Effects of Rigor, Salt, Freezing, Lyophilization and Storage Time on ph, Water-Holding Capacity and Soluble Protein Nitrogen in Beef Muscle 1
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1 316 Journal of Food Protection, Vol. 47, No. 4, Pages ! (April 1984) Copyright, International Association of Milk, Food, and Environmental Sanitarians Effects of Rigor, Salt, Freezing, Lyophilization and Storage Time on ph, Water-Holding Capacity and Soluble Protein Nitrogen in Beef Muscle 1 J. C. KUO 2 andh. W. OCKERMAN* Department of Animal Science, The Ohio State University, Columbus, Ohio and The Ohio Agricultural Research and Development Center, Wooster, Ohio (Received for publication June 23,1983) ABSTRACT The water-holding capacity (WHC) of frozen and reconstituted lyophilized (freeze-dried) beef (both pre- and post-rigor) increased (P<0.05) with the increase in salt levels (0, 2 and 4%). Freezedried and reconstituted beef had lower (P<0.05) WHC than the frozen control at all salt levels tested. The freeze-drying process may damage some of the beef muscle proteins. The WHC of the freezedried beef (both pre- and post-rigor) decreased (P<0.05) with the increase of storage time (10 weeks). Salt (2 and 4%) retarded the glycolysis process in the pre-rigor frozen and freeze-dried beef as indicated by higher (P<0.05) ph values than the post-rigor frozen and freeze-dried beef. The addition of salt (0, 2 and 4%) increased (P<0.05) the extractable soluble protein nitrogen content in the prerigor frozen beef and decreased (P<0.05) the soluble protein nitrogen content in the post-rigor frozen beef. The pre-rigor freeze-dried beef with 2% salt contained (P<0.05) more extractable soluble protein nitrogen than the other two pre-rigor freeze-dried groups (0 and 4% salt). The pre-rigor beef contained more (P<0.05) extractable soluble protein nitrogen than the post-rigor beef at the three different salt levels (0, 2 and 4%) during the 15 weeks of storage. Ockerman (6) reported that addition of salt (NaCl) to meat lowers the isoelectric point toward the acid end of the ph scale. This is accomplished by the chloride ion binding with the positive charges of the muscle to a stronger degree than the sodium ion combines with the negative charges. Salt when added before rigor reduces the effectiveness of the glycolysis system, and a higher ph is observed at the completion of rigor thus increasing the water holding capacity of the tissue. Wierbicki et al. (/1) reported that the ph shift toward alkalinity produced by addition of sodium chloride, decreased after freezing and thawing. However, Deatherage and Hamm (/) reported that ph values of meat were not signific- 1 Salaries and research support provided by State and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Journal Article No Present address: Department of Food Science, The Tunghai University, Taichung, Taiwan. antly affected by freezing and thawing. Suden et al. (10) indicated that the loss of volatile compounds is responsible for most of the changes in ph of lyophilized (freeze-dried) meat. The rise in ph that occurred when pork was freeze-dried indicated that acidic volatile compounds were removed during the freeze-drying process. Deatherage and Hamm (i) reported that the water-holding capacity of post-rigor meat is increased by quick-freezing (- 55 C) and thawing (at room temperature). This increase in water-holding capacity is probably due to formation of tiny ice crystals without destruction of the cell wall. Slow freezing of meat decreases the water-holding capacity. Saffle and Galbreath (8) reported that under most conditions when the ph increased (varied from 5.4 to 6.5), the amount of protein extracted also increased. Freezing beef reduced the salt-soluble protein in comparison with chilled beef. Johnson and Henrickson (5) found that pre-rigor porcine muscle (ph = 6.44) has 69.9% more extractable salt-soluble protein than post-rigor muscle (ph-5.40). They also noted in pre-blending that addition of 1, 2, or 3% sodium chloride to pre- and post-rigor meat (24 h before the extraction of the protein) increases the extractability of the salt-soluble protein in pre-rigor salted muscle and decreases the extractability of the salt-soluble protein in post-rigor salted muscle. Often it is desirable to hold raw material for sausage production, and the objectives of this research were to investigate the effects of salt levels (0, 2, and 4%), storage method (freeze-dried and frozen), and storage time (0, 5, 10, and 15 weeks) of pre- and post-rigor beef on the ph, water-holding capacity (WHC), and soluble protein nitrogen. EXPERIMENTAL DESIGN Preparation of samples and experimental design Three Hereford steers, 21, 22 and 22 months old, (live weights kg, kg and kg, respectively), were used for this research. The animals were slaughtered separately at the Meat Laboratory, The Ohio State University and the semimembranosus muscles (approximately 2.3 kg each) from the right and left sides of each animal were randomly assigned to preand post-rigor treatments. This experiment was replicated three times. The pre-rigor beef muscle was removed (hot-boned) approximately 35 min post
2 KUO AND OCKERMAN 317 bleeding. Samples were quickly ground twice through a 3/8 in. (9.5 mm) plate (Stimpson Grinder Model 5412) in a cooler (3 ± 1 C) and the ground mixed samples were evenly divided into three groups. Each subgroup received one of three different levels of salt (0, 2 and 4%). Following addition of salt (approximately 55 min post bleeding), the ground beef was reground two additional times through the 3/8 in. plate to obtain a more uniform sample. To control the weight, shape and thickness of beef samples during freeze-drying, the salted and unsalted pre-rigor ground beef was made into uniform shaped 100-g patties using a Tastee Ring Burger Press plastic mold (Robinson Co., Inc.). The whole process for making the pre-rigor beef patties was completed within 1-1/2 h after obtaining the pre-rigor muscle. Each subgroup was further divided into 2 samples for freezing or freeze-drying. The beef samples of the frozen group were vacuum packaged (0.6 kg/cm 2 ) with LC Flex film (moisture and oxygen impermeable, Smith Co.) by Super Vac (Model GK/165, Smith Co.) and immediately frozen and stored at -29 C. The beef samples of the freeze-dried group were also quickfrozen in a freezer bag (LC Hex Film by Smith Co.) at -29 C for approximately 24 hand then freeze-dried (ModelNo MR-BA, VirTis, Gardiner, NY) for 48 h (10 JJI of vacuum, shelf temperature 22 C). After freeze-drying, the beef samples were quickly vacuum packaged (0.6 kg/cm 2 in Super Vac with LC Flex film by Smith Co.) and stored at 25 C. The post-rigor beef muscle was obtained (cold-boned) from the beef sides stored at 3 ± 1 C for at least 48 h. The post-rigor groups (0, 2 and 4% salt) were also subdivided into two groups (frozen and freeze-dried). The procedures for preparing post-rigor frozen and freeze-dried beef samples and the storage conditions were the same as the pre-rigor group. Packages of freezedried and frozen beef samples were randomly assigned to be held for 0, 5, loand 15 weeks. The frozen beef samples (pre- and post-rigor) were thawed at room temperature (approximately 4 h) and the freeze-dried samples were removed from storage before completing chemical analysis. Water-holding capacity (WHC) Approximately 0.5 g of meat was used to determine the WHC by the press method according to Ockerman (6). ph The ph of the samples was determined with a Fisher Accumet ph meter (Model 610A) according to Ockerman (6). Ten grams of meat sample and 100 ml of distilled water were blended for 1 min at low speed before determining the ph with the ph meter. The freeze-dried beef samples were rehydrated by adding excessive distilled water (4:1). Ten grams of the rehydrated meat was used to determine the ph values of freeze-dried beef. Nitrogen compounds Approximately 2 g of freeze-dried sample was used to determine the total soluble nitrogen, soluble non-protein nitrogen and soluble protein nitrogen according to a modification of the procedure of Regier and Tappel (7). Soluble nitrogen. Two grams of freeze-dried beef and 2 g of powdered glass were placed in a mortar. Enough 0.5 N KC1 was added to thoroughly wet the material. The mixture was ground, transferred to a 200-ml volumetric flask with 0.5N KC1 and brought to volume with this solution. The extraction was allowed to proceed with occasional shaking. At the end of 2 h, the solution was centrifuged at 2000 x g for 15 min. The total soluble nitrogen in 50 ml of the supernatant fluid was determined by the Kjeldahl method. Non-protein nitrogen. The non-protein nitrogen was also determined using the KC1 extract supernatant fluid. Ten ml of trichloroacetic acid (85 g/100 ml) was added to 50 ml of the supernatant fluid from the soluble nitrogen preparation. After mixing and letting stand for 15 min, the solution was centrifuged at 2000 Xg for 15 min. The soluble nonprotein nitrogen in 50 ml of this supernatant fluid was determined by the Kjeldahl method. This value was multiplied by 60/50 (necessary because of the TCA dilution). The soluble protein nitrogen was equal to the total soluble nitrogen minus the non-protein nitrogen. The sample of frozen tissue was blended with 100 ml of 0.5 N KC1 for 1 min at low speed, and the solution was diluted to a total volume of 200 ml with 0.5 N KC1 and allowed to stand for 2 h. The procedures to determine the total soluble nitrogen, soluble non-protein nitrogen and soluble protein nitrogen for frozen meat were the same as for the freezedried meat except approximately 7-g (same weight equivalent) sample was used. Statistical analysis Data were analyzed by analysis of variance procedures of the Statistical Analysis System (SAS). Individual F-tests were used to determine the significance of rigor state, salt level, storage method, storage time and the interaction effects. Means were separated by the Duncan's New Multiple Range Test (2) technique. RESULTS AND DISCUSSION The water-holding capacity (WHC) of beef was significantly affected (P<0.01) by rigor state, salt level, storage method and storage time. The interaction of rigor state x salt level x storage method and rigor state x storage method x storage time was also significant (P<0.01). Figure 1 shows the interaction of rigor state x salt level x storage method. The WHC of pre- and post-rigor beef (both frozen and freeze-dried) increased (P<0.05) with an increase in salt levels (0, 2 and 4%). Ockerman (6) reported that the WHC of meat tissues increases with the addition of salt due to the shift of the isoelectric point of the meat tissues. The freeze-dried beef (both pre- and post-rigor) had lower (P<0.05) WHC than the frozen control (both pre- and postrigor). This suggests that the freeze-drying process may damage some of the muscle proteins. Deatherage and Hamm (7) reported that the decrease in tenderness and juiciness in freeze-dried post-rigor meat was caused by a loss of WHC of muscle proteins. However, Hamm (4) reported that the WHC of sausages made from freeze-dried pre-rigor meat was identical with that of sausages made from freshly slaughtered pre-rigor meat. Figure 2 shows the WHC of beef as affected by rigor state, storage method and storage time. In the frozen tissues, prerigor samples had higher (P<0.05) WHC than the post-rigor samples at the various intervals of storage time (0, 5, 10 and 15 weeks). In the freeze-dried beef, pre-rigor samples also had higher (P<0.05) WHC than the post-rigor samples at yr I 60.0 ^^ ^ Pre-rigor, Freeze-dried ;.' '.'* ' '"" * Post-rigor, Freeze-dried 4o.o-r;;::.'- ^ 30.0 S ,, Figure. 1 The water holding capacity (WHC) of beef (S.E.=0.74) as affected by rigor state, salt level and storage method (effect of storage time absorbed). JOURNAL OF FOOD PROTECTION. VOL. 47, APRIL 1984
3 318 CHARACTERISTICS OF PROCESSED BEEF MUSCLE 100.0" " «20.0' 10.0 Pre-rigor, Freezedried Post-rigor, Freezedried Pre-rigor, Freeze-dried Post-rigor, Freeze-dried 0 'T IS Figure 2. The water holding capacity (WHC) ofbeef(s.e. =0.86) as affected by rigor state, storage method and storage time (effect of salt levels absorbed). Figure 3. The ph values of beef (S.E, = 0.08) as affected by rigor state, salt level and storage method (effect of storage time absorbed). and 5 weeks of storage, but at 10 and 15 weeks of storage, the differences between them were not significant. The higher WHC in pre-rigor samples is probably due to the higher ph values. Ockerman (6) reported the WHC of muscle tissues increases with the increase of ph values. The WHC in freeze-dried beef (both pre-and post-rigor) decreased (P<0.05) during the 10 weeks of storage. The WHC in frozen beef (both pre- and post-rigor) decreased during the 0-10-week storage period, and increased slightly at the 15- week storage period. The ph values of beef were affected (P<0.01) by rigor state, salt level, storage method, and storage time. In addition, the interactions of rigor state X salt level x storage method and rigor state x storage period x storage time were also significant (P<0.01). In Fig. 3, the ph values for both frozen and freeze-dried beef (pre-rigor) increased (P<0.05) with an increase in salt levels (0,2 and 4%). This agrees with the work by Ockerman (6), who reported that the average ph value of pre-rigor muscle tissue normally increases with the increase in salt level. The ph values for both frozen and freeze-dried beef (postrigor) were not affected by the addition of three different levels of salt (0,2 and 4%). With the addition of 2 and 4% salt (Fig. 3), the pre-rigor meat (frozen and freeze-dried) had higher (P<0.05) ph values than the post-rigor meat (frozen and freeze-dried). However, with no added salt, the pre-rigor meat (both frozen and freeze-dried) and the post-rigor meat (both frozen and freeze-dried) were not significantly different. This indicates that in pre-rigor meat without salt, to retard the glycolysis process, the ph values of this tissue dropped to approximately the same ph values as the post-rigor meat. Figure 4 shows the ph values of beef as affected by rigor state, storage method and storage time. The ph values of the pre-rigor frozen beef ( ) were Pre-rigor, Freezedried Post-rigor, Freezedried Figure 4. The ph values of beef (S.E. =0.09) as affected by rigor state, storage method and storge time (effect of salt levels absorbed). higher (P<0.05) than those of the post-rigor frozen beef ( ) at the various intervals of storage (0, 5, 10 and 15 weeks) at -29 C. The same trend was followed (P<0.05) by the pre-rigor freeze-dried beef ( ) and the post-rigor freeze-dried beef ( ) stored at 25 C. This suggests that glycolysis was retarded in frozen and freeze-dried beef during storage (15 weeks) at -29 C and 25 C, respectively, when the effect of salt levels is not considered. The soluble protein nitrogen (soluble total nitrogen minus soluble non-protein nitrogen) of beef was affected (P<0.01) by rigor state, salt level and storage time, but not by storage method. However, the interactions of rigor state x salt level x storage methods, and rigor state x salt level x storage time, and rigor state X storage method X storage time and salt level X storage method x storage time were also significant.
4 KUO AND OCKERMAN 319 Figure 5 shows the soluble protein nitrogen of beef as affected by rigor state, storage method and salt level. Ockerman (6) reported that the myofibrillar proteins were most soluble in concentrated salt solution (approximately 0.6 TVKC1) and the sarcoplasmic proteins were soluble in a dilute salt (approximately 0.1NKC1) solution. In this research, the salt solution (0.5 N KC1) was used to extract the salt-soluble protein, which comprised most of the beef myofibrillar proteins. The extractable soluble protein nitrogen in the pre-rigor frozen beef (Fig. 5) increased with the increase in salt levels (0, 2 and 4%). This is probably due to the increase of ph values by adding salt to the pre-rigor frozen beef (Fig. 3). Johnson and Henrickson (5) used different materials and found that addition of 1, 2 and 3% salt to pre-rigor pork muscle also increased the amount of extractable soluble protein. Saffle and Galbreath (8) reported that the soluble protein content (extracted with 3% saline solution) could be used as a measure of emulsifying capacity (the higher, the better). This suggests that the pre-rigor frozen beef with addition of 4% salt may be used as a pre-blend to manufacture sausages or other emulsified products. However, further research is probably needed to study the functional and organoleptic properties of sausages made by such a pre-blend. In the pre-rigor freeze-dried beef (Fig. 5), the treatment with 2% salt contained a greater amount of extractable soluble protein nitrogen than the other treatments (0 and 4% salt). Addition of 4% salt to the pre-rigor freezedried beef decreased the amount of extractable soluble protein when compared to that of the 2% salt level. This phenomenon is different from that of pre-rigor frozen beef. The increase in salt levels caused a decrease (<0.05) in solubility of the post-rigor frozen beef (Fig. 5). This indicates that to obtain the greatest amount of extractable soluble protein for sausage preparation, no salt should be added to post-rigor frozen beef. One explanation for these results is that the ph of the post-rigor frozen beef with three different salt levels ( ) was very close to the isoelectric point of the beef muscle protein ( ); thus the meat protein was in a less soluble state. Johnson and Henrickson (5) also found similar results by adding 1, 2 and 3% salt to post-rigor pork muscle. They explained this by stating that the postrigor pork muscle protein was tied up in the actomyosin complex and that the ph of the post-rigor muscle was at the isoelectric point of pork muscle protein. In the post-rigor freeze-dried beef (Fig. 5), the group with 4% salt contained less (P<0.05) extractable soluble protein nitrogen than the other two groups (0 and 2%). The amount of extractable soluble protein nitrogen was not significantly different between the group with no salt addition and the group with 2% salt. More soluble protein nitrogen was extracted from the prerigor samples (both frozen and freeze-dried) at the three different levels of salt (Fig. 5). This probably was due to the higher ph of the pre-rigor frozen and freeze-dried samples. Saffle and Galbreath (8) reported that the amount of salt-soluble protein extracted is greater for pre-rigor frozen meat than for post-rigor frozen meat. Figure 6 shows the soluble protein nitrogen as affected by rigor state, salt level and storage time. The effects of rigor 4> Pre-rigor, 2% salt Pre-rigor, 4% salt Pre-rigor, 0% salt.. Post-rigor, 0% salt,..* Post-rigor, 2% salt..m Post-rigor, 4% salt = A S Pre-rigor, Freeze-dried ^r:=rsrw -«S^-- Post-rigor, Freeze-dried Figure 5. Soluble protein nitrogen (total soluble nitrogen - soluble non-protein nitrogen) of beef (S.E. =0.05) as affected by rigor state, storage method and salt level (effect of storage time absorbed). Figure 6. Soluble protein nitrogen (total soluble nitrogen - soluble non-protein nitrogen) of beef (S.E. =0.06) as affected by rigor state, salt level and storage time (effect of storage methods absorbed). state and salt level were discussed previously. The soluble protein nitrogen in pre-rigor meat (with three different levels of salt) decreased between 0-10 weeks of storage. However, between 10 and 15 weeks of storage, the soluble protein nitrogen in these treatments (except for the pre-rigor meat with 4% salt) increased slightly. The soluble protein nitrogen in post-rigor meat (with three different levels of salt) remained
5 320 CHARACTERISTICS OF PROCESSED BEEF MUSCLE very stable during the 10 weeks of storage. However, between 10 and 15 weeks of storage, the soluble protein nitrogen of the post-rigor meat also slightly increased in the prerigor meat. Regier and Tappel (7) reported that the KCl-soluble protein nitrogen of freeze-dried beef decreased during 16 d of storage at 54.4 C. The reason why the soluble protein nitrogen increased between 10 and 15 weeks of storage is not known, but is probably due to autolysis of the less soluble proteins into the soluble protein fraction during storage. REFERENCES 1. Deatherage, F. E., and R. Hamm Influence of freezing and thawing on hydration and charges of the muscle proteins. Food Res. 25: Duncan, D. B Multiple range and multiple F tests. Biometrics 11:1. 3. Hamdy, M. K., V. R. Cahill, and F. E. Deatherage Some observations on the modification of freeze dehydrated meat. Food Res. 24: Hamm, R The use of freeze-dried pre-rigor beef in sausages. Proceedings of the 14th Meat Industry Research Conference, American Meat Institute Foundation. Arlington, VA. 5. Johnson, R. G., and R. L. Henrickson Effect of treatment of pre- and post-rigor porcine muscles with low sodium chloride concentrations on subsequent extractability of proteins. J. Food Sci. 35: Ockerman.H. W Chemistry ofmeattissue. TheOhioStateUniversity, Columbus, OH. 7. Regier, L. W., and A. L. Tappel Freeze-dried meat. III. Nonoxidative deterioration of freeze-dried beef. Food Res. 21: Saffle, R. L., and J. W. Galbreath Quantitative determination of salt-soluble protein in various types of meat. Food Technol. 18: SAS Ins. Stat. Anal. System SAS user's guide. SAS Ins. Inc., Raleigh, NC. 10. Suden, J. R., A. M. Pearson, and L. R. Dugan, Jr Rehydration of freeze-dried pork as related to ph and protein denaturation. J. Food Sci. 29: Wierbicki, E., V. R. Cahill, and F. E. Deatherage Effects of added sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and citric acid on meat shrinkage at 70 C and of added sodium chloride on drip losses after freezing and thawing. Food Technol. 11: Bullerman, con't. from p. 315 Penicillium roqueforti to sorbic acid. J. Dairy Sci. 66(Supplement 1): Marth, E. H., C. M. Capp, L. Hasenzahl, H. W. Jackson, and R. V. Hussong Degradation of potassium sorbate by Penicillium species. J. Dairy Sci. 49: Hesseltine, C. W Conditions leading to mycotoxin contamination of foods and feeds, pp In J. V. Rodricks (ed.) Mycotoxins and other fungal related food problems. Advances in Chemistry Series, No American Chemical Society, Washington, DC. 15. Jarvis, B Factors affecting the production of mycotoxins. J. Appl. Bacteriol. 34: Mislivec,P. B.,C.T. Dieter, andv. R.Bruce Effect of temperature and relative humidity on spore germination of mycotoxic species of AspergillusandPenicillium. Mycologia67: Olivigni, F. J., and L. B. Bullerman Production of penicillic acid andpatulin by an atypical Penicillium roqueforti isolate. Appl. Environ. Microbiol. 35: Smith, J. E., and D. R. Berry An introduction to biochemistry of fungal development. Academic Press, New York. 326 pp. 19. Scott, W. T., and L. B. Bullerman Influence of carbohydrate and nitrogen source on patulin production by Penicillium patulum. Appl. Microbiol. 30: Yousef, A. E., ande. H. Marth Growth and synthesis of aflatoxin by Aspergillus parasiticus in the presence of sorbic acid. J. Food Prot. 44: Yousef, A. E., and E. H. Marth Incorporation of [ l4 C] acetate by Aspergillus parasiticus in the presence of antifungal agents. Eur. J. Appl. Microbiol. Biotechnol. 18:
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