface of meat treated with acetic acid can turn a brownish yellow color and obtain a strong acetic acid odor, depending on the concentration of the acid utilized and the amount of time of the application. Lactic acid has also shown an ability to decrease the number of microorganisms present on meat surfaces; however, the benefits of utilizing lactic acid are offset by greater discoloration of carcass surfaces as compared to that from acetic acid. In a continual effort to discover practical and more effective methods of treating meat from animal carcasses to reduce or to eliminate the presence of microorganisms thereon, especially pathogenic microorganisms, research has focused on methods in addition to those described above. Smoke preparations, in particular, have received significant attention for their antimicrobial effects. An advantage of utilizing smoke preparations in the fight against microbial infestation is that smoke preparations are natural additives to food and are considered Generally Recognized as Safe (GRAS) under the food additive provisions of the Federal Food, Drug and Cosmetic Act

(FFDCA) . It has been reported that certain smoke preparations have been effective against Staphylococcus aiireus, E. coli and Saccharomyces cerevisiae . (Fretheim, K. , P.E. Granum, and E. Void, 1980, Influence of Generation Temperature on the Chemical Composition, An ioxidative, and Antimicrobial Effects of Wood Smoke. J. Food Sci. 45*. 999-1002.) It has also been recognized

that smoke preparations can have an antioxidant effect on meat to inhibit the development of adverse results from oxidation, such as oxidative rancidity.

As is well known to those in the meat processing field, the treatment of foods with wood smoke for food preservation has been substantially replaced by the use of "liquid smoke", which is a solution comprising liquid reagents capable of imparting a smokey hue or coloration and flavor to a meat exposed to a liquid or vapor phase of the solution. The use of liquid smoke in lieu of wood smoke is now quite conventional in meat processing and can be more fully appreciated in reference to representative U.S. Patent Nos. 3,873,741; 4,250,199; and 4,298,435.

Applying conventional liquid smoke solutions to meat, however, especially raw meat, is not without its drawbacks. Problems associated with applying conventional liquid smoke solutions to meat include adversely affecting the appearance, taste and edible quality of the meat such that the meat is discolored and has a harsh smoke flavoring causing the meat to be generally commercially unsatisfactory.

U.S. Patent No. 5,043,174 to Lindner, assigned to Hickory Specialties, Inc. of Brentwood, Tennessee, discloses a process for curing meat comprising applying an acetic solution derived from liquid smoke to the meat subsequent to peeling and prior to packing to control Listeria monocytogenes re-inoculation thereof. The solution is known as "Code V" manufactured by Hickory

Specialties, Inc. of Brentwood, Tennessee. The liquid smoke derivative solution comprises: acetic acid in a concentration of about 6.5 to 8.0% weight per unit volume (w/v); carbonyl in a concentration of about 1.0 to 8.0% weight per unit volume (w/v) ; phenol in a concentration of about 0.1 to 1.0% weight per unit volume (w/v); and water in a concentration of about 83.0 to 92.4% weight per unit volume (w/v) . The specific application of the liquid smoke solution as disclosed by U.S. Patent No. 5,043,174 to Lindner occurs to cooked meat between the two above- noted peeling and packing steps in a meat processing sequence for curing meat which includes the steps of grinding and blending selected meat, stuffing the meat into casings, applying liquid smoke to the meat, cooking the meat in a smokehouse, chilling the meat, peeling the casings from the meat, and packing the meat for shipment. The disclosure of U.S. Patent No. 5,043,174 to Lindner is incorporated by reference herein.

Despite the methods discussed above which have been developed in an effort to reduce or to eliminate the presence of microorganisms on meat for consumption, there exists substantial room for improvement in the art for providing a practical and more effective method of reducing or eliminating the presence of microorganisms, particularly pathogenic microorganisms, on meat ultimately for consumption, especially raw meat.

Summary and Objects of the Invention

In accordance with the present invention, a meat processing method is provided which is designed to more practically and effectively reduce, inhibit and/or eliminate the presence of host organisms, such as microorganisms, including pathogenic microorganisms, on meat from animals ultimately for consumption. The process herein provided comprises application of a solution derived from liquid smoke, a liquid smoke fraction, to meat to reduce and to inhibit host organism infestation on the meat. The liquid smoke derivative solution comprises: acetic acid in a concentration of about 6.5 to 8.0% weight per unit volume (w/v) ; carbonyl in a concentration of about 1.0 to 8.0% weight per unit volume (w/v); phenol in a concentration of about 0.1 to 1.0% weight per unit volume (w/v); and water in a concentration of about 83.0 to 92.4% weight per unit volume (w/v) .

Most suitably, the liquid smoke derivative solution is produced by processing conventional liquid smoke through an evaporator which separates and condenses the low boiling elements of the liquid smoke to produce the liquid smoke derivative solution. The remaining product from the separator is essentially a more concentrated solution of conventional liquid smoke. Further in accordance with the present invention, and in addition to providing a process for reducing or eliminating bacterial spoilage of meat, a process is provided for reducing or preventing oxidation of meats to reduce off-flavors due to oxidation and to prevent the development of warmed-over or

oxidized flavors on pre-cooked meats upon re-heating when the liquid smoke derivative solution of the present invention is applied to meat.

It is therefore an object of the present invention to provide a novel meat processing method which reduces and inhibits host organism infestation, particularly bacterial, of meat, especially raw meat.

It is another object of the present invention to provide a novel meat processing method which reduces the rate of spoilage of the meat and additionally reduces or prevents oxidation of the meat.

It is yet another object of the present invention to provide a novel meat processing method for reducing and inhibiting host organism infestation of meat and which provides oxidation protection to meat without adversely affecting the taste and edible quality of the meat.

It is still another object of the present invention to provide a novel meat processing method which reduces and inhibits host organism infestation of meat and provides oxidation protection to meat with little or no coloring of the meat.

It is a still further object of the present invention to provide a novel meat processing method for reducing and inhibiting host organism infestation of meat and providing oxidation protection to the meat which can be effectively applied to raw meat.

It is a still further object of the present invention to provide a novel meat processing method for reducing and

inhibiting host organism infestation of meat and providing oxidation protection to the meat wherein the meat processing method can be effectively utilized on meat at refrigeration temperatures. Some of the objects of the invention having been stated, other objects, as well as other advantages, will become evident as the description proceeds below.

Brief Description of the Drawings

Figure 1 is a graph illustrating an example of the effects of distilled water, distilled water with the liquid smoke fraction, and the liquid smoke fraction alone on E. coli attached to beef tissue in accordance with this invention;

Figure 2 is a graph illustrating an example of the effect of a 4% liquid smoke fraction solution on E. coli attached to beef trimmings in accordance with this invention,*

Figure 3 is a graph illustrating an example of the effect of a 6% liquid smoke fraction solution on E. coli attached to beef trimmings and ground beef in accordance with this invention,*

Figure 4 is a graph illustrating an example of the effect of a hot 8% liquid smoke fraction solution on E. coli attached to beef trimmings and ground beef in accordance with this invention; and

Figure 5 is a graph illustrating an example of the effect of a 12% liquid smoke fraction solution on E. coli

attached to beef trimmings in accordance with this invention.

Best Mode for Carrying Out the Invention The present invention is directed to a meat processing method utilizing a solution derived from liquid smoke, referred to herein as a liquid smoke fraction, which is applied to meat to reduce and to inhibit host organism infestation, such as microbial, particularly bacterial, infestation of the meat and which provides oxidation protection to the meat. The liquid smoke fraction, as applied in accordance with this invention, serves as an antimicrobial agent when used as a carcass wash for beef, pork and poultry; as an antimicrobial agent when used as a spray or dip for seafood to retard bacterial spoilage; and as an antimicrobial agent in raw hamburger or other comminuted meat products for the reduction, inhibition and elimination of pathogenic microorganisms. The liquid smoke fraction applied in accordance with this invention can also be combined with a sauce or glaze and applied to meat to reduce the rate of bacterial spoilage thereof. It has further been found that application of the liquid smoke fraction in accordance with this invention serves as a flavoring agent in a rib boil or a shrimp boil to reduce off-flavors due to oxidation and serves as an antioxidant flavoring agent to prevent the development of warmed-over or oxidized flavors in pre-cooked meats upon re-heating.

Liquid smoke is commercially available from Hickory Specialties, Inc., of Brentwood, Tennessee, under the name "Zesty Smoke Code 10". This liquid smoke can be utilized to obtain the liquid smoke fraction (Code V, manufactured by Hickory Specialties, Inc. of Brentwood, Tennessee) used for this invention and used in U.S. Patent No. 5,043,174 to Lindner, assigned to Hickory Specialties, Inc., discussed previously. The specifications of this liquid smoke are set forth below.

ZESTI SMOKE (Code 10) Liquid Smoke Specifications

Acidity 10.5-11.0

Staining Index 69-80 Carbonyl Level (g/100 ml) 15-25

Phenol Level (mg/ml) 12-22

Specific Gravity @ 25°C 1.068-1.079

Density (lbs/gal) 8.90-8.99 pH Level 2-3 Color Amber

The liquid smoke fraction utilized by the present invention can be produced as a derivative or by-product of the liquid smoke described above. The liquid smoke is preferably processed through a separator (for example, an

AVP evaporator) wherein the liquid smoke is fed as a feed stock which is heated and the low boiling acids thereof removed from the top of the evaporator and condensed into the liquid smoke derivative solution. This process yields two products, one being a concentrated liquid smoke having higher acidity, staining index, carbonyl and phenol levels, specific gravity, density and darker color than conventional liquid smoke. The concentrated liquid smoke

produced from this process is sold under the trademark SUPERSMOKE ^® by Hickory Specialties, Inc. of Brentwood, Tennessee for a variety of end uses. The process f rther yields the liquid smoke fraction described in accordance with this invention as a derivative or by-product. The liquid smoke fraction is a low pH, low flavor, low or no stain product. It is believed that the liquid smoke fraction produced as a derivative or by-product during the process for producing the concentrated liquid smoke has in the past been either discarded completely or recycled in some fairly insignificant manner. The only known use of the liquid smoke fraction is by Hickory Specialties, Inc. of Brentwood Tennessee pursuant to its U.S. Patent No. 5,043,174 to Lindner, as discussed previously. The liquid smoke fraction possesses the following specifications:

Liquid Smoke Derivative Solution Specifications

Acidity 6.8-7.8 Staining Index None

Specific Gravity 1.005-1.015

Carbonyl (g/100 ml) 2.0-7.0

Phenol (mg/ml) 1.0-4.0 pH Level 2.0-2.4 Color Amber

Based upon analysis, the liquid smoke fraction utilized in accordance with this invention comprises: acetic acid in a concentration of about 6.5 to 8.0% (preferably 6.8 to 7.8%) weight per unit volume (w/v); carbonyl in a concentration of about 1.0 to 8.0%

(preferably 2.0 to 7.0%) weight per unit volume (w/v);

phenol in a concentration of about 0.1 to 1.0% (preferably 0.1 to 0.4%) weight per unit volume (w/v); and water in a concentration of about 83.0 to 92.4% (preferably 84.8 to 91.1%) weight per unit volume (w/v). According to the meat processing method of this invention, the liquid smoke fraction described above is applied to meat from an animal. It is envisioned that the meat can be raw or cooked meat, and the meat can be comminuted prior or subsequent to application of the fraction to the meat. The meat processing method according to the present invention advantageously has particular application to raw meat, but can also be used effectively on meat during or after cooking. Furthermore, it has been advantageously been found that the meat processing method according to the present invention is effective as applied to meat which is at refrigeration temperatures, for instance from approximately 28° F to 45° F.

The liquid smoke fraction can be applied as a measure to reduce microbial infestation, even to the point of elimination, as well as to inhibit further infestation by providing increased resistance to microbial infestation. It is therefore understood that application of the liquid smoke fraction can be to inhibit primary microbial infestation but can also be applied after primary infestation has occurred to reduce and eliminate microbes and inhibit further infestation.

The present meat processing method can be used with meat from any animal prepared for consumption including vertebrate and non-vertebrate animals. The non-vertebrate animals can be crustaceans such as lobsters, clams, shrimps, and combinations thereof; the vertebrate animals can be cold-blooded or warm-blooded and can be fish, cattle, pigs, birds, and combinations thereof. Application of the liquid smoke fraction according to the meat processing method of this invention can therefore be on meat such as fish, beef, pork, and poultry. Prior or subsequent to application of the liquid smoke fraction to meat according to the meat processing method of this invention, it is envisioned that various processing steps can occur to the meat such as cutting the meat into primals prior to packaging or further processing comminuted meat into dry fermented sausages prior to packaging.

Application of the liquid smoke fraction to meat according to this invention can be accomplished by any conventional method, such as spraying, dipping, atomization, brushing or swabbing. The liquid smoke fraction can also be applied internally to meat by injection or by incorporating it as an ingredient during processing. The method of application of the liquid smoke fraction to meat, the contact time of the liquid smoke fraction with the meat, and the level of concentration of the liquid smoke fraction are important factors in the effectiveness of the liquid smoke fraction in reducing and

inhibiting microbial infestation and providing oxidation protection to the meat, as can be seen below.

Example I At seven different levels of liquid smoke fraction (Code V, Hickory Specialties, Inc., Brentwood, Tennessee) concentration, and three levels of inoculations, factorial design replicated twice was used for survival study of five strains of microorganisms including pathogenic microorganisms Salmonella enteri tidis, Listeria monocytogenes and E. coli , and two strains of non- pathogenic E. coli . Stock cultures were activated three times on agar slants, inoculated to tryptic soy broth, and then incubated at 37°C for 12 hours. This inoculum was used and found to contain 10 ⁸ cell forming units. Inoculations were then carried out to tryptic soy broth containing different percentages of the liquid smoke fraction. The test tubes were then incubated at 37°C for 24 hours and 48 hours. Readings of microbial survival were then recorded with the following results reported in Table 1 below wherein the following abbreviations have been employed:

MO = microorganism

Inoc. = inoculation l.s. = liquid smoke fraction

TABLE 1

* (+) indicates growth in 24 hours

** (-) indicates no growth in 48 hours

*** (-+) indicates no growth in 24 hours and growth in 48 hours

TABLE 1 (continued)

* (+) indicates growth in 24 hours

* * (-) indicates no growth in 48 hours * * * (-+) indicates no growth in 24 hours and growth in 48 hours

Based upon the growth or lack of growth of 12 hour old active cells of the microorganisms in different concentrations of the liquid smoke fraction for 24 hours

and 48 hours incubation at 37°C, it is evident from Table 1 that when the inoculum level was very high, a 2% liquid smoke fraction could be the optimum concentration to inhibit growth of the microorganisms. It is noted herein, however, that inoculum levels of 10 ⁸ and 10 ⁶ are unusually high levels and are unlikely to be found in contaminated foods. An inoculation level of 10 ⁴ is still fairly high, but can be encountered in a highly contaminated food. At this level, a 1.5% liquid smoke fraction was enough to inhibit completely the growth of each of the microorganisms. Recovery study revealed that the cells in the no-growth tubes did not survive. The liquid smoke fraction therefore had a lethal effect on the microorganisms, and for the strains studied, a 1.5% liquid smoke fraction solution was sufficient to inhibit growth for 12 hour active cells of 10 ⁴ concentration.

Example II Using a factorial 3-x-3 design study, the effect of the liquid smoke fraction (Code V, Hickory Specialties, Inc., Brentwood, Tennessee) on beef tissues inoculated with E. coli was studied. Twenty-seven tissues of raw meat (beef) were cut (2-X-2-0.25 inches) from a frozen inside round. The tissue samples were thawed and inoculated with E. coli (Rifampicin resistant) by immersing them in a diluted culture (around 10 ⁷ cell forming units) for 10 minutes at room temperature. The inoculated tissues were then dried at 4°C for 10 hours and treatments of distilled water by itself, distilled water

and the liquid smoke fraction (1:1), and the liquid smoke fraction by itself were applied at three different times (0, 10 and 60 seconds) . Three replications were performed for each treatment. Samples were placed in a filter stomacher bag, and diluent (30 ml; 0.1% peptone water) was added and stomached for 2 minutes. Serial dilutions were prepared in peptone water (0.1%) and spiral plated using a Spiral Plater Model D (Spiral Biotech, Bethesda, Maryland) on tryptic soy agar (TSA, DISCO, Detroit, Michigan) containing Rifampicin (100 ppm; Sigma Chemical Co., St. Louis, Missouri) . The plates were then incubated at 35°C for 24 to 48 hours and enumerated.

As illustrated in Figure 1, microbial analysis of the study, conducted 12 hours after treatment application, indicated that application of distilled water (DW) did not result in reduced E. coli levels on the meat surface for all three application times (0, 10 and 60 seconds). The results showed, however, that the presence of the liquid smoke fraction (LS) resulted in decreased levels of E. coli on the meat surface on both treatment levels. Increased application times resulted in larger reductions of microorganisms in both treatments. A reduction of 1.38 log-sq. in. was obtained when the beef tissues were treated with the liquid smoke fraction for 1 minute. The liquid smoke fraction was therefore more effective than distilled water in reducing E. coli attached to beef tissue. Since the time between treatment application and microbial analysis was 12 hours, it is

contemplated that increased holding periods could result in increased bactericidal action of the liquid smoke fraction or at least prolong the lag phase of the organisms. Example III

The inhibitory concentration of the liquid smoke fraction (Code V, Hickory Specialties, Inc., Brentwood, Tennessee) on foodborne spoilage and pathogenic microorganisms was studied. The foodborne spoilage and pathogenic microorganisms as listed in Table 2 hereinbelow were obtained from a culture collection.

TABLE 2

1. Streptococcus faecalis 21. Listeria monocytogenes

2. Shigella flexneri 22. Listeria innocua

3. Hafnia alvei 23. Listeria innocua

4. Enterobacter aerogenes 24. Listeria monocytogenes

5. Serratia marcescens 25. Listeria monocytogenes

6. Staphylococcus aureus 26. Listeria monocytogenes

7. Pseudomonas aeruginosa 27. Listeria innocua

8. Citrobacter freundii 28. Listeria innocua

9. Klebsiella pneumoniae 29. Listeria monocytogenes

10. Escherichia coli 8-3 Scott A

11. Escherichia coli 30. Salmonella typhimurium

0157:H7 (rif. res.) NR

12. Escherichia coli 31. Salmonella typhimurium

0157 :H7 S-l

13. Escherichia coli 32. Salmonella typhimurium

0157 :H7 PI-376

14. Escherichia coli 33. Salmonella typhimurium

0157:H7 PI-370

15. Escherichia coli 34. Salmonella typhimurium

0157 :H7 PI-375

16. Escherichia coli 35. Salmonella typhimurium

0157 :H7 PI-3711

17. Escherichia coli 36. Salmonella pullorum

0157 :H7 PI-3751

18. Escherichia coli 37. Salmonella typhimurium

0157 :H7 PI-377

19. Escherichia coli 38. Salmonella typhimurium

0157 :H7 PI-372

20. Escherichia coli 39. Salmonella newport

0157 :H7 Salami ATCC 6962 isolate 40. Salmonella typhimurium S-5-3

The cultures were maintained on brain heart infusion agar (BHIA; DIFCO, Detroit, Michigan) slants at 4°C with monthly transfers. In preparation of agar plates, the liquid smoke fraction was diluted in sterile distilled water (10 ml) to give a series of doubling dilutions (1:1 to 1:32). Each of these diluted liquid smoke fraction solutions (10 ml) was mixed with tryptic soy agar (TSA; DIFCO, Detroit, Michigan) tempered at 48°C and poured into 100 x 15 mm petri dishes. The plates were then incubated

at 35°C for four (4) hours to equilibrate the liquid smoke fraction in the agar. The plates were then used for inoculation.

In preparation of the cultures and master template, fresh cultures were prepared by inoculating into brain heart infusion broth (BHI; DIFCO, Detroit, Michigan) and incubated at 35°C for eighteen hours. The cultures (200μL) were dispensed into sterile microtitrer plates (96 wells; Dynatec Laboratories, Inc., Chantilly, Virginia). The organisms were replicated on the agar plates using a 7 x 6 replicator using one well as a control well. The replicator was dipped in alcohol and flamed before its use. Replicas were made from the highest dilution (1:32) to the lowest dilution (1:1) to avoid any carry-over of the smoke components. The plates were incubated at 35°C for 24 hours and observed for growth.

Results from the study showed that the liquid smoke fraction inhibited all of the organisms tested at 1:16 dilution except for Pseudomonas aeruginosa, a spoilage organism. This organism, however, was inhibited at the next lower dilution (1:8) . When converted to a percentage scale, the dilutions would be 2.08% for the 1:16 dilution and 4.17% for the 1:8 dilution; these percentages are within the usual limits of food products. Example IV

The effect of adding 4%, 6% and 8% liquid smoke fraction solutions to beef trimmings inoculated with E. coli 0157:H7 prior to being ground was studied.

Experiment 1

E. COli 0157 :H7 isolates (EDL933, A8959-C7, EC45753, ATCC 43895, and an isolate from a salami outbreak) were used and the cultures stored on tryptic soy agar (TSA, DIFCO, Detroit, Michigan) slants at 4°C. Fresh 24 hour cultures were centrifuged and resuspended in buffered peptone water diluent to a target of 1 x 10 ⁹ CFU/ml. Green food dye (McCormick) was added to the suspension to ensure thorough mixing of the inoculum in the ground beef. Whole carcass beef trimmings were collected and cut into uniform portions of 1.5 cm thick, 4-6 cm long, and 3- 5 cm wide, and the portions were kept at 4°C until use. The test comprised the following treatment steps: l. control (+ inolculum) 2. sodium lactate (2%) (60% syrup)

3. 4% liquid smoke fraction (LS) (Code V, Hickory Specialties, Inc., Brentwood, Tennessee)

4. sodium lactate (2%), 4% liquid smoke fraction (LS) , and fiber (1%) 5. 4% liquid smoke fraction (LS) and fiber (1%) 6. meat control (no inoculum)

The meat portions were weighed into 2043g amounts, placed in a Hobart mixer (Hobart Corp., Troy, Ohio) and mixed for 2 minutes with 100 ml of the inoculum to give a 1 x 10 ⁹ CFU/g. The liquid smoke fraction treatments were added after the culture mixing and mixed for 2 minutes. The remaining treatment ingredients were added and mixed for 4 minutes. The meat and treatment mixture were then

ground through a 1/2 inch plate and then double ground through a 1/8 inch plate. The ground product was then weighed out into 100-125g amounts and made into patties using a manual patty maker. The patties were then vacuum- packed and stored at 4°C until used.

Samples (25g) were collected and diluted in 225 ml buffered peptone water diluent and stomached in a filter stomacher bag (Model 400, Tekmar, Inc., Cincinnati, Ohio) for 2 minutes. Serial dilutions were made and spiral plated (Model 500 D Sprial Plater, Spiral Biotech, Inc., Bethesda, Maryland) onto MacConkey sorbitol agar (MSA, DIFCO) . The plates were incubated at 35°C for 18-24 hours. Data was recorded as log ₁₀ CFU/g.

The graph of Figure 2 illustrates the trend of the organisms and shows that no difference was found between the control and the treatments. A reduction of the E. coli 0157:H7 isolates occurred at the beginning (in the control and the treatments) , but did not decrease after that point. It is believed that the overwhelming number of the organisms (1 x 10 ⁹ CFU/g) on the meat likely inhibited the antimicrobial properties of the liquid smoke fraction utilized as well as the other compounds.

Experiments 2 and 3

A strain of E. coli 0157:H7 (Rifampicin resistant) was stored on a tryptic soy agar (TSA, DIFCO, Detroit, Michigan) slant at 4°C. The organism was grown at 37°C for 20 hours in brain heart infusion broth to a target of 1 x

10 ⁹ CFU/ml. The organism was centrifuged for 10 minutes, resuspended in sterilized distilled peptone water (0.1%), and used to inoculate fresh beef trimmings.

The fresh beef trimmings were inoculated with the E. coli 0157:H7 as the organism was added to the trimmings to give a 1 x 10 ⁷ CFU/g, and the inoculated trimmings were mixed for 4 minutes using a Hobart mixer (Hobart Corp.,

Troy, Ohio) . Two treatments of the liquid smoke fraction

(Code V, Hickory Specialties, Inc., Brentwood, Tennessee) were used. Specifically, a 6% liquid smoke fraction solution and a hot (140°F) 8% liquid smoke fraction solution were used. Additionally, one control was used.

For the treatments, the liquid smoke fraction solutions were added to the beef trimmings 10 minutes after inoculation and mixed for 4 minutes. The control was only inoculated with E. coli 0157:H7. The treatments and the control were placed in separate sterile bags and held at

3-4°C for 48 hours.

Surface samples (25g) were taken from the beef trimmings after inoculation (to check initial microbial population) immediately after treatment (8% liquid smoke fraction solution) , and after 48 hours of storage at 3-4°C. After 48 hours, the treatments and control were coarse ground (1/2 inch) , followed by a double fine ground (1/8 inch) . Patties were made using a patty maker, and they were bagged and stored at 3-4°C for up to 7 days.

Samples (25g) were taken from the patties at different time intervals. The samples were placed in a

filter stomacher bag (Model 400, Tekmar, Inc., Cincinnati, Ohio), and diluent (225 ml; 0.1% peptone water) was added and stomached for 2 minutes. Serial dilutions were prepared in peptone water (0.1%) and spiral plated (Model 500 D Spiral Plater, Spiral Biotech, Inc., Bethesda, Maryland) on MacConkey sorbitol agar (MSA, DIFCO, Detroit, Michigan) containing Rifampicin (100 ppm; Sigma Chemical Co., St. Louis, Missouri). The plates were incubated at 37°C for 24-48 hours and enumerated. Data was recorded as log ₁₀ CFU/g.

The graphs of Figures 3 and 4 show the trend of E. coli 0157:H7 when beef trimmings were treated with 6% and 8% liquid smoke fraction solutions, respectively. Similar results were obtained with the 6% liquid smoke fraction solution treatment and the hot 8% liquid smoke fraction solution treatment. The graph of Figure 3 shows the reduction of the organism in the patties. The graph of Figure 4 includes the E. coli 0157:H7 reduction in the trimmings as well as in the patties. The presence of the liquid smoke fraction solutions resulted in decreased levels of E. coli 0157:H7 attached to the beef trimmings and ground beef. When the beef trimmings were treated with 6% liquid smoke fraction solution, the difference of the means between the control and the treatment was 0.7, 1.1, 0.8, 1.4 and l.l log ₁₀ CFU/g at days 0, 2, 4, 6 and 8, respectively. The reduction of E. coli 0157:H7 treated with hot 8% liquid smoke fraction solution, when comparing the control against the treatment, was 1.0, 1.1, 1.3, 1.2,

0.9 and 1.1 log ₁₀ CFU/g after 48 hours at 3-4°C, after 48 hours at 3-4°C and grinding, at days l, 3, 5 and 7, respectively. The largest reduction of the organism was obtained during the 48 hours of storage at 3-4°C during the grinding of the trimming. This reduction was due to the antimicrobial properties of the liquid smoke fraction and probably to the killing and exposure of the organism to new surface when grinding. Between day 0 (when the patties were made) and day 7, the control and the treatments barely declined, keeping the same trend. It is believed that increasing the holding time (72 hours) and/or increasing the liquid smoke fraction temperature or percentage could even more greatly reduce the E. coli 0157:H7. Example V

The effect of adding a 12% liquid smoke solution to beef trimmings inoculated with E. coli was studied. A strain of E. coli 0157:H7 (Rifampicin resistant) was stored on a tryptic soy agar (TSA, DIFCO, Detroit, Michigan) at 4°C. The organism was grown at 37°C for 20 hours in brain heart infusion broth to a target of 1 x 10 ⁹ colony forming units (CFU)/ml. The organism was then centrifuged for 10 minutes, resuspended in sterilized distilled peptone water (0.1%), and used to inoculate fresh beef trimmings.

The E. coli was added to the fresh beef trimmings to yield a 1 x 10 ⁷ CFU/g and mixed for 4 minutes using a Hobart mixer (Hobart Corp., Troy, Ohio). The test

treatment received a 12% liquid smoke fraction solution (Code V, Hickory Specialties, Inc., Brentwood, Tennessee), and a control was treated with 12% sterile water. The treatment and control were separately mixed for 4 minutes. Excess liquid smoke fraction and water were then drained as 0 ml of the liquid smoke fraction and 50 ml of water were drained from the treatment and control, respectively. The treatment and control were then coarse ground (1/2 inch) followed by a fine ground (1/8 inch) . Patties were made using a patty maker, and they were bagged and stored at approximately 3 to 4°C for up to 3 days.

Surface samples (25g) were taken from the beef trimmings after inoculation (to check initial microbial population) and immediately after treatment. Samples (25g) were taken from the patties at days 0, 1, 2 and 3. The samples were placed in a filter stomacher bag (Model 400, Tekmar, Inc., Cincinnati, Ohio), and diluent (225 ml; 0.1% peptone water) was added and stomached for 2 minutes. Serial dilutions were prepared in peptone water (0.1%) and spiral plated (Model 500 D Spiral Plater, Spiral Biotech Inc., Bethesda, Maryland) on MacConkey sorbitol agar (MSA DIFCO, Detroit, Michigan) containing Rifampicin (100 ppm; Sigma Chemical Co., St. Louis, Missouri) . The plates were incubated at 37°C for 24 hours and enumerated. Data was recorded as log ₁₀ CFU/g.

The graph of Figure 5 illustrates the trend of the E. coli 0157:H7 when the beef trimmings were treated with 12% liquid smoke fraction solution and 12% sterile water in

the treatment and control, respectively. The presence of the liquid smoke fraction resulted in decreased levels of E. coli 0157:H7 attached to beef trimmings and ground beef. The reduction in E. coli 0157:H7 in ground beef treated with 12% liquid smoke fraction solution was 4.1 log ₁₀ CFU/g one day after grinding and 4.8 log ₁₀ CFU/g 3 days after grinding. The difference of the means between the control and the treatment was 1.5, 1.1, 2.3, 3.4 and 3.0 log ₁₀ CFU/g after treatment (12% liquid smoke fraction solution, 12% water) and after grinding at days 0, 1, 2 and 3, respectively.

Example VI The antioxidant effects of the liquid smoke fraction (Code V, Hickory Specialties, Inc., Brentwood, Tennessee) were studied. Various levels (0%, 1% and 2%) of the liquid smoke fraction were mixed with ground meat, forming patties (0.25 lb. each) and fully cooked on a gas grill to an internal temperature of 155°F. After cooking, the meat patties were immediately chilled, wrapped in aluminum foil and then frozen.

After 30 days of frozen storage, the cooked patties were reheated in a microwave oven and tested. They were evaluated for the presence and amount of warmed-over flavor which is the result of oxidation. The 2% treatment level had little or no off-flavors while the control (0%) showed significant levels of warmed-over flavor due to oxidation. The 1% treatment level demonstrated moderate levels of off-flavor due to oxidation. Application of the

liquid smoke fraction at various levels therefore provided an antioxidant effect to protect the meat from the warmed- over flavor resulting from oxidation.

The examples set forth above are merely illustrative of some applications of the meat processing method of this invention and are not intended to limit the scope of the invention as defined by the claims appended hereto. From the examples set forth above, it can therefore be seen that the meat processing method according to this invention can be effectively used to fight microbial infestation, even pathogenic microorganisms.

It is envisioned that the liquid smoke fraction can be applied to meat at various stages from slaughtering of an animal to even being applied to previously cooked meat. Further in accordance with this invention and as can also be seen by the above examples, it has been surprisingly found that the liquid smoke fraction can be effectively applied at very low concentrations, around 1.5%-2% for instance. It can therefore be seen and understood that whether the liquid smoke fraction is applied by dipping, spraying, swabbing or brushing, a broad spectrum of microorganisms on meat are affected by being reduced and/or inhibited. Quite importantly, some of the microorganisms thus affected are pathogenic microorganisms, and the liquid smoke fraction has advantageously shown a lethal effect on selected pathogenic microorganisms. Moreover, it is further understood that the liquid smoke fraction applied

according to the liquid smoke invention imparts oxidation protection to meat.

The meat processing method of this invention is very practical and easy to implement, and although the liquid smoke fraction can be applied to cooked meat, for instance upon re-heating, the meat processing method has shown particular effectiveness when utilized on raw, uncooked meat. It is believed that this novel meat processing method is a significant advancement in the meat processing art and fight to reduce or eliminate microbial infestation and oxidation of meat for ultimate consumption.

It will be understood that various details of the invention may be changed without departing from the scope of the invention. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation as the invention is defined by the following, appended claims.