Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
  • A23B4/00—General methods for preserving meat, sausages, fish or fish products
  • A23B4/10—Coating with a protective layer; Compositions or apparatus therefor
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
  • A23B4/00—General methods for preserving meat, sausages, fish or fish products
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
  • A23B4/00—General methods for preserving meat, sausages, fish or fish products
  • A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
  • A23B4/16—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of gases, e.g. fumigation; Compositions or apparatus therefor

Definitions

• the present invention relates to a method for preserving raw meat and more specifically relates to a method for preserving raw meat and preventing micro growth by exposing the meat to an atmosphere consisting essentially of carbon monoxide.
• an animal may be slaughtered and cut into halves or quarters which are then forwarded to a wholesaler or retailer where they may be divided into smaller cuts such as steaks or roasts.
• a wholesaler or retailer where they may be divided into smaller cuts such as steaks or roasts.
• the meat must be maintained, frequently the meat is frozen in order to preserve its quality.
• the meat After the meat has been divided into cuts for sale to the eventual consumer, it must also be maintained under constant refrigeration in order to preserve its quality. Under this distribution scheme, it can be from a few days to more than a week before the meat is purchased and consumed. It, therefore, becomes evident that this constant requirement for very low temperatures greatly contributes to the cost of meat.
• the first class being aerobic and the second class being anaerobic.
• the anaerobic microorganisms include facultative (having both aerobic and anaerobic metabolic pathways).
• the aerobic microorganisms cannot survive without oxygen while the anaerobic microorganisms have a non-oxygen requirement for metabolism. It is important to note that iron is an essential element for both organisms.
• the pathogenic bacteria are those interfering with the human host metabolism and interfere with normal physiology causing effects ranging from the minor to the lethal.
• the aerobic bacteria are likely to arrive from outside of the host organism. This is in contradistinction from the anaerobic facultative microbes which may be animal borne because they can grow in the host devoid of free oxygen.
• neither bacteria are desirable to have on the meat. It is therefore important to develop ways of eliminating bacterial growth be it anaerobic or aerobic.
• Both of the Hood references disclose methods of exposing an animal protein source to a reducing agent and then an environment of carbon monoxide in order to preserve the bright red color of protein source. Additionally, the Hood et al. references only treat slurries of the protein source as this is required for saturation by the carbon monoxide. The source is then mixed with the remainder of the food stuff to prepare a moist dog food. Further, the references are concerned only with the application of carbon monoxide in order to preserve the color of product and both require subsequent processing, such as canning or heat sterilization, in order to preserve the actual quality and freshness of the product. Additionally, the Hood '983 reference discloses the addition of a sufficient amount of microbiological and bacteriological inhibitors to further preserve the product.
• the Woodruff et al. '835 reference discloses a process for maintaining a good color and the freshness meat by first exposing meat to an atmosphere with a small amount of oxygen and then exposing the meat to a modified atmosphere containing a small amount of carbon monoxide to effect the conversion of myoglobin to carboxymyoglobin.
• a third required step is the maintenance of the meat in an atmosphere of higher than 10% carbon ioxide.
• the Woodruff et al. '040 patent discloses a process for storing or shipping fresh meat in a modified gaseous atmosphere.
• the process requires maintaining refrigerated meat in an artificial atmosphere composed of oxygen, carbon dioxide and carbon monoxide as well as nitrogen.
• the carbon monoxide may be removed from the modified material after the meat has been treated for at least one hour.
• the Woodruff at al. patents teach maintaining the color in meat by treating the meat with a mixture of gases including carbon monoxide. That is, the Woodruff et al. patents teach chemical alteration of the surface of the meat to maintain the color of the meat and utilize refrigeration for meat preservation. Additionally, the Woodruff et al. patents teach the treatment of meat using a gaseous mixture of carbon monoxide, oxygen, carbon dioxide, and nitrogen. This method of treatment results in the creation of a storage environment which has low oxygen concentration and a carbon dioxide concentration of approximately ten percent.
• This type of gaseous mixture creates optimal growth conditions for the growth of microaerophil bacteria such as Helicobacter pylori and Campylobacter jejuni which are known to be pathogens which cause widespread gastroenteritis.
• the Woodruff et al. method of treating meat does maintain the color of fresh meat, however, the Woodruff et al. method has the disadvantage of accelerating bacterial contamination of meat treated by the Woodruff et al. method, thus shortening the storage life of the meat treated thereby.
• the Koch at al. '117 patent discloses a cover useful for treating fresh red meat with carbon monoxide in order to maintain the bright red color of the meat.
• Koch at al. teaches a cover comprised of two films which are sealed together around the edges and which confines a quantity of carbon monoxide gas therebetween. Both film layers are substantially carbon monoxide impermeable when dry, however; when the film is brought into contact with a freshly cut sample of red meat, the moisture in the meat wets the film and transforms the film into a carbon monoxide permeable structure. The carbon monoxide then contacts the meat sample thereby causing the meat to maintain its desired red color.
• the Australian Patent Document No. AU-A-18559/92 to Tamayama et al. discloses a method for maintaining and improving the quality of meat by causing meat to contact and absorb carbon monoxide gas in a sealed container and then requiring removal of the carbon monoxide gas from the container. Exemplifying the criticality of the removal of the carbon monoxide gas from the container, the patent requires that the carbon monoxide gas within the container be sucked and discharge by means of a pump.
• Applicant has developed a single step method for preserving meat by exposing raw meat to an atmosphere consisting essentially of carbon monoxide and, then, storing the meat in a sealed container. Unlike prior art preservation methods, no additional steps, compounds or additives are required in order to prevent the growth of microbiological or bacterial organisms.
• a method for preserving meat by exposing raw meat to an atmosphere consisting essentially of carbon monoxide is shown.
• Meat treated according to the present invention may not require any form of subsequent refrigeration under certain conditions and time constraints and can be stored for long periods of time following treatment with the carbon monoxide without significant bacterial growth, without freezing, and without a loss in meat quality.
• a method of preventing microorganism growth in meat by exposing raw meat to an atmosphere consisting essentially of carbon monoxide is also provided by the present invention.
• FIGURE 1 is a bar graph of the relationship between aerobic bacterial growth on a fresh meat sample stored at 22-30°C over time in either a CO treated environment or an air only environment;
• FIGURE 2a is a histogram illustrating preservation duration CO preserved meats and air treated meat preserved at 5 +/- 3°C as determined by Microaerophil growth;
• FIGURE 2b is a histogram illustrating preservation duration of CO preserved meats and air treated meat preserved at 5 +/- 3°C as determined by total viable aerobic bacterial growth;
• FIGURE 3 is a graph illustrating spectral analysis of the amounts of hemoglobin in the blood of cats that consumed either CO treated meat or air treated meat;
• FIGURE 4 is a photograph illustrating meats, the colors of meat treated with (A) vacuum only, (B) N 2 , (C) air, and (D) CO;
• FIGURE 5 is a photograph illustrating the color change in meat treated without CO (left) and meat treated with CO (right);
• FIGURE 6 is a photograph illustrating the internal color change of meat treated without CO (left) and meat treated with CO (right);
• FIGURE 7 is a photograph illustrating the color change of a piece of fresh CO treated meat stored at 5°C for three days;
• FIGURE 8 is a photograph illustrating the same meat sample shown in Figure 7 stored with CO at 5°C for ten days;
• FIGURE 9 is a photograph illustrating a transverse cut of the meat sample shown in FIGURE 8 made at 7 cm from the edge showing homogenous bright red color
• FIGURE 10 is a photograph illustrating transverse cuts of CO treated (left) and frozen (right) meat samples after twelve days of storage;
• FIGURE 11 is a photograph of the transverse cuts of meat shown in FIGURE 10 after cooking
• FIGURE 12 is a photograph illustrating transverse cuts of the cooked meat samples shown in FIGURE 11 ;
• FIGURE 13 is a photograph illustrating a section of CO treated meat as shown in FIGURE 10, following exposure to open air at 5°C for two weeks, at the end of this two week period, the meat sample was ground, a 200 gram "hamburger-like" sample was cooked and released CO was measured, (top) prior to cooking, (bottom) following cooking;
• FIGURE 14 is a graph showing the growth of E coli on beef under a defined atmospheres having either untreated or high carbon monoxide atmospheres;
• FIGURE 15 is a graph showing the growth of E coli on beef under a defined atmosphere having either untreated or low carbon monoxide atmosphere;
• FIGURE 16 is a graph showing the growth of Pseudomonas fluorescence on beef under defined atmospheres having untreated atmosphere, low carbon monoxide or high carbon monoxide atmosphere;
• FIGURE 17 is a graph showing the growth of Staphylococcus aures on beef under a defined atmospheres, the atmosphere either being untreated or with high carbon monoxide;
• FIGURE 18 is a graph showing the growth of Listeria monocytogenes on beef under a defined atmospheres, the atmosphere is being either untreated, low carbon monoxide, or high carbon monoxide;
• FIGURE 19 is a graph showing the growth of Clostridium prefringens on beef under defined atmospheres as having either untreated, low carbon monoxide or high carbon monoxide atmospheres;
• FIGURE 20 is a graph showing the growth of Salmonella Typhimirium on beef under defined atmospheres, either untreated, low carbon monoxide or high carbon monoxide atmospheres;
• FIGURE 21 is a graph showing the growth of Listeria monocytogens on poultry under defined atmospheres of wither untreated or high carbon monoxide atmospheres;
• FIGURE 22 is a graph showing the growth of Pseudomonas fluorescence on poultry under defined atmospheres of either untreated atmosphere or high carbon monoxide atmosphere;
• FIGURE 23 is a graph showing the growth of clostridium prefringens on poultry under defined atmospheres of either untreated, low carbon monoxide, or high carbon monoxide atmosphere.
• the present invention provides a method for preserving meat by exposing raw meat, processed or not, to an atmosphere consisting essentially of carbon monoxide (CO) and, subsequently, storing the meat in a sealed container.
• the present invention also provides a method for preventing microorganism growth meat by exposing raw meat, processed or not, to an atmosphere consisting essentially of carbon monoxide, and subsequently storing the meat in a sealed container.
• the term "meat” is defined to include all types of fresh meat and fresh poultry such as beef, pork, veal, lamb, chicken, turkey, fish and the like.
• the meat may be in the form of carcasses, primals (e.g., quarters), subprimals (e.g., top round), or retail cuts (e.g., steaks, ground meat and roasts).
• the process is also effective on whole animals including, but not limited to, cattle, chickens, and fish. Unlike prior art methods, the meat need not be slurried or otherwise pretreated.
• “Fresh meat” is defined as a meat article which has not been frozen and subsequently thawed before its sale or consumption.
• preserving it is meant that the meat maintains a pleasing color, does not spoil and develop a foul smell, bacterial growth is significantly inhibited or retarded, and remains completely pleasing, edible and consumable by humans and other animals.
• Preservation is not only maintained on the surface of the meat, but also throughout the entirety of the meat. That is, the meat is preserved throughout the thickness of the meat.
• "Pleasing color” implies that the color of the meat, preserved by the method according to the present invention, is such that it stimulates the appetite to consume the meat. That is, the color and odor of the preserved meat is such that a consumer would be enticed by the meat and would want to consume the meat.
• meat color is also preserved throughout the thickness of the meat.
• without freezing is defined as storing the meat wherein the temperature is kept between approximately -2 to 30°C.
• the term “without freezing” also excludes the use of any device or method for freezing the meat. Such devices include, mechanical or electrical refrigeration devices such as refrigerators, freezers, coolers, and chillers. This term also excludes the preservation of meat by freezing through storage on ice.
• Exposing raw meat to an atmosphere consisting essentially of carbon monoxide is defined as bringing into intimate contact both carbon monoxide gas and the meat being treated.
• the atmosphere preferably consists of carbon monoxide.
• This term also includes the complete conversion of myoglobin present in the meat sample to carboxymyoglobin and, the complete conversion of myoglobin to carboxymyoglobin/carboxyhemoglobin in fish.
• the meat is completely immersed or saturated with carbon monoxide.
• a cross-section of meat is completely immersed in or saturated to its core with carbon monoxide from the exposed surfaces through the entire cross-section (thickness) including its core region and retains the carbon monoxide until the meat is cooked.
• the meat is preserved throughout its thickness.
• Carbon monoxide is inherently a very inert gas. Carbon monoxide is relatively more inert than nitric oxide gas (NO) released from nitrites which have been used as preservatives for meat for several hundred years. Carbon monoxide is a normal metabolite in the body. It is produced indigenously as a product of heme catabolism (mostly the breakdown of hemoglobin). Carbon monoxide is further converted to carbon dioxide and is released from the body in that form. Recently, it has been found that normal metabolism utilizes carbon monoxide as a neurological messenger. (Baranaga, 1993) The high toxicity of carbon monoxide generally stems from its ability to compete with oxygen for binding to hemoglobin.
• Hb/Mb contain iron and divalent oxidation state (Fe +2 ) and only in this form are Hb/Mb capable of binding the gas ligands 0 2 , NO, and CO. Following any change in the iron oxidation state, Hb/Mb loose their CO binding ability. Denaturation of the proteins (e.g. by heat) can also result in loss of CO binding potential (as well as other ligands).
• Hb/Mb are established catalyzers of the oxidation process in biological tissues. Under regular atmospheric conditions, the Hb/Mb in fresh meat, which are in their native form, exist in a O 2 bound form, the so-called oxy-Hb/Mb. Oxy-Hb/Mb tends to undergo autooxidation to met-Hb/Mb namely the oxidation of the Hb/Mb divalent iron to Fe +3 can concomitantly with the formation of superoxide anion O 2 by the reaction oxy-Hb/Mb (Fe +2 . . . 0 2 ) ⁇ Met Hb(Fe +3 ) + 0 2
• the superoxide anion is unstable and further forms hydrogen peroxide (H 2 0 2 ) which together with Hb/Mb acts as a highly active peroxidation system.
• Met-Hb/Mb no longer binds any of the gas ligands including carbon monoxide.
• the Met-Hb/Mb are catalyzers of oxidations.
• Kb/Mb are protected from autooxidation. Therefore, to protect meats from autooxidation, carbon monoxide is best applied to fresh meat.
• meats treated according to the present invention have a longer storage life and remain both viable and edible in a non-contaminated form f or periods longer than those available using current preservation techniques.
• meat samples are placed in an enclosure or container and flushed or exposed to carbon monoxide gas.
• the process consists of two stages:
• the container for treatment, storage, and transportation of meat by the method of the present invention can be constructed of various gas-impermeable material such as plastic, metal, and other materials known in the art.
• the container can be equipped with both gas inlet and outlet channels which can be opened to allow the influx of gas (CO) or closed in order to render the container sealed.
• a suitable container would be capable of maintaining a seal to prevent the escape of carbon monoxide gas from the container.
• the container can be a sealed room in which large amounts of meat may be treated at a given time, the container can also be a smaller sealable container or chamber.
• the container is of larger volume than the volume of meat being treated to allow for a greater volume of carbon monoxide gas to contact the meat sample.
• meat samples are treated and stored within plastic bags constructed of a material which is safe for the storage of food products such as polyvinylidene chloride.
• the plastic bags will be constructed of a material that is impermeable to the passage of gases therethrough.
• the meat is maintained in the carbon monoxide atmosphere within the bag (container) during storage.
• the container is then filled with the CO gas.
• the addition of the CO gas can be accomplished in any suitable manner; however, the preferred methods include first removing the gas atmosphere present in the container (usually air) by using a vacuum pump, as is well known in the art, to remove any gases present and the container.
• the container is then filled with CO from a source such as a gas cylinder.
• the container is connected to the CO containing cylinder and CO is introduced.
• Input and output pressures are measured during the filling process.
• the input pressure is generally maintained within a range of approximately 1.5 to 5.0 atmospheres.
• the preferred pressure is approximately 2.0 atmospheres.
• the gas flow is stopped and excess gas is allowed to escape until the pressure within the container reaches approximately 1.0 to 1.2 atmospheres.
• the preferred gas pressure in the container is approximately 1.1 atmospheres.
• the ambient temperature of the surrounding can be maintained between -2 to 37°C.
• the parameters that govern gas filling or exposure time vary depending on the pressure of the gas input, the dimensions of the inlet and outlet channels, and the dimensions of the container.
• the gas filling time should be long enough to allow for a sufficient amount of CO gas to be completely absorbed (throughout its thickness) into the meat undergoing treatment. That is, enough CO gas is flushed through the container to allow for the complete penetration and protection of the meat being treated.
• the gas filling time generally ranges from approximately one to thirty minutes with the preferred filling time being approximately five minutes.
• exposure time is defined as the gas filling time.
• a larger and heavier meat sample will require a longer period of exposure to the carbon monoxide in order to achieve long-term preservation.
• a larger meat sample will require a longer exposure to carbon monoxide in order to properly preserve the meat sample without the freezing.
• the temperature during the carbon monoxide exposure is preferably between -2 and 37°C and can vary depending on the temperature selected to in order to carry cut the method.
• Meat treated as previously described above generally contains from 5 to 100% by weight or volume of CO gas.
• the preferred volume of CO in the treated meat is approximately 30% of the weight of the meat (e.g. 30 ml for 100 grams of treated meat).
• the meat surface is initially contacted with the CO gas. Since the surface of the meat is the most prominent site for the presence of bacteria, the meat treated by the method of the present invention is immediately protected. Further, while sealed in the container, penetration of the CO gas continues until the entire meat mass has been penetrated and, thereby, protected. This total penetration allows for the complete substitution of both hemoglobin and myoglobin by the carboxy forms of these compounds as is shown in the following examples.
• the total CO treatment of the meat throughout its thickness also enables meat which has been treated according to the present invention to maintain a pleasing color for extended periods of time after the meat has been removed from the packaging or container in which it was treated. That is, as shown in the following examples, meat treated according to the present invention can be transported, unpacked, and then maintained in a fresh form for a further extended period of time without a loss of color or quality.
• Samples of fresh meat (30 grams of beef, veal, or turkey) treated with CO by the method of the present invention were incubated in a suitable container for thirty minutes at a temperature of 15 ⁇ 3°C.
• Control samples were treated identically to CO- treated meats but were treated with air.
• the meat samples were removed from the container and were placed on an open benchtop at 15 ⁇ 3°C, or in an air exposed thermostatically controlled environment at 37°C.
• the color of the air treated meats turned brown gradually (within three hours at 37°C and twelve hours at 15 ⁇ 3°C), indicating non-fresh or spoiled meat.
• the CO-treated meat samples maintained a wine-red color for at. least 24 hours following exposure.
• Fresh meat quarters (beef) were kept for six days at -2°C.
• the meat was then cut into 30 gram pieces (4" X 2" X 0.1") and divided into four groups and treated as follows: (A) vacuumed and (2-4) were introduced into gas tight containers by method previously described above and filled at a pressure of 1.1 atmospheres with gas at a volume which was ten time (10x) the volume of the meat.
• the gases used were: group D filled with CO, group B filled with N 2 , and group C filled with air.
• a portion of the samples was kept at 15 ⁇ 3°C and the rest at 7°C. After twenty-four hours at 15 ⁇ 3°C and 48 hours at 7°C time dependent changes in color were observed.
• the samples of group A (maintained solely under vacuum) were brownish-purple.
• the samples of group B (N 2 treated samples) were brownish-red.
• the samples of group C (air treated samples) were brown.
• the color of the samples of Group D (CO treated) were unchanged remaining bright wine-red as shown in Figure 4.
• Beef chunks of 0.5 - 1.5 Kg were turned into CO-treated meat samples by treating with a 100% meat volume of gas according to the method of the present invention. Control chunks from the same source were treated identically but with air instead of CO. All of the chunks were kept at 4°C. The surface color of the air treated meat became brown after three days. The meat chunks were cut transversely for observation of color changes. Color change propagated with time in all meats from the surface towards the center of the chunk and were brown in air treated samples and wine-red in CO-treated samples. Following eighteen days of incubation, the air treated chunks were completely dark brown. In the CO-treated meat, the color change was 3-5 mm from the surface after one and a half hours. Twelve hours post treatment a two cm ring of color change was observed. Three days post treatment, only five percent of the area of the transverse section remained unchanged. After seven days post treatment the color change was complete.
• Samples of CO treated meat of approximately 30 grams in weight or ground meat samples were stored for up to seven days at a temperature between 4 to 10°C. Samples of the CO treated meat were given to twelve starved cats (4kg per cat). Under these conditions, the meat samples were immediately consumed. No mal- effects were observed in any of the animals within 48 hours post consumption.
• Cats were fed fresh CO treated meat (250 grams per day per cat for one week).
• a control group was fed meat from the same source which was treated identically but with air instead of CO.
• the animals were continuously monitored by animal-tenders and showed regular behavior. The animals were found healthy by a house veterinarian.
• a blood sample was drawn from all the animals and the red blood cells were separated. The state of hemoglobin in these cells was analyzed spectrophotometrically as shown in Figure 3. From the spectra, it was found that the hemoglobin of both groups was completely in the oxy- hemoglobin form indicating no CO in the blood of the animals fed CO-treated meat.
• Meat packing Freshly slaughtered meat (beef) chunks of 0.5-1.5 Kg were cut into 25cm 2 pieces, 0.5-1.0 cm width (12-20 grams). Four samples were immediately submitted to bacterial count (as described below). The rest of the samples were treated with CO according to the method of the present invention. The gas pressure was 1.1 atmospheres and the volume of gas was equal to (100%) of the meat weight. The gas content was either air or 100% CO. The samples were preserved within a predetermined temperature range. Bacterial growth was measured at time intervals determined according to the temperature of preservation.
• the quality of the CO-treated meats depends on the amount of globin fractions converted to CO bound forms and their location. The location is important since bacterial growth starts on the surface of the CO-treated meats as does CO penetration.
• Assessment of the CO bound myoglobin and hemoglobin in the CO- treated meats was carried out using two parameters: (a) measurements of the circumference width of zones which underwent visible color change due to CO binding (CO-treated meats were successively cut transversely and the depth of the color-changed zone was measured with a ruler) and (b) assessment of the CC bound fraction of hemoglobin and myoglobin in CO-treated samples.
• Bacterial growth measurement was made according to international standards and carried out by a ISO 9000/IEC Guide 25 licensed bacteriological laboratory. As bacterial growth is mostly on the meat surface, the routine contamination tests at governmental laboratories relate to bacterial growth on a standard minimal area of 25cm 2 . The bacterial growth is then expressed as the number of bacteria per cm 2 . The most stringent standards allow a growth of up to 5x10 6 (6.7 in log scale) bacteria per cm 2 while the least strict ones consider a growth of up to 1x10 7 (7.0 in log scale) non-contaminated.
• the procedure entails treating a 25 cm surface area sample of the meat with 25 ml of aqueous solution.
• the bacterial content is introduced into the solution using a stomacher apparatus (Seward Lab U.K.). This suspension is then diluted in a ten fold series up to 10 "9 in 0.1 M phosphate buffer, pH 7.0.
• each dilution is applied to each of three types of 60mm growing plates: (a) containing plate count agar (PCA, Difco) incubated at 33°C ⁇ 0.2 for 48 hours to enable total viable aerobic count, (b) containing SPS agar (Difco) allowing clostridium growth, and (c) containing the same medium as a (a) allowing microaerophile growth.
• Type (b) and (c) plates were confined within sealed anaerobic jars supplied with gas generating kits (Oxoid, U.K.) and incubated for twenty four hours at 35°C. Bacterial colonies (up to 200 per plate) were counted using a colony counter.
• the maximal bacterial growth allowed for non-contaminated meat is 1x10 7 /cm 2 total viable aerobic bacteria and 1x10 4 /cm 2 of microaerophils.
• the shelf life of a meat in a non-contaminated form was determined by the duration until the above defined bacteria levels were reached ("preservation duration") .
• Table 2 represents the "preservation duration" under various conditions at which CO-treated meat retained pleasing odors while air preserved meat smelled badly.
• Type of Size Temperature range of Preservation duration meat preservation °C in days
• Meat samples were sealed in plastic bags under 100% CO at a pressure of about 1.1 atmosphere. CO volume was 30 + 20% of the meat volume. The bags were kept at 5°C.
• Figures 7-9 demonstrate a typical experiment in which meat was treated 24 hours after slaughter. A sample of calf meat weighing 18 pounds (about 8 Kg) was cut into two nine pound pieces having maximum length of 30 cm and maximum width of 20 cm. One of these samples was kept frozen at -18°C while the other was preserved in CO at 5°C. After three days, the bag was opened to release the unbound CO and the meat was left in the open air for half an hour. The meat sample was cut transversally at about one third of its length (7 cm from one edge). About 30%- of the meat radius (from surface to core) changed in color from dark to bright red (see Figure 7). Because any CO which reaches Hb/Mb bind quickly, the change in meat color serves as a measure of the CO diffusion rate.
• the meat samples were reassembled and repacked with CO. Following seven additional days at 5°C, the meat samples was again unpacked and cut transversally once in the middle of the sample, about 15cm from the edge (shown in Figure 8), and once closer to the end of the sample, at about one third of the sample's length (shown in Figure 9). As shown in the transverse cut in Figure 8, the meat was almost completely bright red except for a small dark red area shown by an arrow. This indicates a nearly complete diffusion or saturation of the CO from the surface to the core of the meat sample. The cut at one third of the sample's length shows that in this zone, oxy — Hb/Mb were completely converted to their carbomonoxy forms as shown in Figure 9.
• the measure CO level in PPB(m) was translated and expressed as CO PPM(c) released from 2 Kg of cooked meat (a family meal size) into a sealed room of 3.0 square meters (kitchen size dimensions). Considering the volume of one mole of gas at room temperature as 22.4 liters, the expected CO level was calculated as:
• Freshly drawn human blood was used. The red cells were washed and lysed in a hypotonic buffer. The mixture was centrifuged to separate cytosol from membranes. The concentration of hemoglobin in the solution was measured spectrophotometrically and was found to be 16.2 mM. The solution was sealed in a beaker and CO was gently flushed above the solution surface while stirring for 30 minutes. A few grains of dithionite ⁇ was added to consume any residual oxygen. The color of the solution turned bright red typical of carbomonoxy Hb. The solution was then left while stirring (for 15 minutes) to the open air to released dissolved CO. The Hb was identified spectrophotometrically as carbomonoxy Hb.
• the hemoglobin solution was cooked exactly as the meat and the amount of CO in the pot atmosphere was measured. From the concentration of hemoglobin (each heme molecule binds one CO molecule) the amount of CO molecules was calculated in the solution. From this amount and the pot volume, the expected PPM level of totally dissociated CO was calculated. As in the procedure of cooked meats the gas level in the pot was measured by the CO monitor. The ratio of measured to calculated CO level turned out to be 1.06 indicating that, within experimental error, all CO was indeed released from the Hb by cooking. TABLE 3: CO RELEASE FROM COCKED MEATS

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