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The effect of uncooled storage on proteolysis bitterness and apparent viscosity in ultra-high-temperature milk
Can. Can. Insl. Ins!. Food Faad Sei. Sci. Teehnol. Technol. J. J. Vol. 17, No. I. pp. 014-017, 1984
RESEARCH
The Effect of Uncooled Storage on Proteolysis Bitterness and Apparent Viscosity in Ultra-HighTemperature Milk R.C. McKellar, D.A. Froehlich, G. Butler,l H. Cholette and C. Campbell Food Research Institute Research Branch Agriculture Canada, Ottawa, Canada KIA OC6
Abstract
Introduction
Changes in proteolysis, bitterness and apparent viscosity in 2 batches of ultra-high-temperature milk from each of 4 dairies, 2 using the indirect and 2 the direct method of sterilization were examined during storage at 20°C for up to 30 weeks. Proteolysis and bitterness increased with time in milk from all 4 dairies. These changes were more apparent in milk sterilized by the direct method. Milk sterilized by the indirect method exceeded the manufacturer's expected shelflife of 12 weeks while 2 of the 4 batches of directly sterilized milk spoiled within this period. Increases in apparent viscosity and gelation were detected in milk from dairies using the direct method of sterilization between 6 to 10 weeks after significant bitterness had developed. Close correlations (0.822-0.97 (0.822-0.971) I) between proteolysis and developed. time were observed for all 4 dairies. Proteolysis correlated well with bitterness (0.762-0.862) in milk sterilized by the direct method. Lower but highly significant (p < 0.01) correlations (0.477 and 0.507) were obtained with the other 2 dairies. These results suggest that rate of proteolysis could be used to predict shelf-life of ultra-hightemperature milk sterilized by the direct method.
Ultra-high-temperature (UHT) milk, produced by a 130o -lS0 -l50°C treatment of milk at Bo C for 2-8 s, is gaining popularity in Canada. Two UHT processes are currently in use in this country: the indirect process in which milk is heated by a series of plates or tubes and the direct process in which steam is injected directly into the milk. UHT milk can be stored unrefrigerated for up to 3 or 4 m without developing serious offflavour; however, the shelf-life is eventually limited by age gelation and the development of bitterness and other off-flavours during storage. A number of workers have implicated heat-stable extracellular proteinases of psychrotrophic origin in age gelation ( Law et al., 1977) and the development of bitterness (Bengtsson et al., 1973, Richardson and Newstead, 1979) in UHT milk. Close correlations between proteolysis and shelf-life of UHT milk have been obtained (Mottar et al., 1979; Richardson and Newstead, 1979; Mottar, 1981). In a previous study (McKellar, 1981) a high positive correlation was observed between added psychrotroph proteinase and off-flavour development in UHT milk. Conventional methods for the detection of proteolysis such as the determination of non-protein nitrogen and the Hull test are not sensitive enough to predict shelf-life of milk products (Richardson and Newstead, 1979; Richter et al., 1979). In contrast, proteolysis was detected in milk before off-flavour development by measuring the release of tricholoracetic acid (TCA)-soluble free amino groups using trinitrobenzene sulfonic acid (TNBS) (McKellar, 1981). In the present study, detection of proteolysis in stored UHT milk with TNBS was examined and the ability of this method to predict shelf-life was assessed. In addition, the effect of method of sterilization on proteolysis, bitterness and gelation in UHT milk was studied.
Resume
L'aptitude a la conservation de laits sterilises par les methodes directe et indirecte fut etudiee au cours du stockage a 20°C pendant 30 semaines. On a pris deux lots de lait UHT de chacune de 4laiteries, 2 utilisant la methode indirecte, et 2, la methode directe. Les parametres mesures furent les changements en proteolyse, amerI'amertume augmentume et viscosite apparente. La proteolyse et l'amertume thent avec le Ie temps chez tous les laits. Ces changements furent plus terent evidents dans les laits sterilises par la methode directe. Le lait sterilise par la methode indirecte a depasse la duree de conservation de Ie fabricant, tandis que 2 des 4 lots de lait 12 semaines visee par le par la methode directe furent gates durant cette periode. sterilise par Des augmentations de la viscosite apparente et de la gelification furent aussi observees chez les laits sterilises par la methode directe, mais seulement apres un entreposage de 6 a 10 semaines et apres mais Ie developpement developpement d'une amertume significative. De fortes correlale tions (0.822 - 0.971) furent obtenues pour tous les laits entre la Ie temps. Une bonne correlation (0.762 et 0.862) a ete proteolyse et le obtenue entre la la proteolyse et l'amertume I'amertume chez les laits sterilises par la methode directe. Des correlations (0.477 et 0.507) plus faibles, mais hautement significatives (P < 0.01) furent obtenues pour les autres laiteries. Ces resultats suggerent sugghent que les taux de prodeux autres teolyse pourraient servir a predire l'aptitude a la conservation des laits UHT sterilises par la methode directe.
Materials and Methods
IEngineering and and Statistical Statistical Research Institute Research Branch, Agriculture Canada, OtIEnginet:ring tawa, Ontario, Ontario, Canada Canada KIA KIA OC6. tawa,
UHT milk (20/0 (2070 butterfat) was obtained from 4
Copyright ()<> 1984 1984 Canadian Canadian Institute Copyright Institute of of Food Food Science Science and and Technology Technology
14
Canadian dairies (A, B, C and D), 2 using the indirect (A and B) and 2 the direct (C and D) methods of sterilization. All milk was packaged in I1 L Tetra Brik cartons. Two batches each consisting of 140 cartons and produced within 2 weeks of each other were obtained from each dairy. Milk was stored at 20°C and cartons removed as required. At intervals of 2 and 4 weeks, 2 cartons from each batch were opened aseptically and 1 mL was spread on plate count agar (Difco Laboratories, Detroit, Michigan) and incubated at 20°C for 48 h to determine sterility. Duplicate 2 mL samples were also removed removed for the determination of free amino groups. Viscosity measurements were performed on the milk prior to their submission for sensory evaluation. Results were the means of the 2 cartons. TCA-soluble free amino groups were determined with trinitrobenzene sulfonic acid (TNBS) as described previously (McKellar, 1981). Proteolysis was defined as the increase in the concentration of TCA-soluble free free amino groups per mL of milk (.::ljtmoles/mL). (Ajtmoles/mL). Apparent viscosity was measured with a Brookfield synchro-lectric synchro-Iectric viscometer model LVF (Brookfield Engineering Laboratories, Stoughton, MA). A model RVT bob No. No.22 was used for most measurements. Bobs No. No.33 and 4 were substituted when viscosity increased. Four measurements at 2 min intervals were made per carton at 20°C at a speed of 30 rpm and readings were converted to centipoises.
Sensory Evaluation Panelists were initially screened for their ability to perceive bitterness. The 13 most sensitive panelists evaluated samples of both water and milk with 1 to 48 mM leucine (a bitter amino acid) for intensity of bitterness using descriptive analysis with scaling (Larmond, 1977). During training a reference sample of 30 mM of leucine in water was established and anchored at 5.2 cm on a 15 cm scale. Ten trained panelists evaluated the 8 UHT milk samples (4 dairies times 2 batches) during 2 daily sessions, morning and afternoon. In each session, each of the 4 dairies was evaluated with batches being randomly assigned to either of the 2 sessions. Evaluation of the 8 samples was replicated twice over 2 days. Each judge received 60 mL of the leucine-in-water reference and 30 mL of each milk sample, all at 17°C. Numerical values were obtained by measuring the distance in centimeters of the line scores from the left end of the line scale. The first 3 series of evaluations were 4 weeks apart, while the following 6 series of evaluations were 2 weeks apart. Milk from dairies A and B were evaluated throughout the 9 sessions, while milk from dairies C and D were evaluated up to the eighth and sixth sessions, respectively. Gelled milk was not evaluated for bitterness. Statistical Analysis Analysis of variance was performed on the bitterness Can. Insl. lnsi. Food Food Sci. Sci. Technol. Technol. J. J. Vol. Vol. 17, 17. No. No. I, I, 1984 1984 Can.
Table I. Correlation coefficients for the relations between proteolysis, bitternes, and time. Dairy A
Process Indirect Indirect Direct Direct
B C D **P <0.01 INo. of cartons
Proteolysis vs Time 0.822**(64 1) 0.898**(62) 0.926**(57) 0.926* *(57) 0.971**(58) 0.971 **(58)
Proteolysis vs Bitterness 0.477**(35) 0.507**(36) 0.762**(32) 0.862**(24)
scores. The point of significant bitterness was established for each batch by means of t-test comparisons of later sessions to the initial sensory panel sessions. The relationship of bitterness and proteolysis to time was examined by analysis of covariance. Analysis of variance was also performed on background TCA-soluble free amino groups and significance at the 5070 level was determined by Duncan's Multiple Range Test. Throughout, because all effects were nested, the between-batch, within-dairy mean square was used to establish dairy differences and the between-carton, within-batch mean square was used to test batch differences.
Results and Discussion Background TCA-soluble free amino groups were determined for each batch of milk within one week of production date. The values, ranging from 0.631 0.631 to 0.780 {tmoles/mL, jtmoles/mL, were similar for all 4 dairies. A significant difference (P < 0.05) between batches was found for dairy A. Values for dairy A were also significantly different from those for Dairies B, C and
D. Correlations between proteolysis and time-since-production and between proteolysis and bitterness are shown in Table 1. l. Values reported are by dairy since insufficient data was available to warrant calculating correlation coefficients for each batch. Proteolysis in the milk from all 4 dairies was closely correlated with time. In addition, the increase in bitterness correlated well with the increase in proteolysis. Correlations between proteolysis and bitterness were lower for milk sterilized by the indirect (.477 and .507) as compared to the direct method (.762 and .862). However, all correlations were highly significant. Table 2. Relationship of proteolysis and bitterness to time-sincetime·sinceproduction determined by analysis of covariance Slopes Dairy Batch Proteolysis' Bitterness 2 Proteolysis I 0.154 (.0381) A 0.0084 (.0007 3) 1I 2 0.0088 (.0009) 0.049 (.0394) I1 0.0097 (.0008) 0.1 04 (.0355) B 0.104 2 0.0083 (.0008) 0.139 (.0307) I1 0.0216 (.0010) 0.135 (.0440) c 2 0.0302 (.0016) 0.216 (.0403) I1 0.0437 (.0016) 0.224 (.0696) D 2 0.0505 (.0019) 0.330 (.0483) 'Based on 241 observations. 2Based 2Eased on 127 observations. Jstandard error of the slope based on variation between between cartons at each time. McKellar et al. / 15
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Fig. 1 The effect of storage at 20°C 20 0 e on proteolysis in UHT milk. (a) Batch 1, I, (b) batch 2. Dairies; A (0); B (e); e C (D); D (_).
The relationship of proteolysis and bitterness to time-since-production was examined by analysis of covariance. This analysis has been summarized in Table 2. Proteolysis increased significantly (P < .05) with time for all dairies; however, the development of proteolysis during storage varied considerably among dairies (Figure 1). The slopes for dairies A and B, where less proteolysis was observed, did not differ from each other (P < 0.05; Table 2). Extent of proteolysis was much greater in milk sterilized by the direct method (dairies C and D). The milk from dairies A and B experienced a leveling-off of proteolysis after week 15 (Fig. 1).
A closer examination was made of the sensory data to uncover thresholds of bitterness detection in each batch. Mean bitterness scores are given in Table 3. At the initial sensory session, the milks ranged in ages from 2 to 7 weeks from date of production. Initial scores varied between 2.5 and 4.1; however, they were not related to age of the milk. Scores which differed significantly from the mean initial score of 3.33, derived from initial scores from all 4 dairies, are indicated in Table 3. Milk sterilized by the indirect method became significantly bitter after 18 to 19 weeks of storage, while directly sterilized milk suffered loss of flavor after 10 to 18 weeks. The sensory score associated with detectable bitterness appears to be approximately 4.3. Age gelation was observed in milk from dairies C and 0D between 6 and 10 weeks after the milk had become significantly bitter. Gelation was observed at an apparent viscosity of 100 centipoises and was not found in milk from dairies A and B. Increased apparent viscosity was noted for batches 1 and 2 at 24 and 21 weeks for dairy C and 18 and 16 weeks for dairy oD (Figure 2a and b). This study has clearly demonstrated that proteolysis is more extensive in milk sterilized by the direct as compared to the indirect method. This is in agreement with the findings of other workers (Mottar et al., 1979; Mottar, 1981; Snoeren and Both, 1981; Corradini and Pecchini, 1981). The direct method imparts a greater heat intensity to the milk and may therefore inactivate the proteinase more effectively than does the direct method (Corradini & Pecchini, 1981). Alternatively, proteinase activity may be inhibited by the greater amount of denaturated J3-lactoglobulin present in milk sterilized by the indirect method (Snoeren & Both, 1981). The development of bitterness in UHT milk was closely related to proteolysis (Mottar et al., 1979; Mottar, 1981). In this study, highly significant correlations were obtained between proteolysis and bitterness for all 4 dairies; however, correlations were low for dairies A and B suggesting that factors other than proteolysis may be responsible for the development of bitterness in milk sterilized by the indirect method.
Table 3. Mean bitterness scores for UHT Milk stored at 20°C. Sessions I (weeks) Batch Process 0 4 8 12 10 14 16 18 20 S.E.2 A Indirect 3.2 3.5 3.2 4.3* 4.4* 5.8** 5.4** 4.0" .57 I 3.1 4.Q4 3.7 3.2 4.0 4.3* 5.2** 3.3 .57 2 Indirect 4.1 3.4 4.2 B Indirect 4.1 2.8 3.5 2.8 4.5 5.0** 4.4* 1I 4.8** 4.6* .61 2 Indirect 2.5 3.2 3.7 4.7** 4.8** 5.2** 4.4* .59 3.1 4.1 eC Direct 3.5 3.2 2.9 3.8 3.3 .57 I 4.1 5.1** 6.0** ND3 5.1** 6.5** .57 2 Direct 2.6 3.4 3.4 3.3 5.2** 4.1 ND D Direct 3.0 .59 I 2.5 3.8 5.2** 4.8** 4.5* ND ND ND 2 Direct 2.5 5.7*' ND .57 2.7 4.3* 4.3* 6.3** 5.7** ND ND A~~rage scores by session 3.33 2.94 3.69 4.38 4.85 5.66 4.24 4.74 ;\.~~rage 3.61 .21 ~P<0.05 and **P
Dairy
16 / MCKellar McKellar et al.
J. Inst. Can. Sci. Technol. Aliment. Vol. 17, No. I, 1984
terness, although significant, was slight and it is possible that gelation plays a greater role in limiting the shelf-life of UHT milk sterilized by the direct method than does bitterness. Background free amino groups in fresh UHT milk were lower than reported previously (McKellar, 1981). Significant differences were oserved between batches and between dairies; however, the differences were relatively small when compared to the amount of proteolysis necessary for significant bitterness. Background variability of free amino groups may be slight for a given dairy and subsequent changes may serve as an indicator of UHT milk quality. R2 values calculated from the data in Table 1 indicate that 58.1 and 74.3070 of the variation in bitterness in milk from dairies C and D, respectively, can be explained by proteolysis. It may be possible, therefore, to predict degree of bitterness and subsequent shelfclife of milk sterilized by the direct method by monitoring the initial rates of proteolysis in stored milk. Further studies are necessary to establish sensory scores corresponding to significant bitterness and to ascertain if these scores are of commercial significance.
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References
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100
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(weeks' TIME (w.eks'
Fig. 2. The effect of storage at 20°C on apparent viscosity in UHT milk. a, batch I; b, batch 2. Dairies; A (0); B (e); C (0); D (_).
Significant differences in rates of proteolysis were observed between dairies and to a lesser extent between batches within dairies. Dairy differences may be ascribed to processing parameters. Batch differences may be explained by differences in the quality of raw milk or to variations in the quantity of proteinase produced by psychrotrophs (Cliffe and Law, 1982; McKellar, 1982). Milk sterilized by the indirect process had a longer shelf-life as compared to directly sterilized milk. All batches of milk from dairies A and B exceeded the expected shelf-life of 12 weeks. The shelf-life of milk from dairies C and D was reduced as a result of extensive proteolysis; however, milk from dairy C still exceeded the shelf-life of 12 weeks. Milk from dairy D, with a manufacturer's expected shelf-life of 16 weeks had an observed shelf-life of 10-12 weeks. Increased viscosity and gelation were observed with milk sterilized by the direct method. Gelation was detected after significant bitterness had developed. Bit-
Can. lnsf. Vol. 17, No. 1, I, 1984 fnst. Food Sci. Techno!. Technal. J. Val.
Bengtsson, K.L., Gardhage, L. and Isaksson, B. 1973. Gelation in UHT treated milk, whey and casein solution. The effect of heat resistant proteases. Milchwissenschaft 28:495. Cliffe, A.J. and Law, B.A. 1982. Predictive test for gelation in UHT milk using hide powder azure. Dairy Ind. Int. 47:25. Corradini, C. and Pecchini, G. 1981. Effect on proteinases of different UHT treatments. Neth. Milk Dairy J. 35:393. Larmond, E. 1977. Laboratory methods for sensory evaluation of food. Research Branch, Canada Department of Agriculture, Publication 1637. Law, B.A., Andrews, A.T. and Sharpe, M.E. 1977. Gelation of ultra-high-temperature-sterilized milk by proteases from a strain of Pseudomonas fluorescens isolated from raw milk. J. Dairy Res. 44:145. McKellar, R.C. 1981. Development of off-flavors in ultra-hightemperature and pasteurized milk as a function of proteolysis. J. Dairy Sci. 64:2138. McKellar, R.C. 1982. Factors influencing the production of extracellular proteinase by Pseudomonas fluorescens. J. Microbio!. 50:305. Appl. Microbiol. Mottar, J. 1981. Heat resistant enzymes in UHT milk and their influence on sensoric changes during uncooled storage. Milchwissenschaft 36:257. Mottar, J., Waes, G., G" Moermans R. and Maudts, M. 1979. Sensoric sorie changes in UHT milk during uncooled storage. Milchwissenschaft 34:257. 1979. Effect of heat-stable Richardson, B.C. and Newstead, D.F. 1979, proteases on the storage life of UHT milk. New Zeal. J. Dairy Sci. Technol. Techno!. 14:273. Richter, R.L., Schmidt, R.H., Smith, K.L., Mull, L.E. and Henry, S.L. 1979. Proteolytic activity in ultra-pasteurized, aseptically packaged whipping cream. J. Food Prot. 42:43. Snoeren, T.H.M. and Both, P. 1981. Proteolysis during the storage II. Experiments with milk of UHT-sterilized whole milk. 11. heated by the indirect system for 4s at 142°C. Neth. Milk Dairy J. 35:113. Accepted September I, 1983.
McKellar et al. / 17 McKeIlar
RESEARCH
The Effect of Uncooled Storage on Proteolysis Bitterness and Apparent Viscosity in Ultra-HighTemperature Milk R.C. McKellar, D.A. Froehlich, G. Butler,l H. Cholette and C. Campbell Food Research Institute Research Branch Agriculture Canada, Ottawa, Canada KIA OC6
Abstract
Introduction
Changes in proteolysis, bitterness and apparent viscosity in 2 batches of ultra-high-temperature milk from each of 4 dairies, 2 using the indirect and 2 the direct method of sterilization were examined during storage at 20°C for up to 30 weeks. Proteolysis and bitterness increased with time in milk from all 4 dairies. These changes were more apparent in milk sterilized by the direct method. Milk sterilized by the indirect method exceeded the manufacturer's expected shelflife of 12 weeks while 2 of the 4 batches of directly sterilized milk spoiled within this period. Increases in apparent viscosity and gelation were detected in milk from dairies using the direct method of sterilization between 6 to 10 weeks after significant bitterness had developed. Close correlations (0.822-0.97 (0.822-0.971) I) between proteolysis and developed. time were observed for all 4 dairies. Proteolysis correlated well with bitterness (0.762-0.862) in milk sterilized by the direct method. Lower but highly significant (p < 0.01) correlations (0.477 and 0.507) were obtained with the other 2 dairies. These results suggest that rate of proteolysis could be used to predict shelf-life of ultra-hightemperature milk sterilized by the direct method.
Ultra-high-temperature (UHT) milk, produced by a 130o -lS0 -l50°C treatment of milk at Bo C for 2-8 s, is gaining popularity in Canada. Two UHT processes are currently in use in this country: the indirect process in which milk is heated by a series of plates or tubes and the direct process in which steam is injected directly into the milk. UHT milk can be stored unrefrigerated for up to 3 or 4 m without developing serious offflavour; however, the shelf-life is eventually limited by age gelation and the development of bitterness and other off-flavours during storage. A number of workers have implicated heat-stable extracellular proteinases of psychrotrophic origin in age gelation ( Law et al., 1977) and the development of bitterness (Bengtsson et al., 1973, Richardson and Newstead, 1979) in UHT milk. Close correlations between proteolysis and shelf-life of UHT milk have been obtained (Mottar et al., 1979; Richardson and Newstead, 1979; Mottar, 1981). In a previous study (McKellar, 1981) a high positive correlation was observed between added psychrotroph proteinase and off-flavour development in UHT milk. Conventional methods for the detection of proteolysis such as the determination of non-protein nitrogen and the Hull test are not sensitive enough to predict shelf-life of milk products (Richardson and Newstead, 1979; Richter et al., 1979). In contrast, proteolysis was detected in milk before off-flavour development by measuring the release of tricholoracetic acid (TCA)-soluble free amino groups using trinitrobenzene sulfonic acid (TNBS) (McKellar, 1981). In the present study, detection of proteolysis in stored UHT milk with TNBS was examined and the ability of this method to predict shelf-life was assessed. In addition, the effect of method of sterilization on proteolysis, bitterness and gelation in UHT milk was studied.
Resume
L'aptitude a la conservation de laits sterilises par les methodes directe et indirecte fut etudiee au cours du stockage a 20°C pendant 30 semaines. On a pris deux lots de lait UHT de chacune de 4laiteries, 2 utilisant la methode indirecte, et 2, la methode directe. Les parametres mesures furent les changements en proteolyse, amerI'amertume augmentume et viscosite apparente. La proteolyse et l'amertume thent avec le Ie temps chez tous les laits. Ces changements furent plus terent evidents dans les laits sterilises par la methode directe. Le lait sterilise par la methode indirecte a depasse la duree de conservation de Ie fabricant, tandis que 2 des 4 lots de lait 12 semaines visee par le par la methode directe furent gates durant cette periode. sterilise par Des augmentations de la viscosite apparente et de la gelification furent aussi observees chez les laits sterilises par la methode directe, mais seulement apres un entreposage de 6 a 10 semaines et apres mais Ie developpement developpement d'une amertume significative. De fortes correlale tions (0.822 - 0.971) furent obtenues pour tous les laits entre la Ie temps. Une bonne correlation (0.762 et 0.862) a ete proteolyse et le obtenue entre la la proteolyse et l'amertume I'amertume chez les laits sterilises par la methode directe. Des correlations (0.477 et 0.507) plus faibles, mais hautement significatives (P < 0.01) furent obtenues pour les autres laiteries. Ces resultats suggerent sugghent que les taux de prodeux autres teolyse pourraient servir a predire l'aptitude a la conservation des laits UHT sterilises par la methode directe.
Materials and Methods
IEngineering and and Statistical Statistical Research Institute Research Branch, Agriculture Canada, OtIEnginet:ring tawa, Ontario, Ontario, Canada Canada KIA KIA OC6. tawa,
UHT milk (20/0 (2070 butterfat) was obtained from 4
Copyright ()<> 1984 1984 Canadian Canadian Institute Copyright Institute of of Food Food Science Science and and Technology Technology
14
Canadian dairies (A, B, C and D), 2 using the indirect (A and B) and 2 the direct (C and D) methods of sterilization. All milk was packaged in I1 L Tetra Brik cartons. Two batches each consisting of 140 cartons and produced within 2 weeks of each other were obtained from each dairy. Milk was stored at 20°C and cartons removed as required. At intervals of 2 and 4 weeks, 2 cartons from each batch were opened aseptically and 1 mL was spread on plate count agar (Difco Laboratories, Detroit, Michigan) and incubated at 20°C for 48 h to determine sterility. Duplicate 2 mL samples were also removed removed for the determination of free amino groups. Viscosity measurements were performed on the milk prior to their submission for sensory evaluation. Results were the means of the 2 cartons. TCA-soluble free amino groups were determined with trinitrobenzene sulfonic acid (TNBS) as described previously (McKellar, 1981). Proteolysis was defined as the increase in the concentration of TCA-soluble free free amino groups per mL of milk (.::ljtmoles/mL). (Ajtmoles/mL). Apparent viscosity was measured with a Brookfield synchro-lectric synchro-Iectric viscometer model LVF (Brookfield Engineering Laboratories, Stoughton, MA). A model RVT bob No. No.22 was used for most measurements. Bobs No. No.33 and 4 were substituted when viscosity increased. Four measurements at 2 min intervals were made per carton at 20°C at a speed of 30 rpm and readings were converted to centipoises.
Sensory Evaluation Panelists were initially screened for their ability to perceive bitterness. The 13 most sensitive panelists evaluated samples of both water and milk with 1 to 48 mM leucine (a bitter amino acid) for intensity of bitterness using descriptive analysis with scaling (Larmond, 1977). During training a reference sample of 30 mM of leucine in water was established and anchored at 5.2 cm on a 15 cm scale. Ten trained panelists evaluated the 8 UHT milk samples (4 dairies times 2 batches) during 2 daily sessions, morning and afternoon. In each session, each of the 4 dairies was evaluated with batches being randomly assigned to either of the 2 sessions. Evaluation of the 8 samples was replicated twice over 2 days. Each judge received 60 mL of the leucine-in-water reference and 30 mL of each milk sample, all at 17°C. Numerical values were obtained by measuring the distance in centimeters of the line scores from the left end of the line scale. The first 3 series of evaluations were 4 weeks apart, while the following 6 series of evaluations were 2 weeks apart. Milk from dairies A and B were evaluated throughout the 9 sessions, while milk from dairies C and D were evaluated up to the eighth and sixth sessions, respectively. Gelled milk was not evaluated for bitterness. Statistical Analysis Analysis of variance was performed on the bitterness Can. Insl. lnsi. Food Food Sci. Sci. Technol. Technol. J. J. Vol. Vol. 17, 17. No. No. I, I, 1984 1984 Can.
Table I. Correlation coefficients for the relations between proteolysis, bitternes, and time. Dairy A
Process Indirect Indirect Direct Direct
B C D **P <0.01 INo. of cartons
Proteolysis vs Time 0.822**(64 1) 0.898**(62) 0.926**(57) 0.926* *(57) 0.971**(58) 0.971 **(58)
Proteolysis vs Bitterness 0.477**(35) 0.507**(36) 0.762**(32) 0.862**(24)
scores. The point of significant bitterness was established for each batch by means of t-test comparisons of later sessions to the initial sensory panel sessions. The relationship of bitterness and proteolysis to time was examined by analysis of covariance. Analysis of variance was also performed on background TCA-soluble free amino groups and significance at the 5070 level was determined by Duncan's Multiple Range Test. Throughout, because all effects were nested, the between-batch, within-dairy mean square was used to establish dairy differences and the between-carton, within-batch mean square was used to test batch differences.
Results and Discussion Background TCA-soluble free amino groups were determined for each batch of milk within one week of production date. The values, ranging from 0.631 0.631 to 0.780 {tmoles/mL, jtmoles/mL, were similar for all 4 dairies. A significant difference (P < 0.05) between batches was found for dairy A. Values for dairy A were also significantly different from those for Dairies B, C and
D. Correlations between proteolysis and time-since-production and between proteolysis and bitterness are shown in Table 1. l. Values reported are by dairy since insufficient data was available to warrant calculating correlation coefficients for each batch. Proteolysis in the milk from all 4 dairies was closely correlated with time. In addition, the increase in bitterness correlated well with the increase in proteolysis. Correlations between proteolysis and bitterness were lower for milk sterilized by the indirect (.477 and .507) as compared to the direct method (.762 and .862). However, all correlations were highly significant. Table 2. Relationship of proteolysis and bitterness to time-sincetime·sinceproduction determined by analysis of covariance Slopes Dairy Batch Proteolysis' Bitterness 2 Proteolysis I 0.154 (.0381) A 0.0084 (.0007 3) 1I 2 0.0088 (.0009) 0.049 (.0394) I1 0.0097 (.0008) 0.1 04 (.0355) B 0.104 2 0.0083 (.0008) 0.139 (.0307) I1 0.0216 (.0010) 0.135 (.0440) c 2 0.0302 (.0016) 0.216 (.0403) I1 0.0437 (.0016) 0.224 (.0696) D 2 0.0505 (.0019) 0.330 (.0483) 'Based on 241 observations. 2Based 2Eased on 127 observations. Jstandard error of the slope based on variation between between cartons at each time. McKellar et al. / 15
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.... 0."
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Fig. 1 The effect of storage at 20°C 20 0 e on proteolysis in UHT milk. (a) Batch 1, I, (b) batch 2. Dairies; A (0); B (e); e C (D); D (_).
The relationship of proteolysis and bitterness to time-since-production was examined by analysis of covariance. This analysis has been summarized in Table 2. Proteolysis increased significantly (P < .05) with time for all dairies; however, the development of proteolysis during storage varied considerably among dairies (Figure 1). The slopes for dairies A and B, where less proteolysis was observed, did not differ from each other (P < 0.05; Table 2). Extent of proteolysis was much greater in milk sterilized by the direct method (dairies C and D). The milk from dairies A and B experienced a leveling-off of proteolysis after week 15 (Fig. 1).
A closer examination was made of the sensory data to uncover thresholds of bitterness detection in each batch. Mean bitterness scores are given in Table 3. At the initial sensory session, the milks ranged in ages from 2 to 7 weeks from date of production. Initial scores varied between 2.5 and 4.1; however, they were not related to age of the milk. Scores which differed significantly from the mean initial score of 3.33, derived from initial scores from all 4 dairies, are indicated in Table 3. Milk sterilized by the indirect method became significantly bitter after 18 to 19 weeks of storage, while directly sterilized milk suffered loss of flavor after 10 to 18 weeks. The sensory score associated with detectable bitterness appears to be approximately 4.3. Age gelation was observed in milk from dairies C and 0D between 6 and 10 weeks after the milk had become significantly bitter. Gelation was observed at an apparent viscosity of 100 centipoises and was not found in milk from dairies A and B. Increased apparent viscosity was noted for batches 1 and 2 at 24 and 21 weeks for dairy C and 18 and 16 weeks for dairy oD (Figure 2a and b). This study has clearly demonstrated that proteolysis is more extensive in milk sterilized by the direct as compared to the indirect method. This is in agreement with the findings of other workers (Mottar et al., 1979; Mottar, 1981; Snoeren and Both, 1981; Corradini and Pecchini, 1981). The direct method imparts a greater heat intensity to the milk and may therefore inactivate the proteinase more effectively than does the direct method (Corradini & Pecchini, 1981). Alternatively, proteinase activity may be inhibited by the greater amount of denaturated J3-lactoglobulin present in milk sterilized by the indirect method (Snoeren & Both, 1981). The development of bitterness in UHT milk was closely related to proteolysis (Mottar et al., 1979; Mottar, 1981). In this study, highly significant correlations were obtained between proteolysis and bitterness for all 4 dairies; however, correlations were low for dairies A and B suggesting that factors other than proteolysis may be responsible for the development of bitterness in milk sterilized by the indirect method.
Table 3. Mean bitterness scores for UHT Milk stored at 20°C. Sessions I (weeks) Batch Process 0 4 8 12 10 14 16 18 20 S.E.2 A Indirect 3.2 3.5 3.2 4.3* 4.4* 5.8** 5.4** 4.0" .57 I 3.1 4.Q4 3.7 3.2 4.0 4.3* 5.2** 3.3 .57 2 Indirect 4.1 3.4 4.2 B Indirect 4.1 2.8 3.5 2.8 4.5 5.0** 4.4* 1I 4.8** 4.6* .61 2 Indirect 2.5 3.2 3.7 4.7** 4.8** 5.2** 4.4* .59 3.1 4.1 eC Direct 3.5 3.2 2.9 3.8 3.3 .57 I 4.1 5.1** 6.0** ND3 5.1** 6.5** .57 2 Direct 2.6 3.4 3.4 3.3 5.2** 4.1 ND D Direct 3.0 .59 I 2.5 3.8 5.2** 4.8** 4.5* ND ND ND 2 Direct 2.5 5.7*' ND .57 2.7 4.3* 4.3* 6.3** 5.7** ND ND A~~rage scores by session 3.33 2.94 3.69 4.38 4.85 5.66 4.24 4.74 ;\.~~rage 3.61 .21 ~P<0.05 and **P
Dairy
16 / MCKellar McKellar et al.
J. Inst. Can. Sci. Technol. Aliment. Vol. 17, No. I, 1984
terness, although significant, was slight and it is possible that gelation plays a greater role in limiting the shelf-life of UHT milk sterilized by the direct method than does bitterness. Background free amino groups in fresh UHT milk were lower than reported previously (McKellar, 1981). Significant differences were oserved between batches and between dairies; however, the differences were relatively small when compared to the amount of proteolysis necessary for significant bitterness. Background variability of free amino groups may be slight for a given dairy and subsequent changes may serve as an indicator of UHT milk quality. R2 values calculated from the data in Table 1 indicate that 58.1 and 74.3070 of the variation in bitterness in milk from dairies C and D, respectively, can be explained by proteolysis. It may be possible, therefore, to predict degree of bitterness and subsequent shelfclife of milk sterilized by the direct method by monitoring the initial rates of proteolysis in stored milk. Further studies are necessary to establish sensory scores corresponding to significant bitterness and to ascertain if these scores are of commercial significance.
400
1E~ a.
300
e
.2 2
..., .. ... ~
0
(J u
200
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I-
Z
au IU
II: a: 0( C 0( C
100
10
15
20
TIME (weeks'
400 b
!'00
300
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200
References
I-
Z au IU II: a: 0( C
100
10
15
20
25
30
(weeks' TIME (w.eks'
Fig. 2. The effect of storage at 20°C on apparent viscosity in UHT milk. a, batch I; b, batch 2. Dairies; A (0); B (e); C (0); D (_).
Significant differences in rates of proteolysis were observed between dairies and to a lesser extent between batches within dairies. Dairy differences may be ascribed to processing parameters. Batch differences may be explained by differences in the quality of raw milk or to variations in the quantity of proteinase produced by psychrotrophs (Cliffe and Law, 1982; McKellar, 1982). Milk sterilized by the indirect process had a longer shelf-life as compared to directly sterilized milk. All batches of milk from dairies A and B exceeded the expected shelf-life of 12 weeks. The shelf-life of milk from dairies C and D was reduced as a result of extensive proteolysis; however, milk from dairy C still exceeded the shelf-life of 12 weeks. Milk from dairy D, with a manufacturer's expected shelf-life of 16 weeks had an observed shelf-life of 10-12 weeks. Increased viscosity and gelation were observed with milk sterilized by the direct method. Gelation was detected after significant bitterness had developed. Bit-
Can. lnsf. Vol. 17, No. 1, I, 1984 fnst. Food Sci. Techno!. Technal. J. Val.
Bengtsson, K.L., Gardhage, L. and Isaksson, B. 1973. Gelation in UHT treated milk, whey and casein solution. The effect of heat resistant proteases. Milchwissenschaft 28:495. Cliffe, A.J. and Law, B.A. 1982. Predictive test for gelation in UHT milk using hide powder azure. Dairy Ind. Int. 47:25. Corradini, C. and Pecchini, G. 1981. Effect on proteinases of different UHT treatments. Neth. Milk Dairy J. 35:393. Larmond, E. 1977. Laboratory methods for sensory evaluation of food. Research Branch, Canada Department of Agriculture, Publication 1637. Law, B.A., Andrews, A.T. and Sharpe, M.E. 1977. Gelation of ultra-high-temperature-sterilized milk by proteases from a strain of Pseudomonas fluorescens isolated from raw milk. J. Dairy Res. 44:145. McKellar, R.C. 1981. Development of off-flavors in ultra-hightemperature and pasteurized milk as a function of proteolysis. J. Dairy Sci. 64:2138. McKellar, R.C. 1982. Factors influencing the production of extracellular proteinase by Pseudomonas fluorescens. J. Microbio!. 50:305. Appl. Microbiol. Mottar, J. 1981. Heat resistant enzymes in UHT milk and their influence on sensoric changes during uncooled storage. Milchwissenschaft 36:257. Mottar, J., Waes, G., G" Moermans R. and Maudts, M. 1979. Sensoric sorie changes in UHT milk during uncooled storage. Milchwissenschaft 34:257. 1979. Effect of heat-stable Richardson, B.C. and Newstead, D.F. 1979, proteases on the storage life of UHT milk. New Zeal. J. Dairy Sci. Technol. Techno!. 14:273. Richter, R.L., Schmidt, R.H., Smith, K.L., Mull, L.E. and Henry, S.L. 1979. Proteolytic activity in ultra-pasteurized, aseptically packaged whipping cream. J. Food Prot. 42:43. Snoeren, T.H.M. and Both, P. 1981. Proteolysis during the storage II. Experiments with milk of UHT-sterilized whole milk. 11. heated by the indirect system for 4s at 142°C. Neth. Milk Dairy J. 35:113. Accepted September I, 1983.
McKellar et al. / 17 McKeIlar