cm STABMZM‘I‘ON OF PREP‘ACKAGED mom WT EY T‘EJEE USE 5F CARBON MM‘IOMDE Thai: 90? fine Wm 65 M. 3. MICHIGAN NATE COLLEGE William Eben Tawmend I955 masts This is to certify that the thesis entitled The Stabilization of Color of Prepackaged Frozen Meats by the Use of Carbon Monoxide presented by William E. Townsend has been accepted towards fulfillment of the requirements for M.S. Animal Husbandry degree in {yams Major profflr DateM / .5: / 9 5'25” 0-169 COLOR STABILIZATION 0F PREPACKAGED FROZEN MEAT BY THE USE OF CARBON MDNOXIDE By ‘Wllliam Eben Townsend AN ABSTRACT OF A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of ' MASTER OF SCIENCE Department of Animal Husbandry Year 1955 Approved {C}. W ‘ - 1:15:35 w ill 1am Townsend Meat is not a seasonal item such as fruits and vegetables. Freez- ing meat for long storage periods is not recommended under good commercial practices. Indications that prepackaging of fresh meat outs may be changing to prepackaged frozen meat cuts are very prevalent. “- In 1947 (1) only 13 frozen meat processors were in the country. By 1953 the number had increased to 138, an increase of more than 950%. Out- put of frozen meats increased from.five million pounds in 1938 to 125 million pounds in 1952. This indicates that the program for experimental adjust- ment of prepackaged frozen meat and the ground work for the "evolution” of frozen meat is slowly being laid by gradual introduction of frozen steaks, liver, etc. Prepackaging frozen meat will give greater recovery of bones, fat and trimmings which can be converted into mere useful and valuable products. There will be a difference in tranSportation, storage, handling and packag- ing costs in favor of either the packer, producer or processor. Centralized cutting, trimming, freezing and packaging of meat at the packing plant would eliminate shipment of bulk carcasses, save freight costs in favor of the consumer, producer or processor. The color of meat and meat products is of great importance in our system of meat marketing. Changes in color are largely utilized by the retailer and meat trade as indicators of freshness in meat. Consumers are very critical of meat color and discriminate against meat cuts that show discoloration. The method of prepackaging fresh and frozen meat gave rise to many technical problems, one of which.may be the retention of color in prepack- aged meat. Since it has been shown that color is a very important factor William Townsend in prepackaged fresh.meat sales, it would seem that the retention of a desirable red color would be of great importance for frozen meat sales. With this factor in mind the following study was carried out with three objectives in mind. 1. To investigate the stability of color in prepackaged frozen meat when treated with carbon monoxide. 2. To study-the effect of carbon monoxide concentration, temperature of treatment and period of exposure on the stability of color in prepack- aged frozen meat. 3. To study the effect of carbon monoxide on Pseudomonas 32, The longissimus dorsi muscle from.U. S. Good and Choice grade beef ribs was used in this study. These steaks were treated with carbon monoxide and measured for the stability of color during the frozen storage period. The color measurements were made in a matching booth under constant illumination in the freezer by the use of'Munsell disks of known color nota- tion. The color was matched by altering the disk mixture. By knowing the percent of each disk required to make the color match it was possible to calculate the Munsell hue, value and chroma of the:meat. The Nickerson (2) formula for index of fading results in a single number instead of three numbers hue, value, and chroma. Steaks were sprayed with Pseudomonas 32° to study the effect of carbon monoxide on bacteria. The steaks were swabbed before and after treatment with carbon monoxide. Results of the study showed the following : 1. The stability of color of prepackaged frozen meat was increased when treated with carbon monoxide. william Townsend 2. In general, those samples treated with 35-40% carbon monoxide gave the best results on the stability of color of meat. 3. The procedure of a high concentration of carbon monoxide for a short period of exposure was better than a low concentration of carbon monOXide for a long period of exposure i.e. 40% carbon monoxide for 5 minutes was better than 20% carbon monoxide for 15 minutes. 4. Carbon monoxide had no appreciable effect on Pseudomonas 32, References: 1. Anonymous 1954 1954 Packaging Shift Seen Spur to Frozen meats. Quick Frozen Foods, 16 (6)399 2. Nickerson,Dorothy 1946 Color Measurements and It's.Application To The Grading of Agricultural Products. U. S. Department of Agriculture, Miscellaneous Publication 580. COLOR STABILIZATION OF PREPACKAGED FROZEN MEAT BY THE USE OF CARBON MONOXIDE BY “filliam Eben Townsend A THESIS Submitted to the School of Graduate Studies of Michigan . State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Husbandry 1965 ACKNOWLEDGEMENTS The author wishes to express his very sincere thanks to Lyman J. Bratzler, Associate Professor of Animal Husbandry, under whose constant interest and thoughtful suggestions the experimental work and the writing of this thesis were accomplished. H3 is also indebted to Dr. Ralph Costilow, Department of Bacteriology and Public Health, for suggestions and ideas employed in the bacterio- logical phase of this investigation. To Lilly, his'wife, the author greatly appreciates her encouragement and help that has made this thesis a reality. 350651 TABLE OF CONTENTS IntrOdUCtioneeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee ReView 0f LiteraturBOOOOOOOOOO000......00.0.0.0... A. B. C. D. E. F. The Color of Fresh Lean Meat............. The COIOr Of Frozen Lean Meateeeeeeeeeoee Chemistry of Color Change in Meat........ Factors Influencing the Color Change of Frozen Meat.............................o 1) 2) 3) 4) 5) 5) Oxygen Pressure and Penetration..... EffeCt Of Packaging Material........ Effect of Storage in Different AtmospherBSeeeeeeeeeeeeeeeeeeeeeeeeo Effect of Time, Temperature, and Rel‘tive Humidity................... Effect of Freezing Methods.......... Effect Of Biological AgentSeeeeeeeee The Chemistry of Carbon Monoxide Hemo- g10binOOOOOOOOOIO...OOOOOOOOOOOOOOOOO0.0. Bacteria and Carbon Monoxide............. Purpose Of StudyOOCOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO Experimental Procedure............................ Part Ie The Stabilization of Color in Pre- packaged Frozen Meat By the Use of Carbon Monoxide..................... Part II. The Effect of Carbon Monoxide Con- centration, Period of Exposure, and Temperature of Treatment on the Stability of the Color of Prepackag- 0d Frozen Meat...................... 11 11 12 14 14 15 19 22 23 23 23 A. B. C. D. E. F. Part TABLE OF CONTENTS (Continued) Sampling Procedure....................... Gassing Procedure........................ Packaging Procedure...................... Conditions of Storage.................... Method of Obtaining Co1or lbadings During Storage........................... Method Of 0010? M6asurement.............. III. The Effect of Carbon Monoxide on P8°Ud0m0n‘3 if? eeeeeeeeeeeeeeeeeeee Results and Discussion............................ Part I. The Stabilization of Color in Pre- packaged Frozen Meat When Treated With Carbon MOHOXidOeeeeeeeeeoeeeeee Part II. The Effect of Carbon Monoxide Con- Part centration, Period of Exposure and Temperature of Treatment on the ‘ Stability of the Color of Prepackh aged Frozen Meat.................... III. The Effect of Carbon Monoxide on PSOUdomonas‘EE: eeeeeeeeeeeeeeeeeeee Summary-o......................................... BibliograthOOOOOOOOOOQ0....OOOOOOOOOOOOOOOOOOOOOO AppendixOCCOO0.0.....OQOOOOOOOOOOOOOOOOOCO0.00.... Page 23 24 24 24 26 26 27 29 29 32 48 53 57 ii INDEX TO FIGURES, TABLESLAND GRAPES Diagram 1 Diagram 2 Table I Table II Table III Photograph I Figure 1 Figures 2 & 2a Figures 3 & 3a Figures 4 & 4a Figure 5 Structural FormUI‘ Of Heme.......... Structural Formula of Heme Showing Attachment of Carbon Monoxide....... Effect of Temperature on the Stability 0f Carboxyhemoglobin................ Empiric Velocity Constants at Three Different Temperatures.............. Effect of Carbon Monoxide on B. pypcyaneus and Koch's Comma SEirilla...COO...OOOOOOOOOOOOOOOOOOC Front View of Gassing Equipment..... Stability of Color in Prepackaged Frozen Meat From 24 Hours to Nine Months.........................'..... Stability of Color in Prepackaged Frozen Meat Treated for 5 Minutes at 35-40%“ and 60-70°F With 20, 25, 30, 35 and 40% Carbon Monoxide.. Stability of Color in Prepackaged Frozen Meat Treated for 10 Minutes at 35-40°F and 60-70°F'With 20, 25, so, 35 and 40% Carbon Monoxide...... Stability of Color in Prepackaged Frozen Meat Treated for 15 Minutes at 35-40%" and 60-70°F With 20, 25, 30, 35 and 40% Carbon Monoxide...... Comparison of Color Stability of Samples Treated for 10 Minutes Eith 20 and 40% Carbon Monoxide at 35- 400? Hnd 60-700Feeeeeeeeeeeeeeeeeeee iii Page 8 16 18 19 20 25 31 34-35 37-38 40-41 43 Figure Figure Figure Figure Figure Figure 8a 9a 10 INDEX TO FIGURES, TABLES AND GRAPHS (Continued) Comparison of Color Stability of Samples Treated for 15 Minutes With 20 and 40% Carbon Monoxide at 35-400F and 60-700Feeeeeeeeeeeeeee Comparison of Color Stability of Samples Treated for 5 Minutes at 35-40°F and 60-700F With 40% Carbon Monoxide, With Samples Treated for 15 Minutes at 35-400F and 60-70°F With 20% Carbon Monoxide............. The Effect of 40% Carbon Monoxide for The for The for The for The for 90 Minutes on Effect of 20% 15 Mflnutes on Effect of 30% 15 Minutes on Effect of 40% 15 Minutes on Effect of 60% 30 Minutes 0n Pseudomonas £2, ... Carbon Monoxide Pseudomonas 3p. ... Carbon Monoxide Pseudomonas £2: ... Carbon Monoxide Pseudomonas _p. ... Carbon Monoxide Pseudomonas 22: ... Page 45 47 49 49 50 50 51 iv INTRODUCTION Meat is not a seasonal item such as fruits and vegetables. Freez- ing for long storage periods is not recommended under good commercial practice but frozen meat is prepared to be sold and used in a relatively short period of time. Indications that prepackaging of fresh meat cuts may be changing to prepackaged frozen meat cuts are very prevalent. (2) Trade sources point to the tremendous increase in frozen meat processors in the past seven years. In 1947 (2) only 13 frozen meat processors were in the country. By 1953 the number had reached 128, an increase of more than 950%. Out- put of frozen meats increased from five million pounds in 1938 to 125 million pounds in 1952. This indicates that the program for experimental processing of pre- packaged frozen meat has reached beyond the state of adjustment and that the ground work for the "evolution" of frozen meat is slowly being laid by gradual introduction of frozen steaks, livers, etc. Prepackaged frozen meats have many advantages, some of which may be shown as follows: 1. The yield of the packaged product from standard cuts will vary and will depend upon the amount of bone, fat, and trimmings removed. The dinner plate yield from the dressed carcass weight will be less than 60 percent. There is a bone, fat, trimming and cooking loss of more than 40 percent. Under the present system of prepackaged fresh meat the bone, fat and trimmings are mainly utilized as inedible products worth much less than comparable edible products. 2. By prepackaging the meat at the packing plant or centralized units of large retailing organizations, the bone, fat and trimmings may -2- be converted into more useful and valuable products. 3. There would be a difference in transportation, storage, handl- ing and packaging costs in favor of either the consumer, producer or processor. Centralized cutting, trimming, freezing and packaging of meat at the packing plant would eliminate shipment of bulk carcasses, save freight costs and give packers greater recovery of products that otherwise would not be obtained. The color of meat and meat products is of great importance in our system of meat marketing. Changes in color are largely used by the re- tailer and meat trade as indicators of freshness in meat. Consumers are very critical of meat color and discriminate against meat cuts that show discoloration. The methods of prepackaging fresh and frozen meat give rise to many technical problems, one of which may be the retention of color in pre- packaged meat. Other factors that would seem.to improve the sales of frozen meat in transparent wrapping materials are visibility, neatness of cut and tightness of wrap. Since it has been shown (19) that color is a very important factor in prepackaged fresh meat sales, it would seem that the retention of a desirable red color would be of great importance for frozen meat sales. “nth this factor in.mind the following study was carried out with three objectives in mind. 1. To investigate the stability of color in prepackaged frozen meat by treatment with carbon monoxide. 2. To study the effect of various concentrations, temperature of treatment and period of exposure to carbon monoxide on the stability of color in prepackaged frozen meat. 3. To study the effect of carbon monoxide on Pseudomona§_gp. REVIEW OF LITERATURE In reviewing the literature on the color of meat, it becomes apparent that reports dealing with color changes of frozen meat p2£_ §3_are limited. Some work has been done using carbon monoxide to convert the heme pigments to their stable carbon monoxide derivatives. Regier et. al. (30) reported that stability of the color of "freeze-dried" beef during storage was increased by the conversion of the heme pigments to their stable carbon monoxide derivatives. Considerable work'has been done with solutions of hemoglobin and myoglobin, the muscle pigments of meat, treated with carbon mon- oxide in studying the kinetic and physiological reactions of carbon monoxide, but little work has been done in studying the stability of color in meat by the use of carbon monoxide. Kennedy and Whipple (18) made a study to determine if there were any differences between blood and muscle hemoglobin. Theseauthors found that muscle hemoglobin was indistinguishable from.blood hemoglogin. 'While literature reports little information concerning the use of carbon monoxide as a means of stabilizing the color in meat, the author has reported the review of literature from articles on carbon monoxide and hemoglobin which he believes can be applied to it. It:was thought that the reactions of hemoglobin with carbon monoxide may be applied towards a better understanding of meat color changes since muscle hemo- globin and hemoglobin have the same amount of iron content in the mole- cule. A. The Color of Fresh Egan Heat Levers (19) stated that the constituent of meat responsible for its -5- color is known as myoglobin, or muscle hemoglobin. 'While muscle hemo- globin and blood hemoglobin are not identical chemically, they react similarly and for the purpose of this study myoglobin will be used in- stead of muscle hemoglobin in this review; Brooks (4) reported that the reddish color (hue) of fresh lean meat is due to the pigment, myoglobin found within the muscle fibers. The depth of color (which corresponds to two other attributes of color, brilliance and saturation) depended on the concentration of hemoglobin and on the thickness of tissue through which light is reflected to the eye by Optical heterogeneties within the muscle. The thicker the surface layer and the greater the concentration of pigment, the deeper was the color. . Whipple's (43,44) work showed that the amount of myoglobin present appeared to be independent of the degree of blood removed and dependent on the type of muscle in.the animal. Brooks (3) stated that in fresh lean meat exposed to air the pig- ment is present in two forms; cxyhemoglobin which is found in the surfac. layer of the meat, while the underlying tissue contains no dissolved oxygen and the pigment is present as purplish hemoglobin. He also re~ ported that the color (hue) depends on the thickness of the oxygen region, if it is greater than the effective thickness through which light is reflected the color is a bright red. Robertson (32) reported that uncut beef is a deep red—purple, the color of the unoxygenated pigment myoglobin. The desirable red color of beef with which the consumer is familar does not exist at the time of cutting. This red color is a surface effect caused by the reaction of fl -6- the oxygen in the air with the myoglobin of the muscle to form the red colored pigment oxymyoglobin. Andersen(l) stated that myoglobin which has not been exposed to air is purple in color. When exposed to air it takes up a molecule of oxygen forming cxymyoglobin which is scarlet in color. Hoagland (14) stated that the red color of fresh lean meat ex- posed to air was due to oxyhemcglobin, which is one of the constituents of the blood remaining in the tissue. Levers (19) stated that the color of myoglobin is dark red or purple and is responsible for the dark color in the interior of meat when first cut, and that oxymyoglobin is bright red and is responsible for the attractive bright red color of meat. Mbran (23) also noted that the normal red color of meat is due to the presence of the red pigment oxymyoglobin. Anderson (1) noted that met hemoglobin accounted for meat turning brown under certain conditions. Brooks (3) stated that in lean meat the oxidation of myoglobin to the brown pigment motmyoglobin takes place only in the surface layer of the tissue containing dissolved oxygen. Brooks (4) found that the color of lean meat is brownish when roughly 60 percent of the myoglobin present in.the superficial layer has been oxidized to metmycglobin.. B. The Color of Frozen Lean Meat meat packaged in an oxygen permeable wrap has a bright red color. This is due to the fact that meat packaged in an oxygen permeable wrap is able to come in contact with the oxygen of the air and that the myo- glcbin can be changed to oxymyoglobin. In an oxygen impermeable wrap -7- there is not enough oxygen available to combine with myoglobin to give oxymyoglobin and the resulting bright red color. Robertson (32) re- ported that meat progressively darkens while in cold storage. ‘While this surface darkening does not impair the palatability of the meat, it does render many cuts unattractive. Therefore, visibility, freedom from frost and color retention are important aids for frozen meat identity. He stated that the brown color in frozen meat is due to methemcglobin.. Moran (23) noted that simple desiccation, particularly of frozen meat, leads to apparent discoloration. He also stated that the control of ”bloom? is probably the most important and at the same time the most difficult to maintain in the storage of meat. The author has noted in a local grocery store that frozen round steaks, stored for long periods,'were very dark in color. Tressler and Evers (39) noted that the red pigment, hemoglobin in the surface layer of meat exposed to the action of air turned brown due to oxidation of oxyhemoglobin to :methemcglobin causing a gradual change in color from red to brown. Brooks (4) mentioned that the formation of the brown color of’ metmyoglobin in frozen meat is much less important ( unless the time of storage is long) than the loss of color due to excessive drying. C. Chemistgy of Color Change In Heat In order to understand the effect of various conditions on the color ’change of meat it is essential that one have a good knowledge of the chemistry of the color change of meat. Levers (19) stated that hemoglobin consists of two parts: heme, the iron-containing fraction, and glebin, the protein fraction. Von Gettingen (42) showed the structrual formula of home with the glebin attached as -3- indicated in the following diagram: Diagram 1. This compound consists of four pyrrole rings which make up the porphyrin ring in combination with iron (Fe). There are three main forms of myoglcbin which explain the color changes of meat; myoglobin (purple), oxymyoglobin (red), and metmyoglobin (brown). The color of the meat is greatly influenced by the relative concentrations of these compounds. Pirie, according to Haurowitz (13), illustrated the structure of hemoglobin and important reactions of the iron atom by the following formula. The porphyrin ring is symbolized by the four nitrogen atoms of the pyrrole rings. N N ’ N H N N / / / Globin ——-a Fe , Globin /Fe -—- 02 Globin —d’e N N H OH H N N Hemoglobin Oxyhemoglobin Moth emoglobin -9- The iron is in the ferrous (Fe / K ) state in hemoglobin (19). The color of this compound is dark red or purple and is responsible for the dark color in the interior of meat when first out. In oxyhemoglobin the iron is still in the ferrous (Fe ,1 ,1) state, but this compound contains more oxygen than hemoglobin. The oxygen is held only in loose combination. No true oxidation has taken place, only oxygena- tion. Oxyhemoglobin is bright red and is responsible for the attractive bright red color of meat. In.methemoglobin the iron contains no more oxygen than hemoglobin but the iron has been oxidized to the ferric (Fe / / /) state, a true oxida- tion. The material is dark brown in color. Lovers (19) stated that oxyhemoglobin is not an intermediate in the formation of methemoglobin. Methemoglobin and hemoglobin form.a reversible oxidation-reduction system of the ferric-ferrous type. He gave the path of the discoloration reaction as: oxyhemoglobin:;::=§ reduced hemoglobin --‘_=-‘_methemoglobin. D. Factors Influencing the Color Change-of Frozen meat 1. Oxygen Pressure and Penetration. Neill (24) has shown that there was no evidence that mothomoglobin was formed in the complete absence of oxygen since in this condition were there were no oxidizing agents formed to oxidize hemoglobin to methemoglobin. Brooks (5) also showed that when hemoglobin was stored in pure nitrogen storage there was no formation of methemoglobin. From this work it was apparent that oxygen was necessary for the formation of methemoglobin, the dark colored pigment of meat. Robertson (32) stated that since discolora- tion is actually an oxidative reaction, the control of discoloration is the -10- elimination of air. He stated that a low oxygen transmission rate film is very essential to increase frozen fresh meat qualities. He also stated that one of the limiting factors in meat freezer storage life is to be found in the degree of oxygen protection offered by the packaging material. The relation between oxygen pressure and the rate of methemoglobin formation was responsible for the rapid discoloration of tissue stored at 000(320F) in gases containing a small amount of oxygen. Brooks (5) found that tissue had a small oxygen up-take so that given sufficient time a ”steady state" was reached where the depth of oxygen penetration was de- termined by the rate of diffusion of oxygen into the tissues and the oxygen consumption of the tissue. He showed that the depth of oxygen penetration was determined by the oxygen pressure in atmospheres at the surface of the tissue, the diffusion coefficient of oxygen through the tissue and the oxygen uptake of the tissue. Brooks (3) found that the depth of oxygen penetration showed a slow increase with time, and a rise in temperature decreased the depth. He found that the depth of oxygen penetration varied from.2 to 5 mm. The depth of oxygen penetration was proportional to the square root of the oxygen pressure in atmospheres. Brooks (5) gave the following formula for finding the depth of oxygen a=\[2£A-—D—.— where d l depth of oxygen penetration: Co the pressure of oxygen at penetration: the surface of the tissue and 2 and _A_ are respectively the coefficients of its diffusion through the tissue and consumption. Therefore, the dissolved oxygen is present only in a superficial layer a few m thick. Discoloration is confined to the superficial layer. 2. Effect of Packaging Material Robertson (32) stated that visibility and tightness of wrap are necessary requirements for frozen meat. He gave the essential features of a packaging material as follows: low moisture vapor transmission, to prevent dehydration and a low oxygen transmission rate. He stated that a packaging material with a low moisture vapor transmission rate is essen- tial because meat bloom is an effect of surface moisture over red meat color. He further mentioned that various color effects can be observed as surface dehydration proceeds on the packaged product in storage. A gray or dark surface color can be noticed in the later stages of dehydra- tion. Ordinarily this late stage of dehydration is known as ”freezer burn". Robertson (32) stated that the wrapping of the product should be so well done that it is free of air packets, which cause the loss of visibility when moisture condenses in the cavity forming a snow or frost. Moran (23) stated that simple desiccation, particularly of frozen meats, leads to apparent discoloration. Levers (19) stated that in using an oxygen imperme- able wrap the supply of oxygen is cut off and the rate of oxidation to methemoglobin i.e. the rate of discoloration, is greatly increased. Metho- moglobin is formed rapidly when the pressure of oxygen around the meat is reduced by the packaging material. 3. Effect of Storggg in Different Aunthpheres Mangel (20) stored samples under atmospheres of nitrogen, oxygen, and carbon dioxide, with air as the control. She found no significant difference -12- but the methemoglobin formation tended to be slower when the tissue was stored under oxygen, than under nitrogen, carbon dioxide or air. Robertson (32) stated that nitrogen may be used to displace the air in a package and thereby prevent discoloration. Moran (23) used a carbon dioxide treatment and doubled the storage life of meat in the air. His purpose was to inhibit molds rather than to maintain color. He found 40% carbon dioxide an effective concentration for molds. In actual practice he found that carbon dioxide in concentra- tions beyond 20% caused a rapid production of methemoglobin in the surface muscle. Brook: (3) noted that the rate of oxidation of hemoglobin in muscle is not affected to a significant extent by concentrations of carbon dioxide below 20%. Hence, if other conditions of storage are the same the color change of meat in air and in air containing 20% carbon dioxide should be the same. Rikert (31) flushed packaged samples with carbon dioxide and nitrogen before evacuating. This resulted in less initial darkening than that which occurred in samples evacuated without flushing. He also stored samples at atmospheric pressure in carbon dioxide and nitrOgen. This had a detrimental effect on the top surface color but improved the bottom surface color when compared with samples stored in air. 4. Effect of Time, Temperature and Relative Humidity At -10°c (14°F) Brooks (4) found no visible discoloration of lean meat stored for sixteen weeks. .At -1.4°c (29.6°F) there was no discoloration owing to the formation of methemoglobin until 40-50 days from killing. langel (20) stored samples at temperatures ranging from -12°c (lO.4°F) -l3— to - 24°C (-ll.2°F). She found that methemoglobin formation was slower in samples stored at the higher temperature than in samples stored at the lower temperatures. Ramsbottomfs (27) work would indicate the opposite. He stored fresh beef at six different temperatures ranging from -2o°r to 26°F packaged in Du Pont zoo M.S.A.T. #87 cellophane. The product stored at 26°F was discolored in less than 30 days, whereas the product stored at -20°F was still scored good in color and appear- ance after one year's storage. He concluded the lower the temperature of storage the longer the storage life. Tressler and Evers (39) stated that frozen meat changes in color from.red to brown at a higher temperature of storage. At 29°F, d13- coloration of beef was noticeable after eight weeks, whereas at 14°F there was no noticeable change in color until after 12 weeks. Ramsbottom.and Koonz (28) determined the relative concentration of oxyhemoglobin and methemoglobin in the surface of tissues stored for one year fit 109? and '300F. They plotted spectra curves from extracts of the surface tissue from spectrophotometric readings. The curves for steaks stored at 10°F for one year were quite similar to the curve for methemoglobin. The curves for steaks stored at - 30"? indicated a mixturecm' of oxyhemoglobin and methemoglobin. This indicated that a greater oxidation and consequently darker beef occurred in the tissue at lODF than at -30°F. Jensen (16) stated that freezing should be done at a rate which gives the meat a bright color. A temperature of -lO°F or lower results in a satisfactory color when freezing by the blast freezing method. He agreed with Robertson in that slowly frozen.meats are dark in color. Brooks (5) noted that the formation of methemoglobin in frozen meat -l4- is much less important (unless the time of storage is long) than the loss of color due to excessive drying. He said that crystals of ice in the superficial layer evaporate, and the small bulbs of air left behind scatter the incident light. Temperature is very important in relation to the formation of mist or "cavity ice". Robertson (32) gave, for example, the influence of temperature on the condensation of moisture in the cavities of meat. He stated that air at 60°F contains 77.29 grains of moisture per pound of air, whereas at -lOQF the air contains 3.206 grains of moisture per pound of air. He also noted that the moisture content of the entrapped air can fluctuate with changes in the storage temperature. He noted that if the temperature went from.O°F to 59p that there'uas an increase of 5.50 to 7.2 grains of’moisture per pound of air. 5. Effect of Freezing Methods Robertson (32) noted that at -50°F a very rapid loss of surface meat color and appearance when the meat was frozen between metal plates in the air blast freezer. He noted that when still air was used for freezing at 0°F the meet was dark in color. He concluded that the dark color in meat frozen by still air was due to the fact that the rate of freezing was so slow that all of the original meat color was lost. 6. Effect of Biological Agents ‘Voegeli (40) and Butler (6) have summarized the investigations of ‘workers who studied the effect of biological agents on the color of fresh meat. 'Voegeli (40) mentioned that these workers found that Pneumococci reduced methemoglobin to hemoglobin in complete absence of oxygen and -15- that anaerobic bacilli have the ability to oxidize hemoglobin. Butler (6) in his work, found that bacteria commonly found on meat cuts caused discoloration. The main effect, an increase in the rate of metmyoglobin. formation, was the greatest during the logarithmic growth phase. According to Tanner (38) microbial development is markedly retarded but not entirely eliminated by freezing. Sulzbacher (37) stated that the popularity of frozen food during the past two decades has occasioned con- siderable interest in the growth and survival of microorganisms during freezing and frozen storage. Haines (10) recognized that the growfliof Pseudomonas gp at sub- freezing temperatures was likely to play a part in meat spoilage. E. The Chemistry of Carbon Monoxide Hemoglobin Carbon monoxide, CO, has a molecular weight of 28.01, a specific gravity of 0.814 at 4°C and a density of 1.250 grams per liter(at 0p and 760 mm. Hg.). It is a colorless and odorless gas except in high concen- trations (7S-lOQ%) when it has an appreciable garlic like odor. It is not very soluble in water (42) Hemoglobin has the very interesting property of uniting with carbon monoxide. According to von Oettingen (42), Bernard and Hoppe-Seyler found that blood of carbon mononide poisoned cadavers had a cherry red color. They found that this color is caused by a new pigment resulting from the combination of carbon monoxide with hemoglobin. They found this pigment resistant to oxidation and putrefaction. Anderson (1) differed somewhat from Bernard and Happe-Seyler concern— ing the color of carboxyhemoglobin. He stated that carboxyhemoglobin is -15- bluish red in color. Ramsey and Eilman (29) noted that carbon monoxide hemoglobin im- parts to the blood a bright cherry red color both in venous and arterial blood. This also agrees with the report of Penrod and Baker (26). According to von Oettingen (42), Hoppe-Seyler in 1889 assumed that in this reaction one molecule of iron in the hemoglobin molecule reacted with one molecule of carbon monoxide. Carbon monoxide hemoglobin can be prepared by treating hemoglobin with carbon monoxide and is represented by the structural formula in diagram.2. H C HC HhGlobin 131‘ng 2 a The affinity of hemoglobin for carbon monoxide is about 250-300 times greater than the affinity for oxygen (15,22,29,42). This affinity is due to the difference in the velocity of the reaction and the combination of carbon monoxide with hemoglobin which occurs more rapidly than with oxygen (42). Howell (15) noted that hemoglobin can be saturated at very low pres- sures of carbon monoxide and that the rate of reaction of hemoglobin with carbon.monoxide is much slower than with oxygen. He stated that carbon -17- monoxide behaves toward hemoglobin essentially as if it were an isotope of oxygen. Penrod and Baker (26) give the following general reaction of carbon monoxide'with hemoglobin. HbCz / co: ‘Hbco ,1 oz Von Oettingen (42) reported that carbon monoxide is a reversible combination of carbon monoxide with hemoglobin. Rbughton (33) gave the following reaction frmm a mass-action stand- point of the reversible combination of carbon monoxide with hemoglobin. 1 mole C0 / 1 mole Hb \ ‘ 1 mole COHb / 1 mole Oz Hartridge (12) studied the effect of various conditions on carbon monoxide hemoglobin. He found carbon monoxide hanoglobin may become un- stable in sunlight and the carbon monoxide may be split off the heme- molecule. If this occurs, then the hemoglobin may be changed to methemo- globin and the reaction is in this way madeirreversible. Although carbon monoxide hemoglobin may in many respects be more stable than oxyhemoglobin, Haldane and Smith (11) observed that the equilibrium reaction between car- bon monoxide and hemoglobin is displaced markedly to the left by light. Hartridge (12) stated that there is a decrease in saturation of hemo- globin with carbon monoxide of 0.5% for every degree rise in temperature. Using only a portion of Hartridges's (12) table which pertains to the effect of temperature on the stability of carbon monoxide hemoglobin the stability of carbon monoxide hemoglobin is shown in Table I. Roughton (35) studied the kinetics of hemoglobin and carbon monoxide and found values of 1' (an empiric velocity constant) at three different temperatures as shown in table II. -18- Table Ia Effect of Temperature on the Stability of Carboxyhemoglobin Temperature Amount of Saturation (%) 14°C 71 66 57 70°C 41 37 33 Table 11. Temperature °C 1' 7.2 118 17.7 252 33.3 747 Roughton (33) noted that the rate of reaction of carbon monoxide with hemoglobin at pH 7.2 and pH 5.6 are about the same whereas the velocity at pH 10 is about 50% faster. Roughton (34) stated that the great affinity of carbon monoxide with hemoglobin is due to the extreme slowness that carbon monoxide hemoglobin dissocates as compared with oxyhemoglobin. He stated that the rate of dissociation of carbon monoxide hemoglobin is about one thousandth of that for oxyhemoglobin although the affinity is only about 20 times as great. Hemoglobin dissociates at a rate of about one-tenthousandth of that for oxygen, the carbon monoxide affinity being about 250 times that of oxygen. According to Ramsey and Eilman (29), Hill and Barcroft noted that carbon monoxide combines more readily with unsaturated oxyhemoglobin, -19- that is, hemoglobin will take up more carbon monoxide at a given tension if a little oxygen is present than if oxygen is completely absent. Ramsey and Eilman (29) also noted that blood containing carbon mon- oxide hemoglobin may be deprived of this gas by submitting it to diminish- ed pressure or by passing air or oxygen through it for some time. Two interesting studies were carried out by Ramsey and Eilman(29). They carried on an experiment to study the effects of carbon monoxide after death on guinea pigs. Four guinea pigs were sacrificed and then injected with laked blood saturated with carbon monoxide from illuminating gas. Twenty-four hours later the animals were autopsied and showed similar appear- ances to those that had died from inhalation of the gas. In another experi- ment two guinea pigs were embalmed for twenty-four hours and placed in an atmosphere of illuminating gas. The tissue and the blood showed marked evidence of carbon monoxide. These experiments indicated that muscle tissue will take up carbon monoxide even after death of the animal. F. Bacteria and Carbon Monoxide “urburg (45) has shown that carbon monoxide at a high partial pressure inhibits the respiration of yeasts and cocci cells. Fisher (7) stated that such gases as carbon monoxide, hydrogen and nitric oxide arrest the growth of bacteria in agar cultures exposed to a slow'stream of the gas, but that the method has no practical value. Seelander (36) studied the effect of carbon monoxide on Mucor, AsEer- gillus and Penicillum. He concluded in general that it is injurious. The injury manifests itself in the inhibition of spore germination, of vegeta- tive growth and of spore production. Frankland (8) found Pseudomonas_pyocyanea and'Vibrio chlorea were in- -20- hibited by carbon monoxide, although not all of the cells of the vibrio were destroyed even after a week. He used a damp-chamber which was made by using a flat porcelain dish covered over with a glass bell-jar. Mercury'was placed into the dish, thus forming an effectual seal and sterilized water being poured over the mercury. By the means of a hose the air was forced out of the damp—chamber and replaced by the desired gas. He found after three days exposure to air that growth commenced again. He concluded that carbonic oxide effectually stopped development but that the effect was only temporary. Table III, below,is a partial table of the results of Frankland's 'work showing the effect of carbonic oxide on §._pyocyaneusand Koch's Comma Spirilla. Table III Efppyogypneus No. of Colonies from one cc. of mixture Air Plate CO Plato After 4 days After 8 days a) 28,952 0 b) 27,794 0 Koch's Comma Spirilla Air Plate CO Plate After 4 days .After 8 days 100 48 Frobisher (9) stated that Carboxydomonas oligocarbpphila oxidizes carbon monoxide and oxygen to carbon dioxide. He stated they are small, -21- motile rods growing in swamps and are autotrophes. Keilin (17) stated that cytochromes which are present in yeast cells do not combine with carbon monoxide. The ordinary (unbound) hematin which is also present in yeast cells has, on the contrary, a much greater affinity for carbon monoxide than for oxygen. However, when these solu- tions were exposed to light, the carbon monoxide was dissociated and the oxidase became active again. Keilin (17) described cytochrome as an inter- cellular respiratory catalyst common to animals, bacteria, yeasts and higher plants. It is easily oxidized with air and reduced by the normal activity of cells or by a chemical reducer. Mefferd and Matney (21) reported that radiation damage to biological systems can be reduced by manipulations which lower the oxidation state 'within the cell or its enviroment. This may be accomplished by reducing the oxygen. Aerobic cells were markedly protected by carbon monoxide in which there were thirteen times more survivors of carbon monoxide treated aerobic suspensions than nontreated controls at twenty second irradiations. 1:. ‘n .41. or” 1|.Piid‘ .nlhv'hwr.fi PURPOSE OF STUDY The main purpose of this investigation was to study the stability of color in prepackaged frozen meat when treated with carbon monoxide. Finding that carbon monoxide will retain the color in prepackaged frozen meat, further investigation was carried on to determine the effect of the conditions of temperature of treatment, concentration and period of exposure to carbon monoxide on the stability of color in pre- packaged frozen meat. It was not the purpose of this study to give meat an artificial red color, but to maintain the red color of the meat while in storage. In conjunction with this study an investigation was carried out to see what effect carbon monoxide had on Pseudomonas s . EXPERIMENTAL PROCEDURE The longissimus dorsi muscle from U. 8. Good and Choice grade beef ribs was used in this study. All steaks were cut in the cooler at 35°F. PART I The Stabilization of Color In Prepackaged Frozen Meat When Treated'flith Carbon Monoxide. In this part of the study a preliminary trial was run to see if the color of carbon monoxide treated meat would remain stable during the frozen storage period. Two boneless steaks were used. One steak was treated with carbon monoxide and the other served as control. Carbon monoxide treatment of the steaks was accomplished by evacuating some of the air from a closed vessel and replacing the air removed with carbon monoxide. Following the carbon monoxide treatment, the treated and control steak 'were each put into a Cry-OéVac plastic bag. The bags were vacuumized, tied and shrunk in a water bath at 190°F which reduced the size of the bag approximately one-third, thus giving the steaks a tight wrap. The steaks were placed in a freezer at 20F to study the stability of the color during the frozen period. PART II The Effect of Carbon Monoxide Concentrations, Temperature of Treatment and Period of Exposure 0n the Stability of Color in Prepackaged Frozen Meat. A. Samplipg Procedure Boneless rib steaks from one cattle cut 1-l/4 inches thick and approxi- mately 4x6 inches in size were used in this part of the study. Five trials ranging from Trial NAeNE were heed. Six steak: were used -24- per trial. Three steaks in each trial were treated at 35-40°F and three at 60-700F with carbon monoxide. The concentrations of carbon monoxide used per trial was as follows: Trial NA With 20‘% carbon monoxide; Trial NB with 25 % carbon monoxide; Trial NC with 30 % carbon monoxide; Trial ND with 35 % carbon monoxide and Trial NE with 40 % carbon monoxide. Exposure periods were 5, 10 and 15 minutes. One steak served as control for the entire group. B. Gassing Procedure .A desiccator wasxmmd as the gassing chamber. Calculations involving barometric pressure, temperature and desired concentration of carbon mon- oxide were used to determine the amount of air to be evaccuated from the desiccator in order to give the desired concentration of carbon monoxide. The equipment used to treat the meat samples with carbon monoxide is shown in the photograph on page 25. The equipment consists of a desiccator, manometer, gas bag, water aspirator, hose and valves. C. Packaging Procedure Following the treatment with carbon monoxide, the steaks were placed in Cry-O-Vac plastic bags. The bags were vacuumized, tied and quickly dipped in and out of a tank of water at 190%. This shrunk the bag about one-third, and gave the packaged meat a very tight wrap. D. Conditions of Storage The steaks were stored in a freezer at 2°F for twelve weeks with color readings taken at various intervals of time during the storage period. -25.. - .....__ l.-- e..,- 1'4 -26- E. Method of Obtaining Color Readings During_Storage Color readings were taken by the use of a matching booth constructed by Voegeli (40). To eliminate condensation on package surfaces it was necessary to move the matching booth into the freezer. Samples were read by placing the sample on a fitted plywood box on the sample stand. This made it possible to remove the sample and still have the same sample area to be viewed in matching the color of the meat from time to time. This was found to be important as different areas of the sample gave different color readings depending upon the concentration of intramuscular fat. By adjusting the sample so that the top surface of the meat was the same level as the disks equal viewing areas of the sample Iand disk mixture were obtained. Adjustment of the Lhnsell disks was continued until a satisfactory match with the sample was obtained. Upon reaching a satisfactory match of the sample the area of this disk mixture was recorded. F. Method of Color measurement Detailed instructions were given by voegeli (40) on the application of disk calorimetry to color measurements of meat. The Munsell system was applied as described by Nickerson (25). The hue, value, and chroma were combined into one number by the use of the Nickerson (25) formula for index of fading: I: c (2AH)+ 6AV+ disc 5 where: chroma of sample C H : difference in hue between sample and standard V difference in value between sample and standard C = difference in chroma betvmen sample and standard -27.. The standard selected for the plotting of color readings of frozen meat was hue 7.0, value 4.0, and chroma 8.0, which was approximately the color of some very bright steaks obtained by Butler (6) following treat- ment with oxygen under pressure. By using the Nickerson (25) formula it was possible to find the position of subsequent sample readings in relation to the standard. These positions were plotted against time to show the color stability of pre- packaged frozen meat treated with carbon monoxide during storage. PART III The Effect of Carbon MOnoxide on Pseudomonas 22' Four boneless rib steaks were used in each trial. Five trials were run using various periods of exposure and concentration of carbon monoxide. A preliminary test showed no inhibitory effect of carbon monoxide on the microbial population of meat. Therefore, the study was confined to the use of a single species of organism commonly found on meat. This organism.known as Pseudomonas sp was used for the remainder of the study. This was the same organism.that Butler (6) used in his study of the effect of bacteria on the color of pre- packaged fresh retail beef cuts. All steaks were cut in the cooler at 35°F, with the knife bging tho- roughly washed with detergent and finally dipped into boiling water before each repeated use. - The steaks were sprayed with a 24 hour broth culture of Pseudomonas sp_ by the use of a sterile atomizer. Two steaks ( S-l, S-2) were swabbed before and after the treatment -28.- with carbon monoxide in Trials 1, II, and III. In Trials 78 and By-l, three steaks (S-l, 3-2 and S-3) were swabbed before and after gassing while one steak ( 8-4 ) served as control; i.e. that steak was swabbed before but not after the gassing procedure. metal cutouts of one square inch were made in a 3x3 inch piece of stainless steel as used by Voegeli (41) Using sterile cotton swabs, swabbings were taken by throughly rubbing the exposed area through the opening of the metal cutout. These cutouts were sanitized before each repeated use by washing in a detergent followed by dipping in a 0.1 percent solution of mercuric chloride (corrosive sublimate) for 5-7 minutes and rinsed with distilled water. The cotton swabs were placed in dilution blanks containing 10 ml. of sterile saline. These were shaken until the cotton swabs were well fluffed. The samples 'were plated out on Tryptone glucose extract agar (Difco) at various dilu- tions. Incubation was at room temperature 20°C (750F) and at refrigera- tion temperature 1.600 (34°F). Colony counts were made at the end of 72, 96, and 120 hours for those plates incubated at room.temperature and at the end of 7 and 14 days for those plates incubated at refrigeration temperature. The number of organisms counted represent organism.per square inch of surface sampled. The counts obtained after 120 hours and twO'weeks are shown in Appendix H. . The results are shown by the method used by Haines (10) in which the counts were converted to the log of the number of organisms per square inch. -29- RESULTS AND DISCUSSION Conversion of the data in Part I and II to the Munsell renotation and index of fading was done by the method as described by Nickerson (25). In order to determine the relation between successive readings, the readings were plotted using the Nickerson (25) formula for index of fad- ind to express the stability of color in terms of a single number instead of in three values of hue, value, and chroma. Using this formula, sample readings were compared to a standard, thus making it possible to determine the stability of color of prepackaged frozen meat treated with carbon mon- oxide. The.standard selected was hue 7.0, value 4.0, and chroma 8.0, which was approximately the color of some very bright steaks measured after treat- ment with oxygen under pressure. These readings were plotted against time to show the stability of color in prepackaged frozen meat treated with carbon monoxide during storage. Part I. The Stabilization of Color in Prepackaged Frozen Meat When Treated With Carbon Monoxide. The results of part one gave a general indication of the stability of color in prepackaged frozen meat treated with carbon monoxide for a storage period of nine months. Figure 1 illustrates the stability of the color in prepackaged frozen meat treated with carbon monoxide. The results Showed that the stability of the red color of meat was held until about 5-6 months of storage, which is the normal suggested storage period of beef. After six months the treatedgsample darkened at about the same rate as the control sample. The treated sample was closer to the standard all during the storage period than the control sample. Visual observations showed a marked difference between the sample treat- ed with carbon monoxide and the control sample. The initial color reading ~30- of the meat treated with carbon monoxide showed a difference of two units nearer the standard when compared to the control sample. The initial color reading for the treated sample was 15 units from standard as compared to 17 units from standard for the control. The color remained relatively stable until about 5—6 months, at which time it gradually darkened, and changed frmm the original 15 units from standard to 19 units from standard. The control, however, increas- ed from.the original 17 units from standard to 26 units from standard, a difference of 9 units as compared to 4 units for the treated sample. Tabulated data in Appendix A for these two samples show that carbon monoxide had a effect on the chroma (saturation) of meat color. The value of chroma was 4.3 for the initial color reading and 3.4 after the nine month storage period. The value of chroma for the control sample was 3.7 for the initial color reading and 2.1 after the nine month :rtorage period. The value of chroma for the treated sample at the end 01? the storage period was approximately the same as that for the control a1: the beginning of the storage period. sauces ea mane m m b m m e n N T .r x + e x x is t - 0 AUG defines m on 930: «m seam puma mascara eemsxosmmam ma. .SHoo mo hpflapepm u H shaman” 1T 3 \ o\ /O\ \‘l \ \\l\ \l... .\ \ eHmEsm Hoapmoo I. II III ovfinocofi Gopheo 5..."? 60960.3. IT 10m 11 mm 1r Om mm. pJBpuaqs mos; satun 0's/b°v HO'L ‘m t . g. - x .V...‘¢,.¢"‘ .. W Part IIe The Effect of Carbon Monoxide Concentration,Period of Exposure and Temperature of Treatment on the Stability of Color of Prepackaged Frozen Meat. A. Prepackaged Samples Treated For 5 Minutes at 35-4001? and EEO-70°F With 20, 25, 30, 35 and 40% Carbon Monoxide. Results of samples treated for 5 minutes at the two temperature with 20 to 40% carbon monoxide are shown in figures 2 and 2a. The results of these treatments seem to show what one would not expect to find. One would expect that the samples treated with a higher concentration 1' car- bon monoxide would be closer to the standard than those treated with a low concentration of carbon monoxide. This study showed that those samples treated with 20, 30, 40% carbon monoxide were in general closer to the standard than those treated with 35 and 25% carbon monoxide. However, all samples treated at 35-4001“ tended to maintain the color during the storage period. There did not seem to be too much difference in the color of those samples treated for 5 minutes and the control sample. These results would then seem to indicate that the stability of the 001or is the same regardless of concentration of carbon monoxide for those 3531ples treated at the lower temperature. A review of the data in Appendix B and C showed that the chroma of the samples treated with the various concentrations of carbon monoxide tended to remain the same throughout the storage period. The value of hue tended to be in the yelloWbred range at the lower ‘3omicentrations of carbon monoxide than those samples treated with the high- er concentrations of carbon monoxide, which resulted in a hue value in the r°"3~ range. Hartridge (12) stated that there is a decrease in the saturation of -33- ‘ hemoglobin with carbon monoxide as the temperature rises. The results of the samples treated at 60-7001? tended to bear this out. In figure 2 the samples treated at 35-400F tended to be near the control, whereas in figure 2a the sample treated at 60-70°F with 20% carbon monoxide is darker than the control sample. This perhaps can be explained by the fact that as the temperature rose the sample treated with 20% carbon monoxide did not absorb enough carbon monoxide. Tabulated data are found in Appendix B and C. 383 5 05.9 ...+. y.. 0H., w m ¢ N -T .5; T55T-T.5+ 5 5 + 5 + o oamfiem Hoppqoo Ilnllll opwxosofi c8330 Rome 54.”? powwoua llln—._‘V..' .-_., . ----..‘_._.._. . 7/2/27jf/ZZZZ717flW Z/ZZZZ7/Z7‘7777Z7/ W /. W'— ...gs- -H,“.—4-v———.———o—~ w-_..-b- ...—o. .. - ~v—-— v — - .7 - Z/ V l ”777f/ZZZZ/7 ‘7 FWZ/ swmtunfixo JO xeqmnn JO 801 l. 3. 4. SULDIARY The stability of color of prepackaged frozen meat was increased when treated with carbon monoxide. In general, those samples treated with 35-40% carbon monoxide gave the best results on the stability of color of meat. The procedure of a high concentration of carbon.monoxide for a short period of exposure was better than a low concentration of carbon monoxide for a long period of exposure, i.e. 40% carbon monoxide for 5 minutes was better than 20% carbon monoxide for 15 minutes. Carbon monoxide had no appreciable effect on Pseudomonas 22-. BIBLIOGRAPHY 1. Anderson, Arthur 1950 Blood. Essentials of Physiological Chemistry. 3'rd Ed. John Wiley and Sons, Inc. 2. Anonymous 1954 Packaging Shift Seen Spur to Frozen Meats. Quick Frozen Foods, 16 (6):99. 3. Brooks, John 1935 The Effect of Carbon Dioxide on the Color Change or Bloom of Lean Meats. Journal of the Society of Chemical Industry. 50: 17T-19T. 4. Brooks, John 1937 Color of Meat. Food Research, 3: 75-77. 5. Brooks, John 1929 Post-mortem Formation of Methemoglcbin in Red Muscle. Bio- chemical Journal, 23: 1391-1400. 6. Butler, 0. D., Bratzler, L. J., and Mallman, W. L. 1953 The Effect of Bacteria on the Color of Prepackaged Retail Beef Cuts. Food Technology, 7: (10) 397. 7. Fisher, Alfred. 1900 The Structure and Function of Bacteria. Oxford at the Claren- don Press, page 85. 8. Frankland, Percy 1889 On the Influence of Carbonic Anhydride and Other Gasses on the Development of Microorganism. Proceedings of the Royal Society of London, (_B_) 45:292-301. 9. Frobisher, Martin 1941 Fundamentals of Bacteriology.'w. B. Saunders Co. 2'nd Ed., page 235} 10. Haines, R. B. 1931 Growth of Microorganisms on Chilled and Frozen Meat. Journal of the Society of Chemical Industry, 50: 223. 11. Haldane, J. S. and Smith, L. J. 1896 Oxygen Tension of.Arteria1 Blood. Journal of Physiology, 20: 497-521. 12. Hartridge, Ho 1912 The.Action of Various Conditions on Carbon Monoxide Hemoglobin. Journal of Physiology, 44: 22-23. 13. Haurowitz, Felix 1950 Chemistry and Biology of Proteins, Academic Press Inc. New York, New York, 374 pages. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. ~54- Hoagland, R. 1915 Coloring Matter of Raw and Cooked Salted Meats. Journal of Agricultural Research, 3: 211. Howell, H. H. 1916 Howell's Textbook of Physiology, 15'th Ed. W. B. Saunders Company Jensen, L. B. 1949 Meat and Meat Foods. The Ronald Press Co. New York, New York.- Keilin, D. 1925 On Cytochrome, A Respiratory Pigment, Common to Animals, Yeasts and Higher Bacteria. Proceedings of the Royal Society of London, (E) 98: 329. Kennedy, R. P. and Whipple, G. H. 1926 The Identity of Muscle Hemoglobin and Blood Hemoglobin. American Journal of Physiology, 76: 685-692. Levers, G. G. 1946 Discoloration of Prepackaged Red Meat. Modern Packaging, 21(5): January 1948. mangel, margaret 1951 The Determination of Methemoglobin in Beef Muscle Extracts. I. A Study of the Spectrophotometric Method. II. Factors Affect- ing Met-hemoglobin Formation in Frozen Beef. University of Missouri Research Bulletin 474. 24 pages. Mefferd, Roy B. and Matney, Thomas S. 1952 Protection of E. Coli Against Ultraviolet Radiation by Pretreat- ment with Carbon Monoxide, Science, 115 : 116-117. Millikan, c. A. 1939 Muscle Hemoglobin. Physiological Reviews, 19:503-523. Mbran, T. 1935 Post-Mortem.and Refrigeration Changes in Meat. Journal of the Society of Chemical Industry, 54:149T-151T. Neill, James, M. 1925 Studies on the Oxidation-Reduction of Hemoglobin and Methamp- globin. IV. The Inhibition of "Spontaneous“ Methamoglobin Formation. Journal of Experimental Medicine, 41:561-570 Nickerson, Dorothy 1946 Color Measurements and Its.Application To The Grading of Egricultural Products. U. S. Department of Agriculture, Misc.‘Pub1ication 580. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. -55.- Penrod, E. B. and Baker, Merl 1954 Effect of Storage Conditions on Drying and Discoloration of Beef. University of Kentucky Engineering Experiment Station Bulletin, 9: 43 pages. Ramsbottom,J. M. 1947 Freezer Storage Effect on Fresh Meat Quality. Refrigeration Engineering, 54:19-23. Ramsbotton, J. M., and Koonz, C. H. 1941 Freezer Storage Temperature as Related to Drip and to Color in Frozen-Defrosted Beef. Food Research, 6:571-580. Ramsey, L. T. and Eilman, H. J. 1931-32 Carbon Monoxide Acute and Chronic Poisoning and Experimental Studies. Journal of Laboratory and Clinical Medicine, 17: 415. Regier, W. L., Emmerson, M. R., Tappel, A. L., Conroy, A., and Stewart G. F. The Preparation and Storage Stability of Freeze-Dried Beef. 1954 National Provisioner, 131:36. Rikert, J. A. 1952 Color Changes of Fresh Meats as Influenced by Some Antioxidant, Temperature and Atmospheric Variations. Rutgers University Thesis, Typewritten publication. 57 pages. Robertson, E. J. 1950 Prepackaged Frozen Meats. Refrigeration Engineering, 58:771. Roughton, F. J. W. 1934 The Kinetics of Hemoglobin. IV. General Methods and Theo- retical Basis for the Reaction with Carbon Monoxide. Proceedings of the Royal Society of London, (B) 115:455. Roughton, F. J. W. 1930 Reactions With Carbon Monoxide. Proceedings of the Royal Society of London, (A) 126:439. Roughton, F. J.'W. 1934 The Kinetics of Hemoglobin. V. The Combination of Carbon Monoxide‘With Reduced Hemoglobin. Proceedings of the Royal Society of London, (E), 115:470. Seelander, K. 1909 Untersuchungen uber die Whrkung des Kahlon oxyds auf Planzen. Bot. Cent'bl. Beihefte. Abt. 1, 24: 357-393. Sulzbacher, wm. L. 1950 Survival of Microorganisms in Frozen Meat. Food Technology, 4: 386-390. 38. 39. 40. 41. 42. 43. 44. 45. -56.. Tanner, F. W. 1944 The Microbiology of Foods. The Gerrard Press. Champain, 111. 1196 pages. Tressler, D. K. and Evers, C. F. 1947 The Freezing Preservation of Foods. 2'nd Ed. Avi Publishing Company,filnc. Voegeli, marvin 1952 The Measurement of Fresh Beef Muscle Color Change by Disk Colorimetry. Unpublished Ph.D. Thesis. Michigan State College, 125 pages. Voegeli, Marvin M., Bratzler, L. J. and Malmann W. L. 1953 Flow Sheets of Prepackaged Fresh Meats. Journa1.Artic1e 1390, Michigan Agr. Exp. Station., Submitted for publication, 1953. von Oettingen, We F0 1944 Carbon Monoxide: It’s Hazards and the Mechanism.of Its Action. Public Health Bulletin No. 290. Federal Security Agency. U. S. Public Health Service, Wbshington, D. C. Whipple, G. H. 1926 The Hemoglobin of Striated Muscle. I. Variation Due to Age and Exercise. American Journal of Physiology? 76: 693-707. Whipple, G. H. 1926 The Hemoglobin of Striated Muscle. II. Variations Due to Anemia and Paralysis. American Journal of Physiology, 76: 708-7140 Wurburg, 0. ‘ 1926 On the Effect of Carbon Monoxide on the Mbtabolism.of Yeast. Biochem. Zeitschr. 177:471. APPENDIX APPENDIX A Trial CO Prepackaged Sample Treated with Carbon Monoxide Cutting Temperature - 34°F, Freezer Temperature - 2°F Standard 7.0R 4.q/s.o Time of Reading Units from After Frozen Hue Value Chroma Standard 24 hours 7.9R 4.4 4.3 15 6 weeks 7.9R 4.3 4.6 14 3 months 7.5K 4.1 5.2 10 4 months 8.4R 4.2 4.1 15 5 months 9.7R 4.2 4.1 17 8 months 6.2R 5.0 3.3 21 9 months 8.3K 4.6 3.4 19 Trial CO Prepackaged Sample Serving as Control 24 hours 8.4R 4.4 3.7 17 6 weeks 0.5YR 4.4 3.2 21 3 months 2.2YR, 4.3 2.5 23 4 months 2.5YR 4.0 2.5 22 5 months 4.0YR 4.0 2.7 23 8 months 6.5YR 4.6 1.9 25 9 months 4.4YR 4.3’ 2.1 26 APPENDIX B Trial 5 NA-NE Prepackaged Samples Treated for 5 Minutes at 35-40°F With 20, 25, 30, 35, and 40% carbon monoxide. Cutting Temperature - 34°F. Treatment Temperature-35-40°F. Freezer Temperature- 2°}? Standard 7.03 4.q/s.o. Time of Reading Units from After Frozen Hue Value Chroma Standard 30:7: iasbgnjfianaxids 24 hours 6.9R 3.8 2.2 19 2 weeks 6.9R 3.4 2.3 21 6 weeks 7.5R 3.8 1.8 20 12 weeks 7.3R 3.8 1.8 20 35?: Bamakmsnsfids 24 hours 4.1YR 4.4 3.1 24 5 weeks 1 .4YR 4. 8 2. 7 24 8 weeks 0.1YR 4.3 2.4 22 12 weeks 1.9YR 4.6 2.6 25 £02 Easbsnflsnsxid: 24 hours 2 .1YR 3.8 2.5 23 5 weeks 9.33 4.2 3.2 18 8 weeks 8.512 4.2 2.3 19 12 weeks 2.9YR 3.5 1.9 19 APPENDIX_§_(Continued) Time of Reading Units frmn After Freezing Hue Value Chroma Standard -. -4-->- “~“- 33% Carbon Monoxide 24 hours 1.6YR 3.4 2.8 24 6 weeks 1.0YR 3.7 3.9 24 8 weeks 9.4R 3.2 3.2 22 12 weeks 0.7YR 3.4 3.6 22 24 hours 0.5YR 3.8 2.8 20 6 weeks 2.1YR 4.4 3.4 22 8 weeks 1.6YR 4.5 2.8 22 12 weeks 9.4a 4.1 2.9 18 Control Prepackaged Sample and Frozen for Twelve weeks. Cutting Temperature 34°F. Freezing Temperature 20?. Standard 7.03 4.0/8.0. Time of Reading Units from after Freezing Hue Value Chroma Standard 24 hours 0.5YR 3.9 1.9 1 21 2 weeks 1.9Y 3.9 1.3 23 6 weeks 2.1YR 3.7 1.6 24 8 weeks 1.9YR ' 3.7 1.6 24 11 weeks 1.2YR 4.0 1.4 22 12 WGBkS 2.2351 402 1.9 24 APPENDIX C Trial 3 N’A"IQE o Prepacknged Samples Treated for 5 Minutes at 60-70°F With 20, 25, 30, 35 and 40% Carbon Monoxide. Cutting Temperature 34°F. Treatment Temperature 60-70°F. Freezing Temperature 3°F. Standard 7.03 4.0/8.0. Time of Reading Units from After Freezing Hue Value Chroma Standard 3.022 Saabanjfisnsxids 24 hours 3.8YR 5.3 2.6 31 2 weeks 1.5YR 5.0 2.5 27 6 weeks 3.5YR 4.6 2.6 26 12 weeks 3.1YR 4.8 2.6 27 3.521 Easbsnjdgngxids ' 24 hours 6.9R 3.2 2.8 21 2 weeks 7.4R 3.1 4.1 18 6 weeks 9.3K 3.3 3.0 22 12 weeks 0.2YR 3.6 2.4 22 39:7: Easbgnfisnsxids 24 hours 8.5R 4.5 2.3 21 2 weeks. 8.23 4.7 2.0 23 7 weeks 1.9YR 4.6 3.1 24 12 weeks 8.6R 4.6 2.1 23 £52 Easbsnflsnsxids 24 hours 9.2R 3.1 3.7 21 2 weeks 8.8R 3.4 3.3 21 . 7 weeks 8.8R 3.3 3.7 20 12 weeks 1.7YR 3.1 3.3 25 APPENDIX C (Continued) Time of Reading Units from After Freezing Hue Value Chroma Standard 40% Carbon Monoxide 24 hours 7.9R 2.8 4.0 24 2 weeks 7.9R 2.8 4.0 24 7 weeks 8.6R 2.5 4.1 23 12 weeks 902R 2.3 4.7 24 Control Prepackaged Sample and Frozen for Twelve Weeks. Cutting Tempera- ture 34°F. Freezing Temperature 2°F. Standard 7.0R 4.0/8.0. Time of Reading - Units from After Freezing Hue Value Chroma Standard 24 nonrs 0.5m 3.9 1.9 21 2 weeks 1.9YR 3.9 1.3 23 6 weeks 2.1YR 3.7 1.6 24 8 weeks 1.9YR 3.7 1.6 24 11 weeks 1.2YR 4.0 1.4 22 12 weeks 2.2YR 4.2 1.9 24 APPENDIX D Trials NA-NE. Prepackaged Samples Treated for 10 Minutes at 35-40°F With 20, 25, 30, 35 and 40% Carbon Monoxide. Cutting Temperature 34°F. Treatment Temperature 35-400. Freezing Temperature 2°F Standard 7.03 4.0/8.0. Time of Reading Units from After Freezing Hue Value Chroma Standard 20% Carbon Monoxide ——-.---.—---—- 24 hours 6.1R 4.6 2.0 22 2 weeks 7.2R 4.3 1.9 19 6 weeks 1.5YR 4.6 1.7 25 12 weeks 8.1R 4.5 1.4 20 24 hours 9.33 3.4 3.1 21 5 weeks 1.7YR 3.8 3.4 . 20 8 weeks 9.8R 3.2 3.0 23 12 weeks 0.8YR 3.8 3.1 21 30% Carbon Monoxide 24 hours 8.7R 4.5 2.1 22 5 weeks 7.7R 4.7 2.6 21 8 weeks 9.6R 3.8 2.2 21 12 weeks 8.5R 4.3 2.6 20 30% Carbon Monoxide 24 hours 0.4YR 3.3 4.0 21 6 weeks 1.5YR 4.5 3.8 21 8 Weeks 906R 3.8 3.5 19 12 weeks 4.7YR 4.0 2.6 24 APPENDIX D (Continued) Time of Reading Units from After Freezing Hue Value Chroma Standard 40% Carbon Monoxide --—__-——~— 24 hours 8.2R 3.5 3.8 17 6 weeks 9.8R 4.2 3.1 18 8 weeks 8.6R 3.3 3.7 19 12 weeks 8.5R 3.5 3.8 18 Control Prepacknged Sample and Frozen for Twelve'Weeks. Cutting Tempera- ture 34°F. Freezing Temperature 20F. Standard 7.0R 4.0/8.0. Time of Reading Units frmm After Freezing Hue Value Chroma Standard 24 hours 0.5YR ' 3.9 1.9 21 2 weeks 1.9YR 3.9 1.3 23 6 weeks 2.1YR 3.7 1.6 24 8 weeks 1.9YR 3.7 1.6 ‘ 24 11 weeks 1.2YR 4.0 1.4 22 12 weeks 2.2YR 4.2 1.9 24 APPENDIX E Trials NAPNE Prepackaged Samples Treated for 10 Minutes at 60-70°F'With 20, 25, 30, 35 and 40% Carbon Monoxide. Cutting Temperature 34°F, Treatment Temperature 60-700F. Freezing Temperature 2°F. Standard 7.03 4.0/3.0. Time of Reading Units from After Freezing Hue Value Chroma Standard 3.0% Easbgnflsnaxidz 24 hours 9.1R 4.7 2.7 22 2 weeks 0.6YR 4.8 2.6 25 6 weeks 9.4R 4.6 2.4 23 12 weeks 1.4YR 4.8 2.5 25 25% Carbon Monoxide 24 hours 6.3R 4.3 2.2 22 2 weeks 6.6R 4.0 2.0 I 18 6 weeks 8.9R 4.4 - 2.3 21 12 weeks 9.3R 4.2 2.1 21 30% Carbon Monoxide 24 hours 9.0R 3.2 4.2 25 2 weeks 2.5YR 5.0 2.6 27 7 weeks 1.4YR 4.8 3.3 24 12 weeks 6.5YR 4.6 1.8 28 35% Carbon Monoxide 24 hours 0.3YR 3.1 2.8 24 2 weeks 5.1K 3.4 3.9 24 7 weeks 1.6YR 3.9 2.8 22 12 WOOkS 0.7YR 30 5 302 22 APPENDIX 3 (Continued) Time of Reading Units from After Freezing Hue Value Chroma Standard 40% Carbon Monoxide 24 hours 8.8R 3.4 3.4 20 2 weeks 8.8R 3.4 3.3 21 7 weeks 801R 306 306 17 12 weeks 2.4YR 3.8 2.9 23 Control Prepackaged Sample and Frozen for Twelve weeks. Cutting Tempera- ture 35°F. Freezing Temperature 2°F. Standard 7.0R 4.0/8.0. Time of Reading Units from After Freezing Hue Value Chroma Standard 24 hours 0.5YR 3.9 1.9 21 2 weeks 1.9YR 3.9 1.3 23 6 weeks 2.1YR 3.7 1.6 24 8 weeks 1.9YR 3.7 1.6 24 11 weeks -1.2YR 4.0 1.4 22 12 weeks 2.2YR 4.2 1.9 24 APPENDIX F Trials NA-NE. Prepacknged Samples Treated for 15 Minutes at 35-400F With 20, 25,30, and 40% Carbon Monoxide. Cutting Temperature 34°F. Treatment Temperature 35-40°F. Freezing Temperature 2°F. Standard 7.03 4.0/8.0. Time of Reading Units from After Freezing Hue Value €hroma Standard 20% Carbon Monoxide 24 hours 6.1R 4.5 2.0 22 2 weeks 7.0R 3.8 2.2 19 6‘weeks 1.0YR 4.6 1.9 27 12 weeks 7.5R 3.1 2.4 23 25% Carbon Monoxide 24 hours 8.6R 4.5 3.1 20 5'weeks 0.5YR 4.6 2.6 23 8 weeks 0.4YR 4.4 2.3 22 12 weeks 9.9R 4.6 3.0 22 30% Carbon Monoxide 24 hours 7.83 4.0 3.4 15 5 weeks 0.1YR 4.3 2.5 21 8 weeks 7.8R 3.7 3.5 18 12 weeks 7.7R 4.1 3.4 15 24 hours 805R 3.3 3.7 19 6 weeks 0.9m 406 3.8 19 8 weeks 8.5K 3.3 3.7 19 12 weeks 9.6R 3.7 3.5 19 APPENDIX F (Continued) Time of Reading Units from After Freezing Hue Value Chroma Standard 40% Carbon Monoxide 24 hours 9.7R 2.9 3.8 23 6 weeks 8.6R 3.1 3.9 20 8 weeks 8.4R 206 4.3 21 12 weeks 1.9YR 2.9 4.2 26 Control Prepackaged Sample and Frozen for Twelve weeks. Cutting Tempera- ture 34°F. Freezing Temperature 2°F. Standard 7.03 4.0/8.0. Time of Reading Units from After Freezing Hue Value Chroma Standard 24 hours 0.5YR 3.9 1.9 21 2 weeks 1.9YR 3.9 1.3 ' 23 6 weeks 2.1YR 3.7 1.6 24 8 weeks 1.9YR 3.7 1.6 24 11 weeks 1.2YR 4.0 1.4 22 12 weeks ZOZYR 4.2 109 24 APPENDIX G Trials NA-NE. Prepackaged Samples Treated for 15 Minutes at 60-700F'With 20, 35.30.35 and 40% Carbon Monoxide. Cutting Temperature 34°F. Treatment Temperature 60-70°F. Freezing Temperature 2°F. Standard 7.03 4.0/3.0. Time of Reading Units from After Freezing Hue Value Chroma Standard 20% Carbon Monoxide 24 hours 0.2YR 4.5 2.3 23 2 weeks 6.1R 4.1 2.4 18 6 weeks 1.4YR 4.1 2.1 23 12 weeks 1.3YR 3.9 1.7 22 25% Carbon Monoxide 24 hours 6.6R 3.2 3.1 20 2 weeks 8.4a 3.4 3.8 18 6 weeks 1.0YR 4.0 3.0 19 12 weeks 8.7R 3.7 3.0 19 30% Carbon Monoxide 24 hours 0.6YR 4.9 2.7 19 2 weeks 8.5R 3.4 4.1 18 7 weeks 8.0R 3.7 3.8 16 12 weeks 9.1R 3.4 4.0 19 24 hours 0.3YR 3.1 2.8 24 2 weeks 5.1R 3.4 3.9 24 7 weeks 1 o GYR 3o 9 2 o 8 22 12 Weeks 0.7YR 305 302 22 éfPENDIX G (Continued) Time of Reading Units from After Freezing Hue Value Chroma Standard 2.0%. £a£b2n_M2naxid° 24 hours 8.33 5.4 4.2 17 2 weeks 5.1R 3.9 4.2 15 7 weeks 8.6R 3.9 3.7 14 12 Weeks 800R 306 306 17 Control Prepackaged Sample and Frozen for Twelve weeks. Cutting Temperature 35°F. Freezing Temperature 29F. Standard 7.0R 4.Q/8.0. Time of abading Units from After Freezing Hue value Chroma Standard 24 hours 0.5YR 3.9 1.9 21 2 weeks 1.9YR 3.9 1.3 23 6 weeks 2.1YR 3.7 1.6 24 8'weeks 1.9YR 3.7 1.6 24 11 weeks 1.2YR 4.0 1.4 22 12 weeks 2.2YR 4.2 1.9 24 APPENDIX H The Effect of various Concentrations and Period of Exposure to Carbon Monoxide on Pseudomonas 32: 23141;}. 40% Carbon Monoxide Time 90 Minutes Incubation Log of Temperature 1.6°C Count 2 week count Incubation Leg of Temperature 20°C Count 120 hour count Before S-l 88,000 4.9445 4,200 3.6232 S-la 288,000 5.4594 24,600 4.3909 After S-2 0 -- 3,500 3.5441 S-2a 6,000 3.7782 15,000 4.1761 2% 11.. 20% Carbon Monoxide Time 15 Minutes Before S-l 1,300 3.4771 0 -- S-2 24,200 4.3838 3,300 3.5185 After S-la 1,220 3.0864 1,610 3.2068 S-2a 1,300 3.1139 1,000 3.0000 APPENDIX H (Continued) TRIAL III 30% Carbon Monoxide Time 15 Minutes Incubation Log of Incubation Log of Temperature 20°C Count Temperature 1.6°C Count 120 hour count 2 week count Before S-l 60,000 4.7782 13,000 4.1139 8-1 23,000 4.3617 6,000 3.7782 After S-la 90,000 4.9542 32,000 4.5051 S-Za 80,000 4.9031 72,000 4.8573 9.14 18.. 40% Carbon Monoxide Time 15 Minutes Before S-1 400,000 5.6021 100,000 5.0000 S-2 1,000,200 6.0000 1,600,000 6.2041 S-3 1,850,000 6,2672 130,000 5.1139 S-4te 1,870,000 6.2718 140,000 5.1461 After S-la 1,200,000 6.0792 170,000' 5.2304 S-Za 310,000 5.4914 90,000 4,9542 S-3a 1,600,000 6,2041 80,000 4,9031 E ** S-4 is the control APPENDIX H (Continued) 3.12.2.4 save. 60% Carbon Monoxide Time 30 Minutes Incubation Log of Incubation Log of Temperature 20°C Count Temperature 1.6°C Count 120 hour count 2 week count Before S-l 1,010,000 6.0043 1,410,000 6.1492 S-2 960,000 5.9823 1,190,000 6.0755 S-3 1,600,000 6.2041 1,650,000 6.2175 S-4** 1,500,000 6.1761 1,620,000 6.2095 After S-1a 1,230,000 6.0899 1,640,000 6.2148 S—Za 1,120,000 6.0492' 1,430,000 6.1553 S-3a 1,500,000 6.1761 1,180,000 6.0719 ** 8-4 is the control I 'p..4 EIVI'"? g3 {‘i‘h} 13"." i ‘ 131,33. ‘2‘: U“. 551 '55 1 r ' N HUI- ‘Bm-‘n‘lY LOAN Nov ‘3 raw 1 59 mIlmmuulfllfllmulinguutmuunuwmmmun