Progress 10/01/01 to 09/30/04
Outputs
The current method for separation of bone (Hill and Hites, 1968) (HH) uses papain to digest the soft tissue. The samples are autoclaved, incubated for at least 13 h, and washed with acetone, carbon tetrachloride and ethyl ether. Despite long use, this method results in incomplete digestion, a long experimental time, and use of noxious chemicals. A modified method (MM) was developed to ensure more complete digestion of the soft tissue. Two incubation times (2 and 4 hours) and five concentrations of papain and bromelain (0.0025%, 0.025%, 0.25%, 0.5%, and 1.0%) with a fixed concentration of ficin (0.002%) were used. Samples were incubated before autoclaving. The amount of bone fragments obtained with HH was 6.78%. The percentages of bone fragments obtained with samples incubated for 2 h at the five enzyme concentrations were 7.55, 7.35, 8.17, 8.92, and 8.08, respectively. The values obtained with samples incubated for 4 h were 7.87, 7.60, 9.07, 8.18, and 8.36. There were
no differences among these values. The MM is as follows. Samples are incubated for 2 h in a mixture of papain (0.5%), bromelain (0.5%), and ficin (0.002%). After incubation, the supernatant is replaced with water, the material autoclaved and the supernatant discarded. The bone fragment residue is washed with water, then dried. The weight of the dried residue compared to the initial weight indicates the percentage bone. The MM was evaluated for linearity and reproducibility using increasing levels of bone. The regression equation for linearity was y = -.03 + 0.05x, (R2 = 0.72), where y is the weight of dry bone fragments and x the bone content of the mince. The coefficient of variation of bone content was 14.8%. Thirty fryers each were deboned with a belt-and-drum deboner at belt tensions of 16 and 24 bar using a drum with 1.3 mm diameter apertures. There were no differences in bone recovery (72.5 and 72.6%, respectively). Recovery of mince from manually deboned rabbit meat passed
through the deboner at 24 bar was 79.8%. The percentage weights of the bone fragments obtained at 16 bar were 0.009% with HH and 0.02% with MM. At 24 bar, the percentage weights of bone fragments were 0.004 with HH and 0.011 with MM. To determine the effect of belt tension of the deboner and separation method on the numbers and sizes of bone particles, the particles were separated using HH and MM. After separation, images were taken using the Quick View Plus imaging system (Raitech, Inc. Charlotte, NC) which were used to evaluate the numbers and sizes of bone fragments and to determine the percentage of bone fragments in different size groups using computer based image analysis software (ImageTool v2, University of Texas Health Science Center, San Antonio, TX). The number of bone fragments in each size group was highly variable and there was no reduction in the number of 0.5-0.85 mm or >0.85 mm fragments as a result of using MM with three enzymes. The percentage bone recovered from
the samples with MM was numerically higher than with HH but there were still more large bone fragments than allowed by current legislation.
Impacts
Incubation of samples without prior autoclaving and the use of a combination of proteolytic enzymes enhance the digestion of soft tissue and greatly reduce the time required for incubation. This will provide a more reliable and usable procedure for determination of bone content in mechanically separated meat. Elimination of the use of carbon tetrachloride, acetone and ether makes the modified procedure safer for the environment and the laboratory personnel carrying out the tests. The modified procedure improves digestion, reduces incubation time, and is safer to use.
Publications
- 1. J. I. McNitt, Z. Negatu and K. McMillin. 2002. Mechanical deboning of rabbit carcasses. Proc. Segundo Congreso de Cunicultura de las Americas, Jul. 19-22, 2002, Havana, Cuba pp. 236-239.
- 2. J. I. McNitt, Z. Negatu and K. McMillin. 2003. Influence of rabbit age, deboner drum aperture and hind/fore carcass half on mince components of mechanically separated rabbit. Journal of Muscle Foods, 14(1):25-32.
- 3. J. I. McNitt, Z. Negatu and K. McMillin. 2004. Bone particle determination in mechanically separated meat-Preliminary Results. Proc. 8th World Rabbit Congress, Puebla, Mexico, September 7-10, 2004, pp. 1416-1421.
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Progress 01/01/03 to 12/31/03
Outputs
The method currently used for separation of bone fragments in mechanically separated meat uses papain to digest the soft tissue. The samples are autoclaved, incubated for at least 13 h, and washed with acetone, carbon tetrachloride and ether. A mixture of papain, bromelain and ficin reduces the incubation time and ensures more complete digestion of the soft tissue. Two incubation times (2 and 4 hours) and five concentrations of papain and bromelain (0.0025 percent, 0.025 percent, 0.25 percent, 0.5 percent, and 1.0 percent) with a fixed concentration of ficin (0.002 percent) were used. Samples were incubated before autoclaving. The amount of bone fragments obtained with the unmodified method was 6.78 percent. The percentages of bone fragments obtained with samples incubated for 2 h at the five enzyme concentrations were 7.55, 7.35, 8.17, 8.92, and 8.08. The corresponding values obtained with samples incubated for 4 hrs were 7.87, 7.60, 9.07, 8.18, and 8.36. There were
no differences among these values although the value for samples digested with 0.25 percent bromelain and papain was significantly higher than the control. The following modifications were thus made to the method. 1. Samples were incubated for 2 h in a mixture of papain (0.5 percent), bromelain (0.5 percent) and ficin (0.002 percent). 2. Samples were incubated before autoclaving. 3. Water was used to wash the residue. The modified procedure was evaluated for linearity and reproducibility using samples prepared by mixing boneless mince with mince containing approximately 5.15 percent bone to obtain mixtures with increasing levels of bone. The percentages of bone fragments obtained were 1.14, 2.75, 4.40 and 5.15 percent in mixtures containing 25, 50, 75 and 100 percent of the bone-containing mince, respectively. The coefficient of determination was 82 percent. The coefficient of variation of bone content was 14.8 percent. There were no differences in the bone recovery in rabbit meat
deboned with a Baader 603 belt-and-drum deboner using belt tensions of 16 and 24 bar (72.5 and 72.6 percent, respectively). Recovery of mince from manually deboned rabbit meat passed through the deboner at 24 bar was 79.8 percent.
Impacts
Incubation of samples without prior autoclaving and the use of a combination of proteolytic enzymes enhance the digestion of soft tissue and greatly reduce the time required for incubation. This will provide a more reliable and usable procedure for determination of bone content in mechanically separated meat. Elimination of the use of carbon tetrachloride, acetone and ether makes the modified procedure safer for the environment and the laboratory personnel carrying out the tests. The modified procedure improves digestion, reduces incubation time, and is safer to use.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
Federal regulations strictly limit the size and number of bone particles allowed in mechanically separated mince. The objective of this project was to modify the existing accepted laboratory method for separation of bone fragments to increase efficiency of separation, use non-noxious reagents, and reduce the time required. In the existing method, the sample of mince is autoclaved, then digested twice with papain, washed with acetone, and separated by flotation in carbon tetrachloride: acetone (CCl4:C3H6O). Using this procedure, it is not uncommon to find bone fragments with some meat attached in the sediment or suspended in the supernatant in the CCl4:C3H6O mixture. Digestion of the sample without autoclaving was compared to the existing method based on the amount of suspended particles in the supernatant collected after the first and the second incubations, after washing with acetone and after washing with CCl4:C3H6O. The supernatant was filtered, and the residues
dried, weighed and expressed as percentages of the wet weights of the original samples. The percentages of residues (S.E.) obtained with 12 repetitions of the unmodified procedure at each of the four steps were 2.82 (0.23), 1.69 (0.10), 0.51 (0.09), and 7.45 (0.79), respectively. The corresponding values obtained with the modified procedure were 4.66 (0.20), 2.69 (0.15), 0.24 (0.08), and 0.11 (0.04) thus indicating that, without autoclaving, most of the tissue was separated after the incubation steps and washing with acetone and CCl4:C3H6O was unnecessary. The percentages of bone residues from the samples were 5.39 (0.55) and 6.2 (0.35) for the unmodified and modified procedures, respectively. The amount for the unmodified procedure was less because some bone particles with soft tissue attached did not sediment in the CCl4:C3H6O. There was no visually apparent soft tissue attached to the bone particles obtained with the modified method. Separation in water or CCl4:C3H6O were compared
using mince incubated following the modified procedure. Final separation of bone from soft tissue was done with water or with CCl4:C3H6O. Supernatants were filtered and the dried residues expressed as percentages of the wet weights of the samples. The percentages of residues (S.E.) obtained with 15 repetitions of the CCl4:C3H6O separation were 4.32 (0.31), 2.57 (0.23), 0.15 (0.04) and 0.15 (0.03) for the four steps, respectively. With 15 samples incubated and rinsed with water, the percentages of residues were 4.00 (0.19) and 2.22 (0.28) respectively for the two incubations. The percentages of bone residues from the samples were 6.00 (0.34) and 6.65 (0.38) for CCl4:C3H6O and water samples, respectively. Thus, separation of bone from the soft tissue can be achieved by washing with water alone. Current research involves using combinations of enzymes including papain, ficin and bromelain to determine optimal incubation times and enzyme concentrations. The mixture of enzymes is used
because the mixture will greatly enhance digestion of the soft tissue thereby giving more complete separation of bone. Currently we are evaluating incubation for two lengths of time with five concentrations of enzymes.
Impacts
Elimination of the use of carbon tetrachloride should reduce the negative effects of this pollutant on the environment. Reduction of the time required and increase of the accuracy of the laboratory method will provide a more reasonable test for bone content in mechanically separated meats.
Publications
- J. I. McNitt, Z. Negatu and K. McMillin. 2002. Influence of rabbit age, deboner drum aperture and hind/fore carcass half on mince components of mechanically separated rabbit. Journal of Muscle Foods, In press.
- J. I. McNitt, Z. Negatu and K. McMillin. 2002. Mechanical deboning of rabbit carcasses. Proc. Segundo Congreso de Cunicultura de las Americas, Jul. 19-22, 2002, Havana, Cuba pp. 236-239.
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