Fluorescent Dye Tracing of Water Entry and Retention in Chilling of Broiler Chicken Carcasses

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M. KAMIYOSHI AND K.

sequence, time and duration of the release of follicle-stimulating hormone and luteinizing hormone in relation to oestrus and to ovulation in the sheep. J. Endocr. 35: 177-184. Rosenberg, E., and P. J. Keller, 196S. Studies on the urinary excretion of follicle-stimulating and luteinizing hormone activity during the menstrual cycle. J. Clin. Endocr. Metab. 25: 1262-1274. Santolucito, J. A., M. T. Clegg and H. H. Cole, 1960. Pituitary gonadotropins in the ewe at different stages of the oestrous cycle. Endocrinology, 66: 273-279.

TANAKA

Snedecor, G. W., 1956. The comparison of two randomized groups. Statistical Methods, Iowa State College Press, Ames, Iowa, pp. 85-101. Steelman, S. L., and F. M. Pohley, 1953. Assay of follicle-stimulating hormone based on the augmentation with human chorionic gonadotropin. Endocrinology, 53: 604-616. Tanaka, K., and S. Yoshioka, 1967. Luteinizing hormone activity of the hen's pituitary during the egg-laying cycle. Gen. Comp. Endocr. 9: 374379.

Fluorescent Dye Tracing of Water Entry and Retention in Chilling of Broiler Chicken Carcasses D . H.

SANDERS

Market Quality Research Division, A.R.S., V. S. Department of Agriculture, A thens, Georgia 30601 (Received for publication June 23, 196°)

INTRODUCTION

D

URING slush ice chilling of chicken carcasses, water is absorbed by and trapped between tissues. Previous work in this laboratory has shown that bacterial populations in these chillers can reach levels in excess of 8X10 6 organisms per ml total mesophilic and 4.0 X102 organisms per ml. capable of growth at 4°C. Current U.S.D.A. regulations allow 12 percent water pickup by carcasses from the final washer and chillers. In Federally inspected plants approximately 160 gm. of water could thus be picked up by a 1.36 kgm. (3 lb.) carcass. The number and types of microorganisms which might be introduced under the skin and into the muscles in this process are unknown. To determine the sites of penetration of water into the carcass, a tracer-dye technique was developed. The possibility of quantitative determination of water absorption and retention by this technique was also studied. Water is taken up rapidly by broiler chicken carcasses during slush ice chilling

(Ayres et al, 1956; Bailey et al, 1948; Baker, 1959; Bigbee and Dawson, 1961; Brant, 1963; Fromm and Monroe, 1958; Kahlenberg et al., 1958; Klose et al., 1959; Kraft and Ayres, 1965; Lentz and Rooke, 1958; May et al, 1963; Mickelberry et al, 1962; Tarver et al, 1956; Tarver and Armas, 1964; Thomson et al, 1960, 1966). The uptake of moisture from the cooling medium is most rapid during the initial 30 minutes of immersion (Bailey et at., 1948; Baker, 1959; Bigbee and Dawson, 1961). Agitation of the water and ice chilling medium greatly increases the rate of water uptake and the total weight gained by carcasses (Klose et al, 1959). Although all reports do not indicate whether the birds were spray washed prior to chilling, a 3 percent gain in weight has been reported in this process alone (Klose et al., 1959), followed by additional gains during chilling in either still or agitated ice slush. The sites in the carcass which take up and retain water most efficiently have not been pinpointed. Lentz and Rooke (1958) report a 2 percent gain in weight of breast

W A T E R AND B R O I L E R C H I L L I N G

muscle during 24 hours of chilling in still ice water with a 1.5 percent gain b y the leg muscle. Klose et al. (1959) found no difference in muscle moisture content when carcasses chilled in still and in agitated ice water were compared to each other and to dry-chilled carcasses. Dodge and Stadelman (1959), along with Froning et al. (1959), found no u p t a k e b y muscle while Swanson et al. (1962) found significant gains of from 1 to 3 percent by thigh and breast muscle. While time and agitation are important variables in water absorption, ice-to-water ratios have wide limits within which there is no measurable effect (Mickelberry et al., 1962). Kotula et al. (1962) showed t h a t the method of opening the body cavity influenced the trapping of water in the tissues between the skin and the muscle as "blisters" or lenses of loosely held water. F r o m m and Monroe (1958) reported t h a t weight loss of broiler chicken carcasses during storage is related to total water absorption and to time of storage in crushed ice; the amount of absorbed water retained tends to equilibrate at 3 to 4 percent. PROCEDURE A front-loading, tumbler-type home washing machine was modified b y the removal of its electrical components. The motor was replaced with a 1/2 hp. variable-speed motor and a 25:1 right-angle speed reducer was added to give continuously variable drum speeds of from 0 to 4 revolutions per minute. The drum has three internal flights so, a t full speed the carcasses were agitated b y 12 tumbling actions each minute. This modified washer contained 14 liters of ice and water combined. Three carcasses moved freely in the water in the drum with no crowding a t this ratio of carcass to coolant. Preliminary trials showed t h a t during 20

2033

minutes of chilling a t full speed more t h a n 12 percent water was absorbed. In a local plant individual carcasses picked, eviscerated, and with necks removed were selected from a point just prior to the final washer. These carcasses were placed in large polyethylene bags and transported to the laboratory for chilling. This trip required 10 minutes and weighing to the nearest 4 gm., and placing the birds in the laboratory chiller required an additional 2 to 5 minutes. Three carcasses were collected each trip and then chilled to eliminate possible differences due to various delays before chilling. A total of 27 birds was chilled, three at a time, on each of two days, the first day at 14°C. and the second a t 0 ° - l ° C . T h e treatments were: (a) high agitation (12 tumblings per minute), (b) moderate agitation (6 tumblings per minute), and (c) no agitation, each repeated three times in succession. A Rhodamine B solution containing 1 mg. of dye per ml. was added to the chiller water as a concentrate at the rate of 10 ml. of concentrate per liter of coolant, in the chiller. Levels of Rhodamine were determined before and after chilling each group of 3 carcasses and were adjusted when ice was added to return the temperature to its starting point between runs. In each treatment the 3 carcasses were removed after 20 minutes, drained for 5 minutes, and weighed. Each carcass was placed in a polyethylene bag 20.32X12.7 cm., the open end was closed b y twisting, secured with a rubber band, and placed in storage a t 4°C. in a forced-draft refrigerator. T h e storage periods were 1 hour, 24 hours, and 48 hours. Three carcasses from each t r e a t m e n t were stored for each of the time periods. Carcasses were removed from the polyethylene bags and weighed at the end of each storage period. T h e

2034

D. H. SANDERS

drip from each bag was collected in a covered beaker. The 3 carcasses from each treatment after each storage period, with skin intact, were photographed under UV illuminated on AnsoChrome 5001 daylite film, and then were photographed again with skin removed to expose breast and leg muscle surfaces. The carcasses were then divided into: (1) breast, (2) skin, (3) legs, (4) backs, and (5) wings (skin remained on the wings). Breasts and back were divided at the rib junction and the scapula was left attached to the breasts. The respective parts from all 3 birds were pooled and weighed, then ground twice through a #12 Hobart cutter with 3.175 mm. die holes. This both ground and mixed the samples. The fluorescent material was uniformly distributed after the second grinding. Some cartilage failed to pass the cutter head and was discarded. The cutter head was disassembled and washed with water after each set of parts. The ground product was then placed in polyethylene bags and the bag open ends were twisted and held with rubber bands. Ground samples were frozen at — 40° C. in a blast freezer and stored at — 40° for further analysis. The frozen samples were thawed at room temperature, 50 gm. portions were removed from each, and absolute methyl alcohol added to bring the total volume to 200 ml. This mixture was blended, in screw-cap blender jars, for 1 minute at high speed. The suspension was placed in 50 ml. polyethylene centrifuge tubes and subjected to 12,000XG for 10 minutes. The alcohol-soluble portion containing the Rhodamine B and water was decanted and protein and fat discarded. No visible

fluorescence remained in the fat portion. Prior work had shown the ratio of dye added to dye-extracted by methyl alcohol to be linear within limits much wider than those found here. Fluorescence determinations of the methyl alcohol extracts were made using a Turner model 110 fluorometer with a primary wave length of 546 imi. and a secondary narrow-pass filter at 570 m/x. Preliminary trials showed no methyl alcohol soluble fluorescent substance which interferred at these wavelengths. All values were corrected from the 0-100 scale of the instrument to a 0-1000 scale based upon a setting of X30 for comparison. RESULTS AND DISCUSSION

A high degree of agitation, as compared to a moderate degree, did not substantially increase either pickup or retention of moisture under our conditions (Table 1). Both agitation rates significantly increased water pickup and retention when compared to no agitation. The temperature of the water in the chiller did not measurably affect either pickup or retention. Tables 2 and 4 show that the amount of dye taken up and retained by the tissue was directly proportional to the degree of agitation during chilling. Table 3 shows that the percentage of total amount of dye picked up by individual parts was TABLE 1.—Pickup and retention of moisture during chilling1 and storage of broiler chicken carcasses

Chiller agitation

Percent pickup

Percentage retained under storage at 4°C. 1 hr.

High Moderate None

2

12.6 12.1 3.4

10.9 8.0 0.8

3

24 hrs.

48 hrs.

7.3 6.5 0.7

5.8 5.4 1.3

1

Mention of specific products or instruments by trade names is made for identification purposes only and does not imply any endorsement by the U. S. Government.

1

Each carcass chilled for 20 minutes. Each figure for pickup is the mean pickup for 18 carcasses. 3 Retention percentages are means on 6 carcasses. 2

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WATER AND BROILER CHILLING

relatively constant regardless of the degree of agitation. Approximately 50 percent of the dye was found in the skin, about 20 percent each by backs and wings, about 7 percent by breast muscle and about 6 percent for leg muscles. The fact that degree of agitation (between high and moderate) did not influence weight gain but did affect dye pickup and retention (fluorescence values) suggest that there is some binding of the dye by the tissues. Thus the dye penetration is not quite as extensive as the water which carries it into the tissues; however, local dye concentrations probably exceed the water held at the same locations. Visual examination of the individual carcasses for fluorescence showed that the water dye solution penetrated the skin mainly in the feather follicles and injured surfaces. No dye was visible in the nonfollicular areas of the skin and the muscles did not show any general pattern of penetration through the skin even in the areas directly under the follicles. Only rarely was penetration through the skin indicated by concentrations of dye in the surface muscle. The primary route of dye entry into the areas under the skin was through the body-cavity openings. The concentration of dye in the tissues indicates that the penetration of muscle tissues is progresTABLE 2.—Mean fluorescence values of MeOH extracts of carcass parts chilled with various degrees of agitation Chilling agitation High agitation Moderate agitation No agitation 1

Breast

46

2

Back

Wings1 Legs

Chiller agitation High Moderate None

Breast % 6.8 2 8.0 7.1

Back Wings1 Legs % % % 18.6 18.2 18.2

19.8 20.1 18.2

5.0 6.1 5.6

Skin % 49.8 47.4 50.9

1

Skin not removed from wings. Each column the mean of parts from 3 birds after storage for 1 hour, 3 birds after storage for 24 hours, and 3 birds after storage for 48 hours. 2

sive, beginning with the most exposed areas at the tip of the keel and over the clavicle. When the subdermal areas in front of the thigh are not exposed by the opening cut then only severe agitation introduced any water into these areas and this is only a very small quantity. If, as happened on one of our birds, one side is opened by such a cut and the other not, then only that one side accumulates. Visual observation showed that the dye penetrated to a depth of 3 mm. in the severely agitated birds in the clavicle area and near the keel tip. Penetration was less than 1 mm. in the central breast area. Again, because the tissues act as a filter, the total depth of penetration by water may be much greater. The extent of dyed muscle surface and dye penetration beneath that surface depended upon severity of agitation, extent of opening, and location of nearest opening in the body cavity. There was very little penetration through the skin except for occasional spots under severe agita-

Skin TABLE 4.—Partial analysis of variance for data in Table 2

126

134

34

337

38

86

95

29

224

19

49

49

15

137

Skin not removed from wings. Each value is the mean of fluorescence values from 50 gm. portions of 3 groups of 3 birds each. 3

TABLE 3.—Mean fluorescence values of MeOH extracts of chicken parts chilled with various degrees of agitation expressed as a percentage within treatments

Source

df

ss

MS

f

Total Temp. Agitation Part

89 1 2 4

697,694.90 2,444.01 100,867.19 491,020.62

2,444.01 50,433.59 122,758.16

1.9152 39.5219++ 96.1986++

Error

81

103,363.08

1,276.09

Significant at 1% level of probability.

2036

D . H. SANDERS

tion. Very little dye entered via the hock cuts or from the i n t a c t internal areas of the b o d y cavity even with severe agitation. Fluorescent measurements of drip from carcasses did

not

quantitatively

corre-

late with water absorbed during chilling or lost during storage. CONCLUSIONS 1. Within the range of this study, temp e r a t u r e of the chiller water did n o t alter water pickup of broilers during chilling or retention during storage. 2. D y e binding and retention are related to agitation a n d water penetration, but

this

technique

is n o t

suitable

for

q u a n t i t a t i v e s t u d y of water u p t a k e a n d retention. SUMMARY A dye tracer method is described for the visual observation of the p a t h s of water penetration into broiler chicken carcasses. Localized

water

penetration

into

the

muscle tissues is observable. Q u a n t i t a t i v e evaluation of dye bound b y the tissues did not correlate with total water absorbed during chilling or with loss in " d r i p " during

storage

or

with

moisture

retained

after storage for 48 hours. ACKNOWLEDGEMENTS This project is a cooperative effort with the Food Science D e p a r t m e n t of the University of Georgia. T h e work was done in facilities provided b y the Food Science D e p a r t m e n t in Athens, Georgia a n d their cooperation is gratefully

acknowledged.

REFERENCES Ayres, J. C , H. W. Walker, M. J. Fanelli, A. W. King and F. Thomas, 1956. Use of antibiotics in prolonged storage life of dressed chicken. Food Technol. 10: 563-568. Bailey, F. L., G. F. Stewart and B. Lowe, 1948. Ice slush cooling of dressed poultry. Refrigeration Engineering, 55(4): 55-56,369-371.

Baker, R. C , 1959. Moisture uptake of water-cooled poultry carcasses. Poultry Sci. 38: 1186. Bigvee, D. G.,and L. E. Dawson, 1961. Somefactors that affect change in weight of fresh chilled poultry. Poultry Sci. 40: 1380. Brant, A. W., 1963. Chilling poultry—A review. Poultry Processing and Marketing, 69(5): 14-22. Dodge, J. W., and W. J. Stadelman, 1959. Relationships between pH, tenderness, and moisture levels during early post-mortem aging of turkey meat. Food Tech. 14: 43-46. Froning, G. W., M. H. Swanson and H. M. Benson, 1960. Moisture levels in frozen poultry as related to thawing losses, cooking losses, and palatability. Poultry Sci. 30: 373-377. Fromm, D., and R. J. Monroe 1958. Moisture absorption and retention of freshly eviscerated broilers as influenced by holding time in slush ice. Poultry Sci. 37: 329-331. Kahlenberg, O. J., E. M. Funk, L. A. Vcss, L. G. Maharg and N. L. Webb, 1958. Factors affecting poultry flavor. 2. The effect of a mechanical quick-chill colling unit. Poultry Sci. 37: 350-353. Klose, A. A., M. F. Pool, D. De Fremery, A. A. Campbell and H. L. Hanson, 1959. Effect of laboratory scale agitated chilling of poultry on quality. Poultry Sci. 39: 1193-1198. Kotula, A. W., J. E. Thomson and J. A. Kinner, 1960. Weight increase during chilling of broilers and influenced by method of opening the abdominal cavity during evisceration. Poultry Sci. 39: 226-227. Kotula, A. W., G. J. Banwart and J. A. Kinner, 1967. Effect of post-chill washing on bacterial counts of broiler chickens. Poultry Sci. 46: 1210-1216. Kraft, A. A., and J. C. Ayres, 1965. Development of microorganisms and fluorescence of poultry chilled in water containing iron or magnesium. Food Sci. 30: 154-159. Lentz, C. P., and E. A. Rooke, 1958. What weight changes occur in eviscerated poultry during immersion cooling and drainage? Food in Canada, 18(9): 24-27. May, K. N., R. L. Helmer and R. L. Saffle, 1963. Effect of phosphate treatment on carcass weight changes and organoleptic quality of cut-up chicken. Poultry Sci. 42: 24-32. Mickelberry, W. C , D. V. Schwall and W. J. Stadelman, 1962. The effect of ice water collant ratios upon moisture absorption and rate of chilling of eviscerated chicken carcasses. Poultry Sci. 41: 1550-1553. Swanson, M. H., G. W. Froning and J. F. Richards, 1962. Moisture levels on frozen poultry as related to thawing losses, cooking losses, and palatabil-

WATER AND BROILER CHILLING ity. 2. Turkey fryer-roasters. Poultry Sci. 41: 272-277. Tarver, F. R., Jr., G. C. McGhee and O. E. Goff, 1956. The rate of cooling and water absorption of poultry held in various mediums. Poultry Sci. 35: 905-910. Tarver, F. R. Jr., and A. E. Armas, 1964. Water absorption in broilers influenced by carcass to coolant ratio. Poultry Sci. 43: 432-434.

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Thomson, J. E., A. W. Kotula and J. A. Kinner, 1961. The effect of temperature and time of prechill immersion on total moisture absorption by fryer chickens. Poultry Sci. 40: 1139-1140. Thomson, J. E., A. J. Mercuri, J. A. Kinner and D. H. Sanders, 1966. Effect of time and temperature of commercial continuous chilling of fryer chickens on carcass temperatures, weight, and bacterial counts. Poultry Sci. 45: 463-469.

The Performance of Laying Hens Fed Various Levels of Protein and a Non-essential Amino Acid Mixture A. G. MANOUKAS AND R. J. YOUNG Department of Poultry Science, Cornell University, Ithaca, New York 14850 (Received for publication June 23. 1969)

INTRODUCTION

T

HE protein and amino acid requirements of the laying hen have been studied extensively during the past two decades. These studies have provided a better understanding of the balance of essential amino acids required for optimum egg production. The requirements for the essential amino acids for the laying hen have been reported by Johnson and Fisher (1958) and Scott (1962). Moran et al. (1967) have calculated the essential amino acid requirements for the laving hen based on the amounts needed for egg formation and body maintenance. Little information exists concerning the absolute requirement of the laying hen for protein nitrogen when a diet is fed containing an adequate level of well-balanced essential amino acids together with various levels of non-essential amino acid nitrogen. Shapiro and Fisher (1965) and Shapiro (1968), using egg protein and an isonitrogenous mixture of glutamic acid and glycine, reported that the laying hen required 13 to 14 grams of protein per hen per day to support 70% egg production and satisfactory nitrogen retention (800 mg.N per hen per day). They found that

a daily protein intake of 16 to 17 grams per hen was necessary for maximum nitrogen retention (1200 mg.N per hen per day). At both levels of protein, the essential amino acids represented 45% of the total nitrogen. Several reports have been published on the utilization of nonprotein nitrogen compounds such as diammonium citrate, urea and glutamic acid by chickens receiving practical diets (Young et al., 1965; Chavez et al, 1966; Reid, 1967; and Moran et al., 1967). Young et al. (1965) failed to improve either egg production or feed conversion when a low protein diet was supplemented with glutamic acid. Several factors may influence the response of the chicken to nonprotein nitrogen and nonessential amino acids (NEAA). These include: (1) an intake of an adequate quantity of total protein, (2) a deficiency of essential amino acids, (3) an imbalance of some of the essential amino acids, (4) a single source of NEAA which may not be utilized as efficiently as a mixture, and (5) a non-utilizable source of nitrogen or a toxic effect from the nonprotein nitrogen source itself. To control some of the above factors a