kl o is u l -4’ . ' ”m. ‘ * x3 ‘ (éh‘a‘: *- fr ‘1’. am”! ‘ 4 gem; FINES: 25¢ per day per ital ammuc LIBRARY MATERIALS: Place in book return to remove char-901m circu'lation. records »...~ . . , . V V. . .. ... ... ... . L; A....... . T u I . l r..- > .. .....i.::\: :- . T... . . ‘olt..9|u-..? 9...... .. . . . Z .. , . ._ ‘11. .otlru.o~‘..o‘.t{\"|:.l.‘l tlhif .rl.o‘q..,ut.bu synthetic actomyosin - ATP > actin. These results concerning the emulsification capacities of myosin, actomyosin without ATP and actin are in agreement with those previously reported by Hegarty gt gt. (1963). It was also noted that myosin was rapidly removed from solution during emulsifi- cation whereas actin was less readily removed, indicating an increased affinity of the oil for myosin as opposed to actin. The emulsions resulting from myosin were thick and creamy whereas actin stabilized emulsions were described as thin and coarse. Synthetic actomysin in 12 the absence of ATP behaved in the same manner as myosin while the use of synthetic actomyosin + 5 mM ATP resulted in the preferential incorporation of myosin into the emulsion while actin remained in solution. Further studies on chicken muscle proteins reported by Galluzzo and Regenstein (1978c) focused on the emulsifi- cation properties of native actomyosin. This work showed that the emulsification capacity of native actomyosin was less than that previously reported by Galluzzo and Regenstein (l978b) for myosin and synthetic actomyosin + 5 mM ATP, however when 5 mM ATP or 5 mM pyrophosphate were added the emulsification capacities were found to be essentially equal. Again this was attributed to the disassociation of the actin-myosin linkage in actomyosin. During timed emulsification of native actomyosin disassociated with 5 mM ATP, myosin was again preferentially removed from solution as in the case of synthetic acto- myosin + 5 mM ATP which was previously reported by these same workers. Several researchers have studied the emulsification properties of intact and semi-extracted myofibrils from beef semitendinosus (Fukazawa gt gt., l96la,b,c) and from chicken breast muscle (Galluzzo and Regenstein, l978c). Fukazawa gt gt. (1961b) studied the binding properties of intact fibrils, actin, tropomyosin poor fibrils and l3 actin-myosin poor fibrils in an experimental sausage. They reported that sausages manufactured using intact fibrils were quite similar to those sausages manufactured using pre-rigor beef muscle whereas sausages made from synthetic myofibrils had binding properties which resembled those of sausages made from 7 day post-mortem muscle. Galluzzo and Regenstein (l978c), in studies involving the timed emulsification of chicken myofibrils, reported that tropomyosin was not involved to any large extent in emulsion formation and that the troponins were only mar- ginally involved. These conclusions are in agreement with those of Fukazawa gt gt. (1961c) who reported these find- ings in addition to the conclusion that actin was not a major stabilizer as has also been stated by Galluzzo and Regenstein (l978b). Protein Solubilization Research concerning the solubilization of muscle proteins has recently been conducted by several labora— tories. There are several factors which exert influences on the solubilization of proteins and therefore the literature relating to each of these factors will be reviewed separately. 14 The major factors involved in meat protein solubili— zation are: . Mechanical Action Ionic Strength pH 1 2 3 4. Temperature 5 Vacuum 6 Preblending Effects of Mechanical Action Several types of mechanical action are routinely applied to meats during their transformation from raw materials to finished products. Among these are blending, chopping, grinding and massaging and/or tumbling. Blending, chopping and grinding are confined primarily to finely comminuted sausage products whereas massaging and tumbling are most widely used in the production of sectioned and formed meat products. Theno gt gt. (1977) reviewed the past advances and the current state of the art in meat processing systems with a focus on the effects of mechanical action on protein solubilization, in particular meat massaging. These workers defined meat massaging as the process of imparting mechanical work to meat pieces or chunks by mixing or churning them in such a manner that they become Soft and pliable with a creamy, tacky exudate on the 15 surface. This exudate is composed of water, salt and solubilized protein and as such serves as an adhesive in the binding of these meat pieces. Protein solubilization is promoted through the intro- duction of mechanical energy into the meat massaging system (Weiss, 1974). Theno gt gt. (l978a) studied the effects of massaging on the microstructural composition of muscle exudate using conventional light microscopy techniques. This work showed that as massaging time in- creased, up to 24 hours, that muscle fibers were pro- gressively disrupted and extensive quantities of fiber fragments began to appear in the meat exudate. As massaging progressed definite clouds of solubilized pro- tein were also observed. These authors found that over- all increased mechanical action due to massaging resulted in improved binding ability of the product. This was attributed to the increased disruption of the muscle fibers which was observed. Cassidy gt gt. (1978) used the same basic approach in the study of tumbled meat and reported similar results. More in depth studies concerning the nature of the protein exudate, in mechanically worked meat pieces have also been reported (Siegel gt gt., 1978a). These researchers reported that although other factors, such as NaCl and phosphate concentration, greatly influenced the composition 16 of the protein exudate the singular effect of massaging only facilitated the availability of these proteins for solubilization rather than functioning directly in the solubilization process itself. Maesso gt gt.(1970a,b) studied, among other factors, the influence of mechanical mixing on cubes of poultry meat. They reported that as the extent of mechanical mixing increased that the intracellular content of broken muscle cells was released resulting in an increase in bind. Other work concerning the effects of mechanical mixing was conducted by Belohlavy (1975). This work utilized pork muscle flaked using an Urschel "Comitrol" and although no measures of protein solubilization or availability for solubilization were made it was reported that binding ability increased to a maximum at 8 minutes of mixing time and then decreased to a minimum at 14 minutes. Gorbatov gt gt. (1972) and Gorbatov and Gorbatov (1974) have extensively studied the effects of chopping on the rheological properties of sausage meats. These workers characterized the changes in consistency and viscosity which meats experience during chopping and reported that the time of chopping required to achieve a specific degree of viscosity was largely dependent on the water content of the batter as this greatly influences protein solubilization. Likewise, Hamm (1960) stated that 17 protein solubilization during chopping is greatly enhanced by chopping for several bowl revolutions prior to the addition of water. This practice theoretically causes the muscle fibers to resist the knives more and thus ex- perience a greater degree of comminution therefore exposing more proteins for solubilization due to the resulting increase in surface area of the meat particles. Although grinding of meat effectively increases the surface area the relative benefits of grinding in compari- son to chopping regarding protein solubilization are rather small. This is due to the decreased magnitude of the surface area increase as compared to that which is accomplished by chopping (Hamm, 1960). Emulsification as accomplished by high speed mills, such as the Griffith Mincemaster is responsible for a great deal of the mechanical energy which is contributed to meat batters during processing (Saffle, 1968). These machines operate on the same basic principle as meat grinders, however, the perforations in the emulsitator plates are considerably smaller in diameter than those in grinder plates resulting in a greater degree of commi- nution. The basic contribution of emulsitators in the processing of a pseudo-emulsion type product is not one of solubilization but rather one of particle size reduction hence resulting in a more stable emulsion. 18 Recently, machines have been introduced into the United States from Europe which are capable of performing the combined functions of blenders and choppers (Johnston, 1979). These machines are reported to produce increased protein solubilization while saving valuable time as compared to the use of separate machines. At present, this type of machinery is not enjoying wide usage in the meat industry due primarily to poor durability and reliability of the prototype equipment. Effects of Ionic Strength Most of the proteins which are of interest in the stabilization and binding of finely comminuted sausages and other meat products are soluble only in salt solutions (Hamm, 1973). Thus the effect of ionic strength on meat protein solubilization plays a major role in the production of these products. Hamm (1960) discussed the influence of salts on meat protein hydration. In living tissue the effect of ions is primarily due to osmotic effects, whereas in post-rigor tissue the salts freely migrate into the muscle fibers due to the destruction of the semipermeability of the cell membranes. Once inside the muscle fibers these salts exert 1yotropic effects which result in the swelling of the fiber. 19 Hamm (1957) studied the effects of various metal chlorides and of the sodium salts of different strong acids on the hydration of muscle homogenates at various ionic strengths. At ionic strength of less than 0.1 no differences were observed related to the particular salt employed, however, when the ionic strength of 0.1 was exceeded, significant differences between the salt types were observed. It was reported that at the pH of sausage systems the influence of the various anions on hy- dration followed the 1yotropic series quite closely. The effects of the anions on hydration were F-< Cl-< Br-< CNS? I- with all of these anions resulting in increased hydration. Regarding the metal chlorides studied it was reported that the effects of the different metals were KI< Na? Mg++< Ca++< Li+< Ba++ again with all metals improving hydration. It was also reported by Hamm (1957) that the pH of the environ- ment greatly influenced the relative effects of the different salts on hydration. The manner in which salt ions bind to proteins is primarily electrostatic due to the attraction of the salt ions by the positively or negatively charged groups of the proteins (Schellman, 1953). It is also suspected that Van der Waal's forces may play a role with anions of high molecular weight, however, this is merely a hypothesis. 20 Cann and Phelps (1955) stated that in the acidic range of the isoelectric point of muscle the electrostatic repulsion between positively charged groups of the protein is reduced by the binding of anions therefore decreasing the net charge of the protein in the following manner: +A' + + . +- -+ _. R-NH3 H3N-R 2 R-NH3A A NH3 R (repulsion) (no repulsion) Due to this reaction the protein structure therefore tightens resulting in a decrease in hydration. The stronger the ion is bound, therefore the stronger is the effect of dehydration. In the basic range of isoelectric point, however, an opposite effect is exerted by the salt since more negatively charged groups are available for the formation of salt bridges with the positive charges in the following manner: + _ +A' +_ _ R-NH3 OOC-R' > R-NH3A OOC-R' (no repulsion) (repulsion) The result in this case is a loosening of the protein structure and therefore an increase in hydration. The more tightly the anion is bound the stronger the hydrating value of that anion will be. The binding of anions 21 therefore also lowers the isoelectric point of the protein due to increased negative charges. This effect of lowered isoelectric point due to bound ions is primarily responsible for the increased solubilization due to the addition of ions at the pH of processed meat systems. The binding of cations is in the same manner as for anions. However, while it is best to have a strongly bound anion it is preferable to have a weakly bound cation. This is due to the fact that the anions result in a decreased isoelectric point while the binding of cations does not (Weber and Portzehl, 1952). Weber and Portzehl (1952) suggested that at ionic strengths in excess of 1.0 and at pH values greater than 7.0 salts were capable of partially dissociating actomyosin into its respective components. However, this effect does not influence meat systems due to the unusual conditions at which it occurs. Most hydration is maximum at NaCl concentrations of u=0.8 to 1.0 (Hamm, 1957). These figures correspond to 5% NaCl with no water added and approximately 6.5% NaCl with 30% added water. Hamm and Grau (1958) also reported that systems with ionic strengths of 0.8 to 1.0 also exhibited maximum hydration after thermal processing. Numerous reports have recently been published regarding the effects of various NaCl levels on massaged hams 22 (Siegel gt gt., l978a,b; Theno gt gt., l978a,b). These workers reported that as NaCl content increased that the degree of protein solubilization also increased with a NaCl content of 2% producing optimal binding properties in the finished product and no noticeable difference between the binding properties of the 2 and 3% NaCl hams. Theno gt gt.(1978b) clearly showed through the use of scanning electrom microscopy that the presence of NaCl in these hams aided in the disintegration and overall disrup- tion of muscle fiber integrity.These results concur with the statements made by Hamm (1960) regarding the loss of membrane semipermeability due to the presence of NaCl. Theno gt gt. (l978a) reported that the exudate formed on massaged hams showed evidence of higher levels of soluble protein as NaCl level increased and these results were substantiated by the reports of Siegel gt gt. (l978a). The use of phosphates in meat products has also been extensively studied. Reports on intact muscle products have shown that phosphate greatly increases pro- tein solubilization and ultimately product bind (Krause gt gt., l978a,b; Siegel gt gt., l978a,b; Theno gt gt., l978a,b; Ockerman gt gt., 1978; Cassidy gt gt., 1978). Although phosphates are highly ionized it is also suspected 23 that the cleavage of the actomyosin linkage is in part responsible for this increase in solubilization as has been demonstrated by Hamm (1973). Pepper and Schmidt (1975) and Neer (1975) have studied the effects of NaCl and phosphates on comminuted and cooked red meat products. Neer reported that 2% NaCl and 0.25% phosphate produced a product with optimal binding char- acteristics while products containing more than these levels did not show any significant improvements in bind. Effects of pH Due to the fact that proteins are composed of amino acids which are ampholytic molecules, in that they display properties of both acids and bases, proteins are also ampholytes (Lehninger, 1975). As the pH of the environment in which a specific protein is present changes the charge of that particular protein also changes until the pH of the solution reaches a point where the net charge on the protein is zero. At this pH, also known as the isoelectric point of the protein, the protein is no longer soluble and therefore precipitates resulting in a total destruction of protein functionality. A great deal of work has been done concerning the effect of pH on the properties of muscle proteins (Froning and Janky, 1971; Froning and Neelakantan, 1971; Hegarty gt gt., 1963; Hwang and Carpenter, 1975; McCready and 24 Cunningham, 1971; Swift and Sulzbacher, 1963). These workers have all reported that as the pH of the system is moved away from the isoelectric point of muscle prot- teins (25.2)the emulsification properties of the protein are increased. This is probably due to improved solubility of the proteins at these pH's. Hamm (1973) reported that the effect of pH on the protein solubility of a meat system is dependent on the presence of other factors in the system. The most common instance of this is exemplified by the effects of the presence of NaCl. As was discussed previously the pre- sence of NaCl results in a lowering of the isoelectric point of the proteins thus significantly increasing the solubilization of proteins at pH's where without NaCl only a small portion of the protein is solubilized. This theory supports the findings of Swift and Sulzbacher (1963) who reported that the addition of NaCl to sytems containing water soluble proteins resulted in improved emulsification properties. Without exception all reports on the binding proper- ties of meat systems as influenced by pH have shown that as the pH is raised away from the isoelectric point that bind improves indicating increased protein functionality and solubilization. 25 Effects of Temperature Saffle (1968) reported that protein solubilization was maximized at a temperature of 4.40C. This temperature is higher than those which are normally used for the laboratory preparation of protein fractions (0-20C) and no doubt results in the denaturation of some proteins. However, the benefits of increased solubilization at these temperatures more than offsets the slight degree of denaturation which occurs. Final temperatures which are realized during the production of finely comminuted sausages normally fall in the range of 15-220C (Hansen, 1960; Helmer and Saffle, 1963). Although these temperatures are greatly in excess of those reported by Saffle (1968) as resulting in maximum protein solubilization they are necessary in order to maximize the fat binding properties of the proteins. Brown and Toledo (1975) studied the relationships between chopping temperature and the fat and water binding properties of finely comminuted sausage batters. These researchers reported that as temperatures increased in excess of 22°C the fat and water binding properties of the batter decreased. However, these workers also found that when the temperature of the batter was lowered by the addition of dry ice a subsequent increase in binding properties was evident. Although this increase was not 26 sufficient to restore the binding properties to a state equal to that of batters which had not been subjected to adverse treatments it does indicate that some of the thermally abused protein in these batters was capable of recovering at least part of its original functionality. Bard (1965) reported that raising meat temperature from -5 to 3°C resulted in a four-fold increase in protein solubility with approximately 5% of the toal protein being soluble at 3°C. These results coincide with those reported by Saffle (1968). Saffle (1965) stated that the usuage of frozen and thawed meat should be limited in finely comminuted meat products due to poor binding properties. However, work reported by Buttkus (1970) and Johnson (1975) indicated that protein extractability is essentially unchanged by freezing and thawing. This indicates that it is the functionality rather than the solubility of protein which is affected by freezing. Further confirmatory reports of instability due to the excessive use of frozen and thawed meats have also been made (Hargus gt gt., 1970; Froning, 1970; Morrison gt gt., 1971). 27 Effects‘of‘Vacuum The application of vacuum to a meat system during blending and/or comminution has been shown to improve the final product in several ways (Starr, 1979). Increased solubilization of proteins due to vacuum blending has been reported by Schmidt (1979). This work involved the application of vacuum blending to a sausage system and revealed increased solubilization of myofibrillar proteins as a direct effect of vacuum. The underlying theory responsible for this increase in solubilization has been presented by Starr (1979). This report stated that when a vacuum is applied to a meat system the meat particles experience a swelling phenomenon which results in increased surface area available for interaction with the extraction solution and/or knives of the chopper. Although this appears to be a very viable explanation for the effects of vacuum on protein solubilization there is also an enhancement of the functionality of the soluble proteins due to the application of vacuum. This may be partially explained by the fact that when a vacuum is applied to the system free air and air which is entrapped within the blend is partially removed. This air, if not removed, would require soluble proteins to stabilize its position in the sausage matrix therefore decreasing the 28 amount of soluble protein which would be available for the stabilization of fat particles (Saffle, 1968). Effects of Preblendigg Very little research has been conducted on the influence of preblending on protein solubilization in meat systems. Webb (1968) defined preblending as the process of presalting some or all of the meat ingredients and allowing the resulting blend to undergo a passive extraction at cooler temperatures for some time prior to final product manufacture. Increased efficiency of fat emulsification properties has been reported for proteins which were extracted from preblended beef check muscles (Borton gt gt., 1968). The results of studies which were reported by Johnson gt gt. (1977) also indicated that preblending for 24 hours sig- nificantly improved the water binding properties of beef and pork blends as compared to samples which were not preblended. Froning and Janky (1971) and Acton (1973) have studied the effects of preblending on poultry muscle and have reported noticeable increases in emulsion stability and binding properties in products made from these tissues due to preblending. 29 Although preblending is widely employed in industry the mechanisms involved in product improvement through preblending are poorly understood and more research is needed in this area (Webb, 1968). METHODS AND MATERIALS This study was conducted in two parts, the first of which was a model systems experiment designed to ascertain the effects of NaCl type, blending time and post-blending storage time on protein solubility and binding properties of sausage blends. The second part of this study was then designed to study the applications of the results of part I on a pilot plant sausage system. Part I Source of Meat All meats utilized in this study were obtained in the fresh, unfrozen state from local meat packers. The pork was obtained as boneless picnics and the beef as boneless cow fronts. Meats were delivered to the meat laboratory three days prior to processing and immediately stored at 4°C. Samples were taken for protein, moisture and fat analysis upon arrival (AOAC, 1970). The mean proximate composition of these meat sources is shown in Table 1. Meat ingredients were always obtained from the same sources in order to minimize variations in composition. 30 31 Table 1. Means and standard deviations for the proximate composition of raw materials (Part I). MEAT SOURCE Boneless C0w Fronts1 Boneless Picnics1 Moisture (%) 67.06 i 0.34 58.51 i 1.19 Fat (%) 12.94 i 0.56 24.39 i 1.56 Protein (%) 18.96 t 0.23 15.95 i 0.32 l 32 Spurce of NaCl Three types of commercially available NaCl were used in this study. The three types were: (1) Flake (2) Dendritic (3) Evaporated and granulated Samples of these were supplied by the Diamond Crystal Salt Col, St. Clair, MI. Information regarding typical analyses is given in Appendix A. ProceSsing A flow diagram of the processing sequence is shown in Figure 1. The boneless picnics and boneless cow fronts were ground through 9.5 and 4.8 mm plates, respectively. This difference in grind size was utilized in order to maximize the surface area of the lean portion of the blend as is normally done commercially. Appropriate amounts of the two meats were then weighed and placed in a Reitz blender (capacity 2 80 kg) equipped with twin ribbon shafts, so that the final meat block represented 68 kg of meat containing 17% protein. At this time the blender was started and 1700g of one of the three experimental salts and 8.2 g of NaNOz, dissolved in 25 m1 of 17°C tap water, were added. This resulted in a blend containing 2.5% NaCl and 120 ppm NaNOZ, The mixture was blended for 30 seconds in order to distribute the NaCl and NaNOz. At this time the blender BONELESS FRESH PICNICS GREE)THNIK}IA 9.51nnITATE 33 BGWHESS FNEHICOWIHKNTS Figure 1. FOmflflfiflE GKDE>THMIK¥IA TO 177. PROTEIN 4'8 ”m PLATE ADD 2.5% NaCl NMDIZOIHTINde BLBm) .—————-SNfiflEIAT 0,2,1% 5,E3& H) MUNHES SKRE HTll C 0. l. 2. 3. 4&5 DAYSOFSTORAGE Processing flow chart (Part I). 34 was stopped and a 1.8 kg sample was removed and placed in a polyethylene bag. Blending was then resumed and subse- quent samples were collected, in the same manner as the in- itial sample, at 2, 4, 6, 8 and 10 additional minutes of blending. All blending was accomplished at room tempera- ture utilizing a ribbon speed of 45 rpm. Following the completion of blending the samples were placed in a 4°C cooler prior to analysis. All samples were analyzed for protein solubility, pH, blend stability and soluble protein composition via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing in polyacrylamide gels (IFPA). These analyses were performed initially and following 1, 2, 3, 4 and 5 days of post-blending storage at 4°C. Determination of Soluble Phase In order to extract the soluble phase from the blend samples 25 g of blend were homogenized for five seconds in a Waring blendor containing 25 ml of 4°C 4% NaCl solution (w/v) (Reagent Grade). The resulting slurry was decanted into a 75 ml polypropylene centrifuge tube and subjected to centrifugation at 20,000 x g for 30 minutes in a Sorvall RC-ZB automatic refrigerated centrifuge equipped with a Sorvall SS-34 fixed angle rotor and operated at a rotor temperature of 4°C. 35 Following centrifugation the soluble phase (Figure 2) was decanted by loosening the fat cap with a micro-spatula and inverting the centrifuge tube into a Nalgene funnel lined with two layers of cheesecloth. The resulting filtrate was collected in a graduated 15 m1 conical centrifuge tube. Following 30 minutes of filtration the volume of filtrate was noted as the volume of the soluble phase. This filtrate was then subjected to protein determination and 1 ml ali- quots were frozen with an equal volume of glycerol for later use in SDS-PAGE and IFPA. Determination of pH 10 g of blend sample were homogenized for 30 seconds with 50 ml of distilled water in a Waring blendor. The re- sulting slurry was subjected to pH determination using a Beckman model 3560 pH meter equipped with a combination electrode, which had previously been standardized with pH 7.00 and 4.01 standard buffers. pH determinations were performed in duplicate for each blend with the average of the two determinations being recorded as the blend pH. Blend Stability A modification of the emulsion stability procedure described by Townsend gt gt. (1971) was used. 35 g of blend was tightly packed into a 50 ml polypropylene dispos- able syringe which was then stoppered using the neoprene piston supplied with the syringe. This neoprene piston Figure 2. 36 FAT it— SOLUBLE PHASE SOLIDS Cross-sectional view of a soluble phase preparation following centrifugation at 20,000 X g for 30 minutes. 37 had previously had small sections of the rim removed in order to accomodate venting during heating. Duplicate samples of each blend were transferred to a preheated 70°C water bath and heated to an internal temp- erature of 69°C as indicated by a thermometer inserted in- to the geometric center of an identical blend sample which had been prepared specifically for this purpose. The cooked blend samples were immediately removed and decanted into Pyrex funnels, the free fluid resulting from this step was collected in 15 ml graduated conical centrifuge tubes. Following 15 minutes of draining the total fluid volume, fat volume and water volume present in the released fluid were recorded. Determination of Protein in the Soluble Phase The micro-biuret procedure described by Goa (1953) was used to determine protein concentration in the soluble phase (Appendix B ) . One ml of soluble phase was diluted with 49 m1 of 4°C 4% NaCl (w/v) (Reagent Grade). Two m1 of this dilution were placed in each of two test tubes containing 2 ml of 6% NaOH (w/v) and 0.2 m1 of biuret reagent. Following in- cubation at room temperature for 15 minutes the absorbance of the solutions at A = 540 nm was determined using a Beck- man model 24 spectrophotometer utilizing distilled water as a blank. Protein concentration was determined using a 38 standard curve prepared with bovine myofibrils (Figure 3). Myofibrils were prepared according to Goll gt gt. (1964) (Appendix C). Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS—PAGE) SDS-PAGE was performed using a modification of the procedure outlined by Porzio and Pearson (1977) (Appendix D). Samples of the soluble phase were thawed at 4°C and aliquots sufficient to yield a final protein concentration of 0.8 ug/ul were diluted with a pH 7.2 SDS tracking dye composed of 1% SDS (w/v), 1 mM EDTA, 0.5 M Tris HCl, 0.5% 2-mercaptoethanol (v/v), 20% glycerol (w/v) and 0.01% pyronin Y (w/v). The ratio of tracking dye to protein was always maintained in excess of 4:1 in order to insure proper de- naturation. The diluted protein samples were heated at 100°C for five minutes and allowed to equilibrate to room temperature. 25 pl of this solution containing 20 ug of protein was applied to the gel and overlayed with chamber buffer (0.2 M Tris glycine, 0.1% SDS (w/v), 0.1 mM EDTA). The acrylamide solution utilized in the preparation of the gels consisted of the following: 10 ml stock acryl- amide, 5 ml of 2.0 M Tris:g1ycine, 2.5 ml glycerol (50% w/v), 1 ml of 2.5% SDS:2.5 mM EDTA, 10 ul of TEMED (N,N,N',N' tetramethylenediamine), 5.5 m1 of deionized distilled water and 1.0 ml of 1% ammonium persulfate (w/v). The ammonium persulfate was added to the solution immediately prior to casting. 540 nm) «— ABSORBANCE (A .075 .250'—' .225'_' .200"' .175'—' .150'—' .125"' .100"' 39 ’1 1 1 L I 1 1 L l J 0.1 0. 2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 PROTEIN CONCENTRATION (mg/m1) Figure 3. Standard curve used in the determination of protein by the micro-biuret method. 40 Casting was accomplished by pouring the gel solution into 5 x 90 mm glass tubes to a level of 80 mm. The sol- ution was overlayed with distilled deionized water and al- lowed to polymerize. The resulting gels were 10.1% acryl— amide as a percentage of gel volume and 0.01% bis as a percentage of total acrylamide (10.1% T:0.01% C). The loaded gels were placed in an electrophoresis chamber and both the anodic and cathodic reservoirs of the chamber were filled with a chamber buffer composed of: 0.05 M Tris, 0.15 M glycine, 0.1% SDS (w/v) and 0.1 mM EDTA (Figure 4). Subsequent electrophoresis was conducted by applying a current of 0.2 mA/gel for a period of 16 hours. At this time the amperage was increased to 0.5 mA/gel until the dye front was approximately 0.5 cm from the end of the gel. Identical samples of a bovine myofibril preparation (Appendix C) were co-electrophoresed in order to serve as molecular weight standards for use in the identification of specific proteins in the experimental samples (Figure 5). The electrophoresed gels were removed from the tubes by rimming with water and were immediately stained in a solution containing: 0.03% Coomassie Blue R250 (w/v) (Sigma), 50% methanol (v/v) and 7% glacial acetic acid (v/v). Fol- lowing 24 hours of staining the gels were destained by dif- fusion for 24 hours in a 30% methanol (v/v), 10% glacial acetic acid (v/v) solution and subsequently stored in a 7.5% glacial acetic acid solution prior to densitometry. 41 z/Q7Q/Z’Q/Q/Q/Q/Q/Q”2/)/2/}{}Z27/mp mumm mcfipcmae .CHE Canon mcflpcman .CwE qumm mwmnoum mkmp qnmm mmmH0um amp Hamm mcflpcwan .CHE wnmm unwpcman .CflE Numm mwmnoum mkmp mnqw mmmHOuw mmmp ouam mcprmHn .CHE on m mchcan .CME ouam anH __ om mm «m mm mm Hm om mm mm mm m em mm N Homz amHm Homz UHHHmozma Homz mx mmmza mHLDHom o u o . Q NNo o nmo 0 new m moa o m Mm pmumascmuu owuwupcmo oxmam pmumuoam>m mmzh Homz .Homz mo ma%u kn poocmsHmafl mm mmflumeOMQ mwmfio mHQDHOm new we pecan pom mammE mmnmswm ummmq .n mHLmH 63 the moisture in the blends manufactured using flake NaCl was more tightly bound within the blend structure than was the moisture in the blends produced with the other two NaCl types. This phenomenon can most probably be attrib- uted to a greater degree of hydration of the muscle proteins present in the flake NaCl blends. This theoretical improve- ment in muscle protein hydration is directly related to the increased pH of the flake NaCl blends which resulted in the protein molecules being more highly charged in these samples; therefore allowing for the increased hydration of these proteins. Theoretically this should result in improved binding of moisture and fat in the finished product. Blends manufactured with dendritic NaCl produced less soluble phase than samples of blends produced with the evaporated and granulated NaCl (P<0.01) (Table 7). This result is the opposite of what would be expected from com- paring the blend pH values and the precise difference is not fully understood. Although no data are available to sub- stantiate this theory it is very possible that the evaporated and granulated NaCl is less pure than the dendritic NaCl thus resulting in the presence of numerous ions as impurities therefore decreasing the hydration of the muscle proteins. Protein concentration in the soluble phase as affected by NaCl type are shown in Table 7. Protein concentration was higher in those blends which were made using flake NaCl 64 as compared to blends manufactured with dendritic NaCl (P<0.05) and blends produced using evaporated and granulated NaCl (P<0.01). Those blends prepared with dendritic NaCl also had a greater protein concentration in the soluble phase than blends manufactured with evaporated and granulated NaCl (P<0.05). During the course of data collection it was also noticed that the soluble phases extracted from blends pro- duced using flake NaCl were thicker and more glue-like in nature than those from blends produced with dendritic or evaporated and granulated NaCl. These protein concentration data correspond quite closely to their respective soluble phase volumes which were pre- viously discussed in that blends with lower soluble pro- tein concentrations exhibited greater soluble phase volumes. This relationship supports the theory that as soluble phase volume decreases the binding properties of the batter will be increased due to increased efficiency of protein utilization through the improvement of protein solubiliza- tion properties. Although the total amounts of protein in the soluble phase correspond quite closely to one another there was probably a great deal of soluble protein which was entrapped within the batter matrix. This entrapped protein, although solubilized, could not be extracted in the soluble phase due to the improved hydration properties of these proteins thus 65 causing them to be strongly bound within the matrix of the batter. This theory represents one possibility for the ex- planation of the inverse relationship between soluble phase volume and protein concentration within the soluble phase. Blend stability data as influenced by NaCl type are listed in Table 8. Blends produced using flake NaCl were found to be superior to the other blends regarding all as- pects of stability which were measured. Water loss was greater for those blends manufactured using evaporated and granulated NaCl as compared to dendritic and flake NaCl (P<0.01). Flake NaCl blends also exhibited a lower water loss than those blends made using dendritic NaCl (P<0.01). These data concur with and reinforce the projections made earlier concerning the improved binding properties which could be expected due to increased pH and the resulting increase in soluble protein concentration and subsequent decrease in soluble phase volume. Fat loss was significantly less for the blends produced using flake NaCl as compared to those produced with dendritic NaCl (P<0.05) and evaporated and granulated NaCl (P<0.01). Blends manufactured utilizing the dendritic NaCl also ex- hibited improved fat binding properties as compared to those blends produced using evaporated and granulated NaCl (P<0.01). Total loss data again revealed that blends produced using the flake NaCl were superior to those produced with 66 .Qodvmv unmnmwmfiu mangoflmwcwwm mum. 36M m 55E muawuomumgm ucgmmmwp :33 mqmflammp .Amodvmv ucemwmfiu zaugowficwwm ohm 30H m 5533 muawuomhmdsm ucmnmwmwp 5E mcmmznm 85 63a 334 88.0 @335 663 H88 8.0 bad Be: 663.0 33.35 6.6.3 bum 8.0 we: um: . 62.0 @235 66.3 4843 m6 8838.6 633689 $65 pmumHOdm>m 8% Swz .Homz mo damp he poocmsamafl mm mumqumHma xuwaflnmum pcwan Mom mammE mopedvm unwed .m mHan 67 dendritic NaCl (P<0.05) and blends manufactured with evap- orated and granulated NaCl (P<0.01). The dendritic NaCl blends were again superior to those produced using evapor- ated and granulated NaCl (P<0.01). These stability results suggest that the binding poten— tials of the inherent meat proteins in blends manufactured with flake NaCl were more efficiently utilized than were those in the blends produced using dendritic or evaporated and granulated NaCl samples. These data coincide quite closely with the soluble protein, soluble phase and pH data discussed earlier and lend credibility to the interpretation of those data. The relative proportions of several myofibrillar pro- teins in the soluble phase as affected by NaCl type are shown in Table 9. There were no significant differences between the various NaCl types studied regarding the relative per- centages of either myosin heavy chains, actin or d-actinin which were present in the soluble phase of the meat blends. The proportions of soluble phase protein in various isoelectric point ranges as affected by NaCl type are shown in Table 10. These data again suggest that the relative composition of the soluble phase protein was essentially constant as no significant differences were noted. These electrophoretic data in combination with data discussed previously show that it is the amount of soluble protein which is important in developing blend stability 68 no.4 mm.m mm.u Nr.e Ase .eeeeube-u Hd.~ 4N.NH um.me me.mH Axe eeuu< em.m rm.eH Ne.mm mm.om Axe s>dthwmmwez um m m lm WNWMWMMWMW UHUHHUd—mh— QXMHHH dame Howz .Homz mo mama %n pmoCmDHmcH mm mmmsa mHnDHOm msu CH mewmuoua umaawpnwmo%8 pmuomamm mo mCOfiumHDCmocoo .m mHan How mammfi mmHmSUm ummmd 69 kw.o Hm.wm me.am ue.o~ Axv w - a me.o mo.eH ad.ma ed.mu Axv a - e Hm.o mu.Hm or.Hm mw.me Asv e - m em.o 6N.m mm.n mm.m Axe m - u «u wwwwwwmmww enumerate dream Arev etude ueuuudeuouH were 4662 .Homz mo mahu kn poocmsamcfi mm mmmna mHnDHom m£u Cw mawmuoua mo coauspflhumwp owuuumHmOmw .OH mapmH How mammE mmpmsvm ummma 70 rather than changes in the type of protein which is present. Since the relative proportions of the various proteins did not change it must therefore be the effect of protein con- centration in the soluble phase which manifests itself in terms of improved blend stability through the improved ef— ficiency of available protein utilization. Effect of Blendinngime The effects of blending time on blend pH and the char- acteristics of the soluble phase are shown in Table 11. The pH's of the meat blends were not influenced by blending time. pH values remained constant at approximately six throughout ten minutes of blending. As blending time increased the volume of the soluble phase decreased to a minimum at eight minutes of blending and then increased slightly at the ten minute sampling interval (Figure 12). All blend times produced signifi- cantly different soluble phase volumes (P<0.05) with the exception of the comparison between four and six minutes of blending (Table 11). The concentration of protein in the soluble phase in- creased in a linear manner from zero to four minutes of blending (Figure 13). As was the case for soluble phase volume this trait again leveled off between four and six minutes of blending and then exhibited an increase at the eight minute sampling interval with a decrease obvious at 71 .Amo.ovmv pampmmmwc waucmoHMchHm mum muafiuomumasm ucmpmmwwp zufiz mumps mpunw 00.0 oqo.om pmm.om 00¢.mm omo.mm noq.om mwm.HN AHE\wEV ckuoua mmmna mHnDHom HN.o m.m q.m 0.0 o.n H.0H m.qH AHEV mESHo> m p o o n m mmmfia mHnDHom 000.0 00.0 N0.0 No.0 H0.0 No.0 00.0 00 x um OH m o e N o A.ceev bane uceucdfim .mEHu wcflpcman kn pmuSmDama mm mmflupmaopa mmmna mansHow pcm ma pcmam .HH panda SOLUBLE PHASE VOLUME (ml) 15 H N KO 0‘1 72 Figure 12. 4 6 8 10 BLENDING TIME (min.) Influence of blending time on soluble phase volume. SOLUBLE PHASE PROTEIN (mg/m1) 73 21 L 1 I E J 2 4 6 BLENDING TIME (min.) Figure 13. Influence of blending time on soluble phase protein concentration. 74 ten minutes of blending time. The mean comparisons revealed that all means were significantly different with the excep- tion of the comparisons between four and six minutes, four and ten minutes and six and ten minutes (P<0.05). When Figures 12 and 13 are compared it becomes appar- ent that soluble phase volume and protein concentration in the soluble phase responded to blending time in opposite but similar fashions. This is shown by the fact that as soluble phase volume decreased the protein concentration in this phase increased with both parameters reaching optimum levels at eight minutes of blending. This quite clearly illustrates the relationship between protein solubilization and the water retention properties of the blend, as has also been reported by Hamm (1960). Blend stability parameters as influenced by blending time are shown in Table 12 and graphically in Figure 14. As blending time increased the amount of water lost during cooking decreased in a manner quite similiar to that which was previously discussed for the responses of solu- ble phase volume and protein concentration in the soluble phase. As in the case of these two parameters water loss during cooking reached a minimum at eight minutes of blend- ing time. Unlike soluble phase volume and protein concen- tration in the soluble phase, however, water loss exhibited a significant (P<0.05) decrease when the blending time was extended from four to six minutes with no significant 5 7 .AHo.0vmV ucmummwwp maucmowmwcwfim mum 300 m C03003 muawuomumasm unmummmwp £003 made: 000 .Amo.0vmv quummmap hauchHmwcwwm ppm 300 0 :05003 muawuomhmasm ucmnmwmwp £003 mammzonm 00.0 0000.0 0000.0 0600.0 000.0 0000.0 000.0 0000\000 0060 00060 00.0 600.0 600.0 600.0 000.0 000.0 000.0 0000\000 0060 000 00.0 0000.0 0000.0 0000.0 0000.0 000.0 000.0 0000\060 0060 00603 00 0 0 0 0 0 0.0060 6500 00000000 .0500 wcwpcman %n poucmsamcfi mm mnoumfimuma xuflawnmum pcmap How mammE mmhmscm ummmq .00 00000 LOSS (ml) 76 TOTAL LOSS G <3 WATER LOSS 0"“““'0 FAT LOSS O—"—"O Figure 14. BLENDING TIME (min.) Influence of blending time on blend stability. 77 differences being found between the six, eight and ten min- ute blending times. Fat loss responded to blending time by exhibiting sig- nificant decreases between zero and two and four and six minutes of blending (P<0.05). Again, as in the case of water loss, no differences were found to exist between the six, eight and ten minute blending times. Total loss data showed the same basic pattern of change as that which was found for water loss. Decrease in water loss was at a maximum between zero and two minutes of blend- ing. Following two minutes of blending the decrease in tot- a1 loss occurred at a slower rate until a minimum total loss was reached at eight minutes of blending (P<0.05) and a sub- sequent increase in total loss was experienced between the eight and ten minute blending times (P<0.05). Tables 13 and 14 show the breakdown of the soluble phase proteins by type of protein and isoelectric point range, respectively. As was the case concerning the effects of NaCl type no differences were found among the blending times studied regarding any of these parameters. This again implies that although more protein was solubilized as blending progressed the relative composition of the soluble proteins was basically unchanged. These results regarding the categorization of the proteins by isoelectric point range is probably due in part to the static condition which blend 78 00.0 00.0 00.0 00.0 00.0 00.0 00.0 000 0000060-0 00.0 00.00 00.00 00.00 00.00 00.00 00.00 000 00060 . . . . . . . . 000000 00 0 00 00 00 00 00 00 00 00 00 00 00 00 000 00000 00060: um -0 00 0 0 0 0 0 0.0000 0000 00000000 .0500 wchC0Hn he p0oc0samc0 00 00030 0HLDH00 050 CH mCH0uOHa 00H00030m005 00000000 mo 05000000500coo .MH 00309 How mC00E m0umnvm 0000A 79 00.0 00.00 00.00 00 00 00.00 00.00 00.00 000 0 - 0 00.0 00.00 00.00 00.00 00.00 00.00 00.00 000 0 - 0 00.0 00.00 00.00 00.00 00.00 00.00 00.00 000 0 - 0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 000 0 - 0 00 00 0 0 0 0 0 0000 60060 60006000600 0.0000 0000 00000000 .0500 wchC0H0 00 0000030000 00 00050 0HDDH00 000 CH mcH00OHQ mo 000050000000 000000H0000 000 05005 0000500 00000 .0H 0Hnme 80 pH maintained throughout the range of blending times studied. All of these results concerning blending time strongly suggest that the optimum time of blending which is required to maximize protein solubilization and binding properties is in the range of six to ten minutes. Some of the data, in particular the total loss data, further suggest that in these model systems this optimum time exists at eight minutes of blending. The decrease in the level of quality of most parameters studied in the blending time range of eight to ten minutes may be due to the increased level of mechanical energy which has been introduced into the system by further blending. This increased input of energy would theoretically result in a certain proportion of the proteins experiencing varia- ble degrees of denaturation and therefore a subsequent de- crease in solubilization and functionality as was seen in the previously discussed data. These results concerning the effects of blending time parallel those results previously reported concerning variable degrees of chopping (Brown and Toledo, 1975). Results on the electrophoretic separation of the soluble proteins are also in agreement with those results previously reported by Siegel et al. (l978a). 81 Effects of Post-Blending Storage’Time The means for the effects of post-blending storage time on the pH and solubility properties of sausage blends are shown in Table 15. The pH values of the blends did not change as storage time increased but rather maintained a constant value of approximately six. A small increase in pH value was always noted between days zero and one of storage, however, this increase was not large or consistent enough to be significant. The volume of soluble phase (Figure 15) (Table 15) decreased in a linear fashion from zero to four days of post-blending storage. Following the minimum value which was obtained at four days of storage the blends exhibited an increase in soluble phase volume at day five of storage (P<0.05). All mean comparisons were found to be signficant (P<0.05). The concentration of protein in the soluble phase (Figure 16) exhibited a linear increase from zero to three days of storage at which time protein concentration remained unchanged through day four (P<0.05). Protein concentration in the soluble phase then increased to a maximum at five days of post-blending storage (P<0.05). The relationship between soluble phase volume and sol- uble protein concentration is less obvious in this case than it was in the case of blending time. However, as soluble 82 000000000 0000000000w00 000 3o0 0 000003 000000000000 000000000 0003 00002 .Amo.0v00 000000 00.0 000.00 000.00 000.00 0m0.0N 000.00 000.00 000\000 0000000 00000 00000om 00.0 00.0 00.0 00.0 00.0 00.00 00.00 0000 000000 00000 00000om 000.0 00.0 00.0 00.0 00.0 00.0 00.0 00 00 m 0 m N 0 o A0000v 050B 0&00O0m 0000000mu00o0 .0500 0m00O00 w0000000u00om >0 0000000000 00 0000000000 00000 00000O0 000 00 00000 0o0 00005 0000000 00000 .m0 0000B 83 15 A12 ' r—1 5 E S 0 o > 01 9 - U2 < m 0 m 0 DO a 8 (L6;- 3 j 1 1 41 O 1 2 3 4 POST-BLENDING STORAGE TIME (days) Figure 15. Influence of post-blending storage time on soluble phase volume. SOLUBLE PHASE PROTEIN (mg/m1) 84 Bhfl' 22 00$ 1 I l I O l 2 3 4 5 POST-BLENDING STORAGE TIME (days) Figure 16. Influence of post-blending storage time on soluble phase protein concentration. 85 phase volume decreased the protein concentration increased and vice versa. An exception to this trend may be demon- strated by comparing the response of soluble protein con- centration and soluble phase volume between four and five days of post-blending storage. Although the soluble protein concentration increased significantly (P<0.05) there was no corresponding decrease in soluble phase volume. This fail- ure to behave in the previously established fashion may well have been due to a partial denaturation of the solubilized proteins during storage thus resulting in a decrease in functionality on a per unit protein basis therefore caus- ing the unexpected increase in soluble phase volume. However, it should be noted at this point that this is merely conjecture and it should in no way be misconstrued as fact. The effects of post-blending storage time on the bind- ing properties of the meat blends as established by blend stability tests are shown in Table 16. These same effects are also graphically illustrated in Figure 17. Total loss decreased from zero to one day of storage (P<0.01) and then remained unchanged throughout the remainder of the five day storage period. Total losses after one, two, three, four and five days of post-blending storage were significantly less (P<0.05) than for the initial storage time studied. 86 .000.ov0v 000000000 0000000000w00 000 3o0 0 000003 000000000000 000000000 0003 00002 0w Amo.ov0v 000000000 0000000000w00 000 3o0 0 000003 000000000000 000000000 0003 0000200 00.0 000.0 0m0.0 00m.0 000.0 000.0 wwo.N Awmm\05v 00O0 000OH wo.o 0m0.o 0mm.o 0mm.o 0m0.o 0mm.o 0N0.o Awmm\050 0000 000 no.0 0NN.0 0ON.0 00N.0 0N0.0 000.0 000.0 Ammm\05v 0000 00003 M0 m 0 m N 0 o A0000v 050B 0w00O0m w0000000u0000 .0500 0&00O00 w0000000u00o0 >0 0000000000 00 0000050000 000000000 00000 000 00005 0000000 00000 .00 0000B LOSS (ml) 2. O. 5 O 87 r TOTAL LOSS G 43 WATER LOSS O- -------- .0 FAT LOSS o—--—-_o 9"--~o\ .\~ " ‘O\~ \‘ ‘O\~ \M. ‘O‘- —._..__O 1 l I J J O l 2 3 fl ’3 POST-BLENDING STORAGE TIME (days) Figure 17. Influence of post-blending storage time on blend stability. 88 Water loss decreased (P<0.01) between zero and one day of storage and then remained constant through five days of post-blending storage. Whereas fat loss was unchanged after two days of post-blending storage and then experienced a latent linear decline from two to four days of storage. As in the case of total loss there was no difference between the fat losses for four and five days of storage (P<0.05). These stability data indicate that water retention prop- erties are the initial trait which is improved by post-blending storage with improved fat retention properties occurring at a later point in time. This phenomenon was quite possibly due to an increase in hydration in time basic range of the isoelectric points of the major muscle proteins caused by the binding of Cl- ions from the NaCl as described by Hamm (1960). Once this muscle protein hydration had reached its maximum at one day of storage as indicated by the water loss data the proteins became more readily soluble in the actual meat system and therefore exerted the latent improve- ment shown in the fat retention properties of the meat blends. The comparison of specific myofibrillar proteins and the partitioning of the soluble protein components by isoel- ectric point range are shown in Tables 17 and 18, respect— ively. As in the previous discussion of these data concern— ing NaCl type and blending time effects, no differences were noted for any of the traits evaluated. This once again 89 00.0 No.0 00.0 Nw.q Nn.0 ww.q wo.m ANV 0000000.”0 00.0 0¢.m0 0m.m0 00.00 00.m0 mm.m0 om.m0 00v 0000< . . . . . . . o 000000 00 0 HM ON om ON M© ON 0m ON ON ON NM ON A$V %>mmm CHWO%Z Mm m .0 m N 0 o 00000v 050B 0w00O0m w000000mu00o0 .0500 0w00O00 w0000000s00o0 00 0000000000 00 00000 00000O0 000 00 00000o00 000000000o05 00000000 0o 00O00000000000 .00 00009 0O0 00005 0000000 00000 90 mm.o nw.w~ mm.wm m0.wm Nn.wm ~0.mm mo.mm ANV w u n mm.o mm.m0 qm.m0 mm.m0 mn.m0 am.m0 0w.m0 00v 0 u o Nw.o mq.om 0m.om no.0m Nw.om mm.om mm.om ANV o u m 00.0 om.m mm.m 0m.m mm.m mm.m om.m ANV m u 0 Mm m 0 m N 0 o 000v 00000 00000000000 A0000v 050B 0w0000m w000000mu0000 .0500 0w00o00 w0000000u0000 00 0000000000 00 00000 0000000 000 00 00000000 00 000000000000 00000000000 000 00005 0000000 00000 .w0 0000B 91 reinforces the theory that soluble protein composition is relatively constant. Therefore it is the concentration of solubilized protein which is important in determining the binding properties of the system rather than the type of protein which is solubilized. This in no way implies that all muscle proteins have the same ability to bind fat and water but rather that the overall binding ability per unit of total soluble protein is essentially constant due to consistent composition of the solubilized proteins. As one compares all of the data presented regarding the effects of post-blending storage time it is evident that although soluble protein concentration was greatest following five days of post-blending storage that the ad- ditional soluble protein in this case lacked the ability to improve binding properties as evidenced by the blend stab- ility data. Therefore, the optimum post-blending storage time appears to be four days in order to optimize the func- tionality of the available protein present in the system. BlendinggTime by Post-Blending Storage Time Interaction A graphical illustration of the effects of the signif- icant (P<0.01) blending time by post-blending storage time interaction on protein concentration in the soluble phase is shown in Figure 18. U.) H N \l SOLUBLE PHASE PROTEIN (mg/ml) H KC 92 BLENDING TIME (min.) O-O——O 2-0- ----- 4D 4JD----4:l. Heat at 100°C for 5 minutes. 117 APPENDIX I) (continued) Electrophoresis I. Remove the water from the gels and place them in the electrophoresis chamber. II. Fill the lower buffer chamber with a solution consisting of: 100 ml reagent II, 40 ml reagent III and 860 m1 of deionized distilled water. III. Place prepared samples onto the gels. IV. Overlay the samples with the solution prepared in Electrophoresis step 11. V. Fill the upper chamber with the solution prepared in Electrophoresis step II. VI. Electrophorese at 0.5 mA/gel until the tracking dye is 0.5 cm from the end of the gel. VII. Remove the gels from the tubes. 118 APPENDIX EL Isoelectric focusing in polyacrylamide gels procedure. Reagents I. Dissolve 5.7 g of urea in distilled,deionized water. When urea is dissolved add 0.2g of Triton X-100, 0.5 m1 of carrier ampholytes and 0.5 ml of 2-mercaptoethanol. Bring to a volume of 10 ml with water and store frozen. II. Disperse 10 g of Triton X-100 in distilled,deionized water. Bring to 100 ml with water and store frozen. III. Dissolve 7.08 g of acrylamide and 1.25 g of DATD in distilled, deionized water and bring to a volume of 25 ml with water and store in a sealed amber container at 40C. IV. Dissolve 1.0 g of ammonium persulfate in deionized, distilled water and bring to a volume of 10 ml with water. Prepare fresh daily. Gel Prepgration I. Dissolve 5.5 g of urea in a solution consisting of 2.0 m1 of solution II, 1.33 ml of solution III, 0.5 m1 of carrier ampholytes and 2.0 ml distilled, deionized water. Swirl until urea is completely dissolved. II. Bring volume to 10 ml with water. III. Add 7.0 ul of TEMED. IV. Deaerate using a water aspirator. V. Add 10.0 ul of solution IV and quickly transfer to prepared gel tubes. VI. Overlay the gels with water and allow to polymerize. 119 APPENDIX E. (continued) Sample PreparatiOn I. Combine protein sample with an aliquot of solution I sufficient to yield a final protein concentration of less than 1 mg/ml. II. Vortex briefly. ‘Electrophoresis I. Place polymerized gels in the apparatus and fill the anodic chamber with 0.02 M H3P04 and the cathodic chamber with 0.04 M NaOH. II. Pre-run the gels using the following schedule: 200 V for 30 minutes 300 V for 15 minutes 400 V for 15 minutes. III. Remove the upper electrolyte and apply the protein samples. IV. Refill the upper reservoir with the appropriate electrolyte. V. Electrophorese for 16 hours at 400 V. VI. Remove gels from tubes. VII. Stain and/or profile. APPENDIX F. 1. 120 protein concentration DEGREES OF SOURCE FREEDOM TOTAL 215 Due to Main Plot Units 5 NaCl Type (NT) 2 Error A 3 Due to Sub-Plot Units 210 Blending Time 5 Post-Blending Storage 5 BT x PBS 25 BT x NT 10 PBS x NT 10 BT x PBS x NT 50 Error B 105 *denotes values of P < 0.05 **denotes values of P < 0.01 SS 18568. 16798. 16466. 322 1769. 102 117. 302 87. 89. 350. 720. 3480 7090 5880 .1216 6390 .2796 3812 .0336 1779 9236 0844 7620 8233. 110. 20. 23. 12 oxxxoooo Analysis of variance for soluble phase 2944 7072 4559 4762 .0813 .7178 .9924 .0017 .8644 74. l—‘boN I’TJ 37‘ 7': .98 7': 7': .42 .76 .27 .31 .02 APPENDIX F.2. 121 DEGREES OF SOURCE FREEDOM TOTAL 215 Due to Main Plot Units 5 NaCl Type (NT) 2 Error A 3 Due to Sub-Plot Units 210 Blending Time 5 Post-Blending Storage 5 BT x PBS 25 BT x NT 10 PBS x NT 10 BT x PBS x NT 50 Error B 105 *denotes values of P< 0.05 **denotes values of P< 0.01 128. 59. 59. .7776 68 33 LJ'INl—‘J-‘N 8885 9063 1287 .9822 .8864 .3726 .5865 .9228 .1344 .5566 18. 5220 25. OOOOOOO‘ Analysis of variance for water loss. 9644 .2592 .7773 .4745 .1835 .1922 .2134 .1111 .1764 I’Ti 114. 38. .69 .04 .09 .21 Or—‘r—‘HN 06 42** .63 APPENDIX F.3. 122 DEGREES OF SOURCE FREEDOM TOTAL 215 Due to Main Plot Units 5 NaCl Type (NT) 2 Error A 3 Due to Sub-Plot Units 210 Blending Time 5 Post-Blending Storage 5 BT x PBS 25 BT x NT 10 PBS x NT 10 BT x PBS x NT 50 Error B 105 *denotes values of P < 0.05 **denotes values of P < 0.01 58 56 NWO‘WW 12 SS 7421 .2019 .0075 .1944 .5401 .4675 .0413 .9696 .7555 .3270 .7872 24. 1920 0000000 Analysis of variance for fat loss. .0038 .0648 .6935 .6083 .2788 .3756 .2327 .2557 .2304 PT] 15. H‘ P4 r4 H‘ h) k» 49’ .01 .64 .21 .63 .01 .11 APPENDIX F. 4. 123 DEGREES OF SOURCE FREEDOM TOTAL 215 Due to Main Plot Unit 5 NaCl Type (NT) 2 Error A 3 Due to Sub-Plot Unit 210 Blending Time 5 Post-Blending Storage 5 BT x PBS 25 BT x NT 10 PBS x NT 10 BT x PBS x NT 50 Error B 105 *denotes values of P < 0.05 **denotes values of P < 0.01 82. 26 56 13. .1890 \Jl—‘NU'IO‘ SS 7333 .6406 24. 8910 .7496 .0927 9987 .5211 .4044 .7988 .4802 18. 7005 12. OOOOOl—‘N Analysis of variance for total loss. 4455 .5832 .7997 .2378 .2208 .2404 .1799 .1496 .1781 I’TJ 21. 15. .95 .24 c) rd ta H‘ 0‘ 34 72 .35 .01 .84 ** APPENDIX F.5. 124 DEGREES OF SOURCE FREEDOM TOTAL 215 8 Due to Main Plot Units 5 0 NaCl Type (NT) 2 0 Error A 3 0 Due to Sub-Plot Units 210 7 Blending Time 5 0 Post-Blending Storage 5 0 BT x PBS 25 0 BT x NT 10 0 PBS x NT 10 0 NT x PBS x BT 50 2 Error B 105 3 *denotes values of P < 0.05 **denotes values of P < 0.01 SS .00012 .81061 .70606 .10455 .18951 .31860 .21635 .25075 .26590 .30001 .65850 .17940 0 0000000 Analysis of variance for pH of blend. .35303 .03485 .06372 .04527 .01003 .02659 .03000 .05317 .03028 I’TJ 10. f—‘l—‘OOF—‘N 13 .10 .50 .33 .88 .00 .76 APPENDIX E. 6. 125 DEGREES OF SOURCE FREEDOM TOTAL 215 466. Due to Main Plot Units 5 128. NaCl Type (NT) 2 122. Error A 3 5 Due to Sub-Plot Units 210 338. Blending Time 5 27 Post-Blending Storage 5 25. BT x PBS 25 39. BT x NT 10 9 PBS x NT 10 8 BT x PBS x NT 50 61 Error B 105 166. *denotes values of P < 0.05 **denotes values of P < 0.01 SS 7292 1764 6468 .5296 5528 .7365 0105 0975 .4361 .7492 .8250 6980 61. HHOOHU‘U‘ 3234 .8432 .5473 .0021 .5639 .9436 .8749 .2365 .5876 33. C) (D C) CD (3 U) Analysis of variance for soluble phase volume. PT] 27** ** .49 .15 .99 .59 .55 .78 APPENDIX F. 7. 126 DEGREES OF SOURCE FREEDOM TOTAL 215 1 Due to Main Plot Units 5 NaCl Type (NT) 2 Error A 3 Due to Sub-Plot Units 210 Blending Time 5 Post-Blending Storage 5 BT x PBS 25 BT x NT 10 PBS x NT 10 BT x PBS x NT 50 Error B 105 *denotes values of P < 0.05 **denotes values of P < 0.01 1519. 2065 1152. 9454. 243. 236. .7802 466. 986 491. 2117. 4912. SS 8053 .3474 912. 7498 5976 4576 7155 5734 4934 1020 3050 4881 Analysis of variance for % Actin 456. 384. 48. 47. 39. 46. 149. 42. 46. 3749 1992 7431 3146 4712 6493 1102 3461 7856 c: rd P4 <3 L. r4 I '11 .19 .04 .01 .84 .00 .05 .91 127 APPENDIX F. 8. Analysis of variance for % myosin heavy chains. DEGREES OF 1 SOURCE FREEDOM SS MS E TOTAL 215 10253.466 Due to Main Plot Units 5 4838.3986 NaCl Type (NT) 2 2085.5002 1042.7501 1.14 Error A 3 2752.8984 917.6328 Due to Sub-Plot Units 210 5415.0677 Blending Time 5 168.7465 33.7493 1.16 Post-Blending Storage 5 107.4925 21.4985 0.74 BT x PBS 25 716.3575 28.6543 0.98 BT x NT 10 311.5372 31.1537 1.07 PBS x NT 10 220.4789 22.0479 0.76 BT x PBS x NT 50 828.6551 16.5731 0.57 Error B 105 3061.8000 29.1600 *denotes values of P < 0.05 **denotes values of P < 0.01 APPENDIX F. 9. 128 DEGREES OF SOURCE FREEDOM s_s_ TOTAL 215 2202.8017 Due to Main Plot Units 5 354.5815 NaCl Type (NT) 2 125.4271 Error A 3 229.1544 Due to Sub-Plot Units 210 1848.2202 Blending Time 5 31.5892 Post-Blending Storage 5 52.1655 BT x PBS 25 225.1052 BT x NT 10 89.6110 PBS x NT 10 85.3111 BT x PBS x NT 50 456.8601 Error B 105 907.5781 *denotes values of P < 0.05 **denotes values of P < 0.01 62. 76. ooxoooooxo Analysis of variance for % a-actinin. 7135 3848 .3178 10. 4331 .0042 .9611 .5311 .1372 .6436 ha <3 P‘ rd P‘ <3 I'd .82 .73 .21 .04 .04 .99 .06 129 APPENDIX F. 10. range of pH 4-5. DEGREES OF SOURCE FREEDOM TOTAL 215 4086. Due to Main Plot Units 5 390. NaCl Type (NT) 2 199 Error A 3 190 Due to Sub-Plot Units 210 3695 Blending Time 5 73 Post-Blending Storage 5 85. BT x PBS 25 461 BT x NT 10 161 PBS x NT 10 199. BT x PBS x NT 50 965. Error B 105 1747 *denotes values of P < 0.05 **denotes values of P < 0.01 SS 1231 6218 .7642 .8576 .5013 .2442 7290 .9376 .4700 7572 4912 .8721 99. 63. 14. 17. 18. 16. 19. 19. 16. 8821 6192 6488 1458 4775 1470 9757 3098 6464 Analysis of variance for % protein in pI P4 rd C) :A F‘ <3 I'Ti .57 .88 .03 .11 .97 .20 .16 130 APPENDIX F. 11. Analysis of variance for % protein in pI range of pH 5-6. DEGREES OF SOURCE FREEDOM SS MS F TOTAL 215 5328.6656 Due to Main Plot Units 5 268.3187 NaCl Type (NT) 2 126.6011 63.3005 1.34 Error A 3 141.7176 47.2392 Due to Sub-Plot Units 210 5060.3469 Blending Time 5 107.7185 21.5437 0.89 Post—Blending Storage 5 122.2423 24.4485 1.01 BT x PBS 25 732.2436 29.2897 1.21 BT x NT 10 242.0630 24.2063 1.00 PBS x NT 10 213.0163 21.3016 0.88 BT x PBS x NT 50 1101.3912 22.0278 0.91 Error B 105 2541.6720 24.2064 *denotes values of P < 0.05 **denotes values of P < 0.01 131 APPENDIX F. 12. range of pH 6-7. DEGREES OF SOURCE FREEDOM TOTAL 215 1048. Due to Main Plot Units 5 142 NaCl Type (NT) 2 57 Error A 3 85 Due to Sub-Plot Units 210 905. Blending Time 5 38. Post-Blending Storage 5 35. BT x PBS 25 133. BT x NT 10 60. PBS x NT 10 46 BT x PBS x NT 50 192. Error B 105 399 *denotes values of P < 0.05 **denotes values of P < 0.01 SS 8174 8841 1537 7304 9333 2784 4218 3084 5600 .0861 3444 .9242 28. WWDOU'INN .5767 5768 .6557 .0844 .3323 .0560 .6086 .8469 .8088 Analysis of variance for % protein in pI rd rd H‘ r4 rd n: I ’11 .00 .01 .86 .40 .59 .21 .01 APPENDIX F. 13. 132 Analysis of % protein in pI range of pH 7-8 DEGREES OF SOURCE FREEDOM SS MS TOTAL 215 4654.3662 Due to Main Plot Units 5 385.8373 NaCl Type (NT) 2 222.3469 111.1735 Error A 3 163.4904 54.4968 Due to Sub-Plot Units 210 4268.5289 Blending Time 5 180.3334 36.0667 Post-Blending Storage 5 200.4770 40.0954 BT x PBS 25 580.3281 23.2131 BT x NT 10 274.3369 27.4337 PBS x NT 10 193.7624 19.3763 BT x PBS x NT 50 824.9292 16.4986 Error B 105 2014.3620 19.1844 *denotes values of P < 0.05 **denotes values of P < 0.01 c: rd P4 I'TJ .04 .88 .09 .21 .43 .01 .86 APPENDIX F. 14. Analysis of variance for yield of experimental bolognas. DEGREES OF SOURCE FREEDOM TOTAL 17 Due to Main Plot Units 5 Blending Time 2 Error A 3 Due to Sub-Plot Units 12 Post-Blending Storage 2 BT x PBS 4 Error B 6 *denotes values of P < 0.05 **denotes values of P < 0.01 '88 10 OD—‘NJ-‘H-L‘O‘ .8979 .5209 .5607 .9602 .3770 .6836 .1750 .5184 2.2804 0.6534 1.3418 0.2938 0.0864 | "11 3.49 15.53* 3.40 134 APPENDIX F. 15. Analysis of variance for moisture content of experimental bolognas. DEGREES OF SOURCE FREEDOM SS TOTAL 1 7 Due to Main Plot Units 5 3.4977 Blending Time 2 2.5455 Error A 3 0.9522 Due to Sub-Plot Units 12 23.2180 Post-Blending Storage 2 11.1551 BT x PBS 4 6.0113 Error B 6 6.0516 *denotes values of P < 0.05 **denotes values of P < 0.01 1.2728 0.3174 5.5776 1.5028 1.0086 I’Tl 4.01 * 5.53 1.49 135 APPENDIX F. 16. Analysis of variance for fat content of experimental bolognas. DEGREES OF SOURCE FREEDOM SS TOTAL 17 53.5427 Due to Main Plot Units 5 11.3517 Blending Time 2 8.8875 Error A 3 2.4642 Due to Sub-Plot Units 12 42.1910 Post-Blending Storage 2 22.8426 BT x PBS 4 8.0588 Error B 6 11.2896 *denotes values of P < 0.05 **denotes values of P < 0.01 4.4438 0.8214 11.4213 2.0147 1.8816 l’fl 5.41 6.07* 1.07 136 APPENDIX F. 17. Analysis of variance for protein content of experimental bolognas. DEGREES OF SOURCE FREEDOM TOTAL 17 Due to Main Plot Units 5 Blending Time 2 Error A 3 Due to Sub-Plot Units 12 Post-Blending Storage 2 BT x PBS 4 Error B 6 *denotes values of P < 0.05 **denotes values of P < 0.01 g HONU‘IOOHO‘ .7509 .6088 .8150 .7938 .1421 .9154 .9270 .2996 IE 0.4075 0.2646 1.4577 0.2318 0.2166 I ’11 1.54 * 6.73 1.07 137 APPENDIX F. 18. Analysis of variance for moisture protein of experimental bolognas. DEGREES OF SOURCE FREEDOM TOTAL 17 Due to Main Plot Units 5 Blending Time 2 Error A 3 Due to Sub-Plot Units 12 Post-Blending Storage 2 BT x PBS 4 Error B 6 *denotes values of P < 0.05 **denotes values of P < 0.01 _s_§_ OOOOOOOH .0840 .2574 .1083 .1491 .8266 .1992 .4192 .2082 0.0542 0.0497 0.0996 0.1048 0.0347 I’d 1.09 2.87 3.02 ill 5958 W” L“ Y" ”I” W! WWII mm" 129 "murmur 3 03082 3