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Proteolysis and storage stability of UHT milk produced in Turkey
ARTICLE IN PRESS
International Dairy Journal 16 (2006) 633–638 www.elsevier.com/locate/idairyj
Short communication
Proteolysis and storage stability of UHT milk produced in Turkey Ali Topc- u, Eren Numanog˘lu, I˙lbilge Saldamlı Department of Food Engineering, Hacettepe University, 06532 Beytepe, Ankara, Turkey Received 23 May 2005; accepted 26 August 2005
Abstract The effect of raw milk quality (total and psychrotrophic bacterial and somatic cell counts, proteinase and plasmin activity) and UHT temperature (145 or 150 1C for 4 s) on proteolysis in UHT milk processed by a direct (steam-injection) system was investigated during storage at 25 1C for 180 d. High proteinase activity was measured in low-quality raw milk, which had high somatic cell count, bacterial count and plasmin activity. The levels of 12% trichloroacetic acid–soluble and pH 4.6-soluble nitrogen in all milk samples increased during storage, and samples produced from low-quality milk at the lower UHT temperature (145 1C) showed the highest values. Bitterness in UHT milk processed from low-quality milk at 145 1C increased during storage; gelation occurred in that milk after 150 d. The RP-HPLC profiles of pH 4.6-soluble fraction of the UHT milk samples produced at 150 1C showed quite small number of peaks after 180 d of storage. Sterilization at 150 1C extended the shelf-life of the UHT milk by reducing proteolysis, gelation and bitterness. r 2006 Elsevier Ltd. All rights reserved. Keywords: UHT milk; UHT temperature; Proteolysis; Plasmin; Bacterial proteinase; Somatic cell count
1. Introduction Ultra-high temperature (UHT) processing involves heating milk at a high temperature for a short time in order to obtain a product with a long shelf-life at room temperature. During the process, most bacteria are inactivated but heat-stable enzymes of native or bacterial origin can survive and cause serious defects during storage of the milk (Valero, Villamiel, Miralles, Sanz, & MartinezCastro, 2001). Proteolysis in UHT milk can cause the development of bitter flavour and leads to an increase in viscosity, with eventual formation of a gel during storage, which is a major factor limiting its shelf-life and market potential (Datta & Deeth, 2003). The enzymes responsible for the proteolysis are the native milk alkaline proteinase, plasmin, and heat-stable extracellular bacterial proteinases produced by psychrotrophic bacterial contaminants in raw milk (Datta & Deeth, 2003; Kelly & Foley, 1997). Corresponding author. Tel.: +90 312 297 71 07; fax: +90 312 299 21 23. E-mail address: [email protected] (A. Topc- u).
0958-6946/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2005.08.018
Grufferty and Fox (1988) reported that proteolysis of casein caused by plasmin is responsible for gelation and bitterness of UHT milk during storage. Plasmin is associated with casein micelles in milk and degrades bcasein to g-casein and proteose-peptones (Datta & Deeth, 2001; Fox & McSweeney, 1996). Plasmin activity is higher in mastitic milk than in normal milk (Bastian & Brown, 1996). Politis, Lachance, Block, and Tumer (1989) reported that as the somatic cell count (SCC) of milk increased from o250,000 to 41,000,000, the concentration of plasmin and plasminogen, its zymogen, increased from 0.18 to 0.37 mg L 1 and from 0.85 to 1.48 mg L 1, respectively. Proteolytic enzymes of bacterial origin occur in milk due to the growth of psychrotrophic bacteria. It has been observed that storing raw milk for more than 72 h at refrigeration temperatures might result in the formation of thermoresistant bacterial proteinases that significantly limit the shelf life of the milk after UHT treatment (Mottar, 1984). A psychrotroph population as low as 104 cfu mL 1 may be sufficient to produce proteinases in milk (Renner, 1988). Sørhaug and Stepaniak (1997) reported that a raw milk psychrotroph population of 6.9–7.2 log cfu mL 1
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caused gelation and bitter flavour development in UHT milk. In Turkey, 1.5–2% of total raw milk production is utilized in UHT milk processing (Anonymous, 2001). Generally, the raw milk is produced in small, primitive family farms. Raw milks collected from these farms do not have to meet standard quality criteria from both bacteriological and SCC standpoints. This situation leads to gelation and sedimentation defects in UHT milk products. In this study, the influence of raw milk quality and UHT temperature on the proteolysis and shelf-life of UHT milk were investigated during storage at room temperature. 2. Materials and methods 2.1. Milk samples Low-(A), medium-(B) and high-quality-(C) raw milks were obtained from three different regions and collected in three different storage tanks according to their quality. Analyses were performed on the bulk raw milk in each storage tank. The raw milk samples (A–C) were processed by direct (steam-injection) UHT treatment in a commercial dairy plant. They were heated to 145 1C for 4 s to produce A1, B1 and C1 UHT milks, and to 150 1C for 4 s to produce A2, B2 and C2 UHT milks. The UHT milks were aseptically packed in continuously-formed 1 L Tetra Pak containers and stored at room temperature (25 1C). Samples of the UHT milks were analysed after processing and at 30-d intervals thereafter for 180 d. Two packs of each batch were opened and analysed in duplicate on each occasion. Results were recorded as means of duplicate analysis and two replications. 2.2. Chemical analysis and microbiological assessment Total solids (TS) and fat content (Gerber method) were determined according to Bradley et al. (1992). The total nitrogen (TN) content of milk samples was determined by the Kjeldahl method (AOAC, 1990). Total and psychrotrophic bacteria were enumerated using the pour-plate procedure as described in IDF standards (IDF, 1991a,b). SCC was determined using a Fossomatic (Foss Electric, Hillerod, Denmark). 2.3. Determination of proteinase and plasmin activity The proteolytic activity in milk samples was determined by the azocasein method; the absorbance was read at 345 nm. Enzyme activity was defined as the increase in absorbance h 1 mL 1 of milk under the described conditions (Bendicho, Marti, Herna´ndez, & Martin, 2002; Christen & Marshall, 1984). Plasmin activity was measured by a method based on the release of a yellow compound (measured at 405 nm) from a synthetic substrate (D-valyl-Lleucyl-L-lysyl p-nitroanilide dihydrochloride; catalog number V 7127; Sigma Chemical Co., St. Louis, MO, USA)
(Rollema, Visser, & Poll, 1983); plasmin activity was defined as mmol p-nitroaniline h 1 L 1 of milk. 2.4. Assessment of proteolysis The 12% trichloroacetic acid–soluble nitrogen (TCA–SN) fraction was prepared by adding 10 mL of 24% (w/v) TCA to 10 mL of milk and the pH 4.6-soluble nitrogen (pH 4.6-SN) fraction was prepared according to Kelly and Foley (1997). The acidified milks were allowed to stand for 30 min at room temperature. The mixtures were centrifuged at 10,000g for 15 min and the supernatants were filtered through Whatman No. 42 filter paper. The nitrogen contents were determined on aliquots of the filtrates by the micro-Kjeldahl method (AOAC, 1990). The pH 4.6-soluble extracts were diluted with an equal volume of 0.2% trifluoroacetic acid (TFA) in HPLC-grade water and filtered through 0.45 mm filters before HPLC injection. Reversed phase (RP)-HPLC analysis was carried out using a Thermo Finnigan HPLC system (Thermo Finnigan, San Jose, California, USA) consisting of an autosampler, temperature control unit for the column (SpectraSystem AS3000), a degasser system (SpectraSystem SCM1000), a quaternary gradient pump (SpectraSystem P4000) and a photodiode array detector (SpectraSystem UV6000LP). A computer with a software package for system control and data acquisition (ChromQuest 4.0) was used for analyses. Separation was achieved on a Phenomenex Jupiter C18 wide-pore analytical column (5 mm, 300 A˚, 250 4.6 mm, Torrance, CA, USA) equipped with a Phenomenex cartridge holder containing a wide-pore C18 guard cartridge (4 3 mm). Separations were conducted at 30 1C using a mobile phase of two solvents at a flow rate of 1 mL min 1. Eluant A was 0.1% (vol/vol) TFA (HPLC grade, J.T. Baker, USA) in deionized water, and eluant B was 0.1% (v/v) TFA in acetonitrile (ultragradient grade; Merck, Darmstadt, Germany), starting with 100% eluant A for 5 min, and continuing with a linear gradient to 50% B over 45 min, holding at 50% B for 5 min, increasing to 60% B over 5 min, and finally holding at 60% B for 5 min. The column was finally eluted with 100% eluant B for 2 min. An aliquot (100 mL) of the filtrate was injected into the HPLC system. The absorbance of the eluate was monitored at 214 nm (Kelly & Foley, 1997). 2.5. Sensory analysis Sensory evaluation of the UHT milk samples was carried out by five experienced panelists during the storage period at 1, 30, 60, 90, 120, 150 and 180 d. The samples were evaluated for appearance, consistency, colour, odour and flavour. Each sensory property was scored on a 4-point scale ranging from extremely unacceptable (1) to extremely acceptable (4). The intensity of bitterness was scored on a scale from 0 (not bitter) to 4 (very strongly bitter). Gelation
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The effect of storage period, heating temperature and raw milk quality on the chemical composition and the level of proteolysis was assessed using analysis of variance (ANOVA) by the general linear model procedure of the SPSS 10.0 statistical package programme. Significance level was established at p o0.05. 3. Results and discussion The properties of the raw milk samples are given in Table 1. The samples were categorised according to their SCC, proteinase, plasmin activity and total bacteria count as A (low quality), B (medium quality) and C (high quality). After standardization and heat treatment, the compositions of all UHT milk samples were not significantly different. Means of total solids (%), protein (%) and fat (%) content were 12.59, 2.99 and 3.42, respectively. Proteinase and plasmin activity were not detected in the UHT milk samples since the spectrophotometric assays were not sensitive enough to detect low levels of activity. The pH values of all samples produced at 145 and 150 1C fluctuated between 6.59–6.72 and 6.62–6.79 during storage, respectively. The changes of pH in all samples were not statistically significant. Total solids, protein and fat contents of all samples were almost constant during the storage period. The effects of raw milk quality, storage period and heating temperature on chemical composition were not significant. The changes of soluble nitrogen values are given in Figs. 1 and 2. The pH 4.6-SN and TCA–SN in all UHT milk samples increased during storage (po0.05). The pH 4.6-SN of UHT-A1, UHT-B1 and UHT-C1 samples increased by 74.7%, 53.8% and 33.8%, respectively at 180 d compared with 1 d of storage. The high-SCC milk had higher levels of proteolysis during storage than the low-SCC milk due to both bacterial proteinase and plasmin Table 1 Properties of raw milk used in UHT milk production
A B C a
SCCa (mL 1)
TBCb (cfu mL 1)
PBCc (cfu mL 1)
Proteinased Plasmine activity ( 10 4) activity
621,000 315,000 212,000
2.2 106 1.8 106 3.5 105
4872 5100 2121
7.1 5.2 4.2
SCC, Somatic cell count. TBC, Total bacteria count. c PBC, Psychrotrophic bacteria count. d Changes in absorbance units h 1 mL 1of milk. e mmol p-nitroaniline h 1L 1 of milk. b
45.6 31.9 23.8
pH 4.6-SN (%TN)
2.6. Statistical analysis
30
20
10
0 0
50
100 Storage time (days)
150
200
0
50
100 Storage time (days)
150
200
(a) 30
pH 4.6-SN (%TN)
was defined as a custard-like gel in the lower portion of the container, which was weak and easily broken. Sedimentation was defined as dense particles settling to the bottom of the package.
635
20
10
0 (b)
Fig. 1. Changes in pH 4.6-soluble nitrogen of UHT milk samples during storage at room temperature, expressed as percentage of total nitrogen (%TN): (a) direct heating at 145 1C for 4 s and (b) direct heating at 150 1C for 4 s. UHT milk was produced from low-quality (~), medium-quality (’) or high-quality (m) raw milk. Results shown are means of duplicate analyses and two replications.
activity (po0.05). The results are in agreement with the findings of Kelly and Foley (1997) and Auldist et al. (1996). The pH 4.6-SN of UHT-A2, UHT-B2 and UHT-C2 samples produced at the higher temperature (150 1C for 4 s) increased by 57.9%, 31.3% and 17.1%, respectively at 180 d compared with 1 d of storage. The levels of pH 4.6SN and TCA–SN in UHT milk samples produced at 150 o C were low due to greater inactivation of proteolytic enzymes. The effect of sterilization temperature on pH 4.6SN and TCA–SN level was significant (po0.05). RP-HPLC chromatograms of the pH 4.6-SN fraction of UHT milk samples after 180 d of storage at room temperature are given in Fig. 3. The retention time of tryptophan was used to define the hydrophobic zone. The hydrophobic peptide portion consisted of the peptides that eluted after tryptophan (from 24 min). The peptides that eluted between 45 and 52 min were a-lactalbumin and blactoglobulin, respectively. The retention times of tryptophan and serum proteins were identified by comparison with those of standard solutions injected separately under the same conditions (Gomez, Garde, Gaya, Medina, & Nunez, 1997). The peptide profiles of the pH 4.6-SN fractions were quite different both qualitatively and quantitatively at
ARTICLE IN PRESS A. Topc- u et al. / International Dairy Journal 16 (2006) 633–638
636
6
14
UHT-A1
10
4 mVolts
TCA-SN (%TN)
12
2
8 6
UHT-B1
4 2 UHT-C1
0
0 0
50
(a)
100 Storage time (days)
150
200
0
5
(a)
6
10 15 20 25 30 35 40 45 50 55 60 Minutes
14 UHT-A2
10
4 mVolts
TCA-SN (%TN)
12
8 6
UHT-B2
2 4 2 0
(b)
UHT-C2
0 0
50
100 Storage time (days)
150
200
0 (b)
5
10 15 20 25 30 35 40 45 50 55 60 Minutes
Fig. 2. Changes in 12% trichloroacetic acid–soluble nitrogen (TCA–SN) of UHT milk samples during storage at room temperature expressed as percentage of total nitrogen (%TN): (a) direct heating at 145 1C for 4 s and (b) direct heating at 150 1C for 4 s. UHT milk was produced from lowquality (~), medium-quality (’) or high-quality (m) raw milk. Results shown are means of duplicate analyses and two replications.
Fig. 3. RP-HPLC chromatograms of pH 4.6-soluble nitrogen fraction of UHT milk samples after storage for 180 d at room temperature: (a) direct heating at 145 1C for 4 s and (b) direct heating at 150 1C for 4 s. UHT milk was produced from low-quality (UHT-A), medium-quality (UHT-B) or high-quality (UHT-C) raw milk.
180 d of storage. The number and corresponding concentration of peptides were low in UHT milk produced at 150 1C (Fig. 3b). There was also little proteolysis in the UHT-C1 milk sample produced with high-quality raw milk at 145 1C (Fig. 3a). The hydrophobic peptide level is highly related with hydrolysis of b-casein (Lemieux & Simard, 1991; Marcos & Esteban, 1999) and the extent of hydrolysis of b-casein is positively correlated with the intensity of bitterness (Lemieux & Simard, 1991). Undesirable bitter flavours in UHT milk have been associated with late-eluted, hydrophobic peptides (Lemieux & Simard, 1992). In the present study, an extremely bitter flavour was detected in UHT-A1 milk sample at 180 d storage (Table 2). The casein degradation level can be attributed to the activity of heat-stable proteinases of native and/or bacterial origin (Lopez-Fandino, Olano, San Jose, & Ramos, 1993). The pH 4.6-SN fraction shows peptide peaks originating from bacterial proteinases and plasmin. The peptides produced by bacterial proteinases are less hydrophobic and elute early in the RP-HPLC profile, while the peptides produced by plasmin are more hydrophobic and elute later (Datta & Deeth, 2003). The pH 4.6 extract of UHT-C1
milk exhibited only minor peaks up to 24 min but large peaks between 24 and 45 min. However, the chromatogram of UHT-A1 contained peaks in both the hydrophilic and hydrophobic regions, indicating activities of both bacterial proteinase and plasmin (Fig. 3a). The sensory evaluation results are shown in Table 2. Colour and odour changes in milk samples sterilized at 145 1C during storage were limited. UHT-A1 samples showed a tendency to bitterness at 60 d which increased considerably during the storage period. Gelation was observed in the UHT-A1 and UHT-B1 samples at 150 and 180 d, respectively. A high level of sedimentation was observed in the UHT-A1 sample during storage. Sensory changes were not detected in the UHT-C1 sample. Changes in appearance and consistency scores of UHT milk samples sterilized at 150 1C during the storage period were small. Colour scores of the same samples were lower than those of the UHT milk samples sterilized at 145 1C. UHT-A2, UHT-B2 and UHT-C2 samples showed cooked odour and flavour at 1 d, which generally decreased on storage. However, gelation was not observed in the UHT-B2 and UHT-C2 samples at 180 d. It has been reported that more severe heat treatment delays gelation (Datta & Deeth,
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637
Table 2 Sensory scoresa of UHT milk samplesb during storage at room temperature
Overall acceptancec (d) 1 60 120 180
UHT-A1
UHT-B1
UHT-C1
UHT-A2
UHT-B2
UHT-C2
19.5 19.1 15.7 12.9
19.5 19.5 17.7 13.6
19.8 20.0 18.1 17.6
17.2 19.0 18.0 16.4
17.3 18.9 18.9 17.4
17.6 19.2 19.3 18.6
0.0 0.5 1.5 3.0
0.0 0.0 1.0 1.8
0.0 0.0 0.7 1.2
0.0 0.0 1.0 1.5
0.0 0.0 0.2 0.5
0.0 0.0 0.1 0.0
Bitterness (d) 1 60 120 180
UHT-A2, UHT-B2, and UHT-C2: UHT milk samples produced with low-, medium- and high-quality raw milk, respectively, and subjected to direct heating at 150 1C for 4 s. a Values are means of duplicate analyses of two replications allocated by 5 panelists. b UHT-A1, UHT-B1, and UHT-C1: UHT milk samples produced with low-, medium- and high-quality raw milk, respectively, and subjected to direct heating at 145 1C for 4 s. c Calculated by sum of appearance, consistency, colour, odour and flavour scores.
2001). Bitterness was clearly identified in the UHT-A2 sample at 180 d. Sedimentation levels were generally low in the milk samples treated at 150 1C. 4. Conclusion UHT milks produced in a direct system from low-quality raw milk with high SCC and psychrotroph counts showed high levels of proteolysis during storage at room temperature. This caused bitterness, gelation and sedimentation which reduced the shelf-life of the milk. The proteolysis appeared to be due to both bacterial proteinase and plasmin. The proteolysis and defects were reduced by processing the milk at a higher temperature (150 rather than 145 1C). This caused a more intense cooked flavour in the UHT milk but the overall acceptability was not affected. Acknowledgements This study was supported by the research fund of Hacettepe University (Project no: 04.01.602.004) and Pınar Food Inc. The authors wish to thank Pınar Food Inc. staff for their help. References Anonymous (2001). Sekizinci Bes Yillik Kalkinma Plani Gida Sanayi Ozel Ihtisas Komisyonu Raporu Sut ve Sut Urunleri Sanayi Alt Komisyon Raporu. The progress planning of eighth five years. No: DPT: 2636OIK: 644, Ankara, Turkey. AOAC (1990). Official methods of analysis (15th ed.). Washington, DC, USA: Association of Official Analysis Chemists. Auldist, M. J., Coats, S. J., Sutherland, B. J., Hardham, J. F., McDowell, G. H., & Rogers, G. L. (1996). Effect of somatic cell count and stage of
lactation on the quality and storage life of ultra high temperature milk. Journal of Dairy Research, 63, 377–386. Bastian, E. D., & Brown, R. J. (1996). Plasmin in milk and dairy products: An update. International Dairy Journal, 6, 435–457. Bendicho, S., Marti, G., Herna´ndez, T., & Martin, O. (2002). Determination of proteolytic activity in different milk systems. Food Chemistry, 79, 245–249. Bradley, R. L., Arnold, E., Jr., Barbano, D. M., Semerad, R. G., Smith, D. E., & Vines, B. K. (1992). Chemical and physical methods. In R. T. Marshall (Ed.), Standard methods for the examination of dairy products (16th ed., pp. 433–529). Washington, DC, USA: American Public Health Association. Christen, G. L., & Marshall, R. T. (1984). Selected properties of lipase and protease of Pseudomonas fluorescens 27 produced in four media. Journal of Dairy Science, 67, 1680–1687. Datta, N., & Deeth, H. C. (2001). Age gelation of UHT milk—A review. Transactions of the Institute of Chemical Engineers C. Food and Bioproducts Processing, 79, 197–210. Datta, N., & Deeth, H. C. (2003). Diagnosing the cause of proteolysis in UHT milk. Lebensmittel Wissenschaft und Technologie, 36, 173–182. Fox, P. F., & McSweeney, P. L. H. (1996). Proteolysis in cheese. Food Reviews International, 12, 457–509. Gomez, M. J., Garde, S., Gaya, P., Medina, M., & Nunez, M. (1997). Relationship between level of hydrophobic peptides and bitterness in cheese made from pasteurized and raw milk. Journal of Dairy Research, 64, 289–297. Grufferty, M. B., & Fox, P. F. (1988). Milk alkaline proteinase. Journal of Dairy Research, 55, 609–630. IDF (1991a) Enumeration of microorganisms. IDF Standard 100B. Brussels, Belgium: International Dairy Federation. IDF (1991b). Enumeration of psychrotrophic microorganisms. IDF Standard 101A. Brussels, Belgium: International Dairy Federation. Kelly, A. L., & Foley, J. (1997). Proteolysis and storage stability of UHT milk as influenced by milk plasmin activity, plasmin/b-lactoglobulin complexation, plasminogen activation and somatic cell count. International Dairy Journal, 7, 411–420. Lemieux, L., & Simard, R. E. (1991). Bitter flavour in dairy products, I. A review of the factors likely to influence its development, mainly in cheese manufacture. Lait, 71, 599–636.
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Lemieux, L., & Simard, R. E. (1992). Bitter flavour in dairy products, II. A review of bitter peptides from caseins: Their formation, isolation and identification, structure masking and inhibition. Lait, 72, 335–382. Lopez-Fandino, R., Olano, A., San Jose, C., & Ramos, M. (1993). Application of reverse-phase HPLC to the study of proteolysis in UHT milk. Journal of Dairy Research, 60, 111–116. Marcos, A., & Esteban, M.A. (1999). Iberian cheeses. In P.F. Fox, (Ed.), Cheese: Chemistry, physics and microbiology, Vol. 2 (2nd ed., pp. 173–219), Maryland, USA: Aspen Publishers. Mottar, J. (1984). Influence de la dure´e de conservation sous re´frige´ration du lait cru sur la conservabilite´ du lait UHT. Lait, 64, 29–45. Politis, I., Lachance, E., Block, E., & Turner, J. D. (1989). Plasmin and plasminogen in bovine milk: A relationship with involution. Journal of Dairy Science, 72, 900–926.
Renner, E. (1988). Storage stability and some nutritional aspects of milk powders and ultra high temperature products at high ambient temperatures. Journal of Dairy Research, 55, 125–142. Rollema, H. S., Visser, S., & Poll, J. K. (1983). Spectrophotometric assay of plasmin and plasminogen in bovine milk. Milchwissenschaft, 38, 214–217. Sørhaug, T., & Stepaniak, L. (1997). Psychrotrophs and their enzymes in milk and dairy products: Quality aspects. Trends in Food Science & Technology, 8, 35–40. Valero, E., Villamiel, M., Miralles, B., Sanz, J., & Martinez-Castro, I. (2001). Changes in flavour and volatile components during storage of whole and skimmed UHT milk. Food Chemistry, 72, 51–58.
International Dairy Journal 16 (2006) 633–638 www.elsevier.com/locate/idairyj
Short communication
Proteolysis and storage stability of UHT milk produced in Turkey Ali Topc- u, Eren Numanog˘lu, I˙lbilge Saldamlı Department of Food Engineering, Hacettepe University, 06532 Beytepe, Ankara, Turkey Received 23 May 2005; accepted 26 August 2005
Abstract The effect of raw milk quality (total and psychrotrophic bacterial and somatic cell counts, proteinase and plasmin activity) and UHT temperature (145 or 150 1C for 4 s) on proteolysis in UHT milk processed by a direct (steam-injection) system was investigated during storage at 25 1C for 180 d. High proteinase activity was measured in low-quality raw milk, which had high somatic cell count, bacterial count and plasmin activity. The levels of 12% trichloroacetic acid–soluble and pH 4.6-soluble nitrogen in all milk samples increased during storage, and samples produced from low-quality milk at the lower UHT temperature (145 1C) showed the highest values. Bitterness in UHT milk processed from low-quality milk at 145 1C increased during storage; gelation occurred in that milk after 150 d. The RP-HPLC profiles of pH 4.6-soluble fraction of the UHT milk samples produced at 150 1C showed quite small number of peaks after 180 d of storage. Sterilization at 150 1C extended the shelf-life of the UHT milk by reducing proteolysis, gelation and bitterness. r 2006 Elsevier Ltd. All rights reserved. Keywords: UHT milk; UHT temperature; Proteolysis; Plasmin; Bacterial proteinase; Somatic cell count
1. Introduction Ultra-high temperature (UHT) processing involves heating milk at a high temperature for a short time in order to obtain a product with a long shelf-life at room temperature. During the process, most bacteria are inactivated but heat-stable enzymes of native or bacterial origin can survive and cause serious defects during storage of the milk (Valero, Villamiel, Miralles, Sanz, & MartinezCastro, 2001). Proteolysis in UHT milk can cause the development of bitter flavour and leads to an increase in viscosity, with eventual formation of a gel during storage, which is a major factor limiting its shelf-life and market potential (Datta & Deeth, 2003). The enzymes responsible for the proteolysis are the native milk alkaline proteinase, plasmin, and heat-stable extracellular bacterial proteinases produced by psychrotrophic bacterial contaminants in raw milk (Datta & Deeth, 2003; Kelly & Foley, 1997). Corresponding author. Tel.: +90 312 297 71 07; fax: +90 312 299 21 23. E-mail address: [email protected] (A. Topc- u).
0958-6946/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2005.08.018
Grufferty and Fox (1988) reported that proteolysis of casein caused by plasmin is responsible for gelation and bitterness of UHT milk during storage. Plasmin is associated with casein micelles in milk and degrades bcasein to g-casein and proteose-peptones (Datta & Deeth, 2001; Fox & McSweeney, 1996). Plasmin activity is higher in mastitic milk than in normal milk (Bastian & Brown, 1996). Politis, Lachance, Block, and Tumer (1989) reported that as the somatic cell count (SCC) of milk increased from o250,000 to 41,000,000, the concentration of plasmin and plasminogen, its zymogen, increased from 0.18 to 0.37 mg L 1 and from 0.85 to 1.48 mg L 1, respectively. Proteolytic enzymes of bacterial origin occur in milk due to the growth of psychrotrophic bacteria. It has been observed that storing raw milk for more than 72 h at refrigeration temperatures might result in the formation of thermoresistant bacterial proteinases that significantly limit the shelf life of the milk after UHT treatment (Mottar, 1984). A psychrotroph population as low as 104 cfu mL 1 may be sufficient to produce proteinases in milk (Renner, 1988). Sørhaug and Stepaniak (1997) reported that a raw milk psychrotroph population of 6.9–7.2 log cfu mL 1
ARTICLE IN PRESS 634
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caused gelation and bitter flavour development in UHT milk. In Turkey, 1.5–2% of total raw milk production is utilized in UHT milk processing (Anonymous, 2001). Generally, the raw milk is produced in small, primitive family farms. Raw milks collected from these farms do not have to meet standard quality criteria from both bacteriological and SCC standpoints. This situation leads to gelation and sedimentation defects in UHT milk products. In this study, the influence of raw milk quality and UHT temperature on the proteolysis and shelf-life of UHT milk were investigated during storage at room temperature. 2. Materials and methods 2.1. Milk samples Low-(A), medium-(B) and high-quality-(C) raw milks were obtained from three different regions and collected in three different storage tanks according to their quality. Analyses were performed on the bulk raw milk in each storage tank. The raw milk samples (A–C) were processed by direct (steam-injection) UHT treatment in a commercial dairy plant. They were heated to 145 1C for 4 s to produce A1, B1 and C1 UHT milks, and to 150 1C for 4 s to produce A2, B2 and C2 UHT milks. The UHT milks were aseptically packed in continuously-formed 1 L Tetra Pak containers and stored at room temperature (25 1C). Samples of the UHT milks were analysed after processing and at 30-d intervals thereafter for 180 d. Two packs of each batch were opened and analysed in duplicate on each occasion. Results were recorded as means of duplicate analysis and two replications. 2.2. Chemical analysis and microbiological assessment Total solids (TS) and fat content (Gerber method) were determined according to Bradley et al. (1992). The total nitrogen (TN) content of milk samples was determined by the Kjeldahl method (AOAC, 1990). Total and psychrotrophic bacteria were enumerated using the pour-plate procedure as described in IDF standards (IDF, 1991a,b). SCC was determined using a Fossomatic (Foss Electric, Hillerod, Denmark). 2.3. Determination of proteinase and plasmin activity The proteolytic activity in milk samples was determined by the azocasein method; the absorbance was read at 345 nm. Enzyme activity was defined as the increase in absorbance h 1 mL 1 of milk under the described conditions (Bendicho, Marti, Herna´ndez, & Martin, 2002; Christen & Marshall, 1984). Plasmin activity was measured by a method based on the release of a yellow compound (measured at 405 nm) from a synthetic substrate (D-valyl-Lleucyl-L-lysyl p-nitroanilide dihydrochloride; catalog number V 7127; Sigma Chemical Co., St. Louis, MO, USA)
(Rollema, Visser, & Poll, 1983); plasmin activity was defined as mmol p-nitroaniline h 1 L 1 of milk. 2.4. Assessment of proteolysis The 12% trichloroacetic acid–soluble nitrogen (TCA–SN) fraction was prepared by adding 10 mL of 24% (w/v) TCA to 10 mL of milk and the pH 4.6-soluble nitrogen (pH 4.6-SN) fraction was prepared according to Kelly and Foley (1997). The acidified milks were allowed to stand for 30 min at room temperature. The mixtures were centrifuged at 10,000g for 15 min and the supernatants were filtered through Whatman No. 42 filter paper. The nitrogen contents were determined on aliquots of the filtrates by the micro-Kjeldahl method (AOAC, 1990). The pH 4.6-soluble extracts were diluted with an equal volume of 0.2% trifluoroacetic acid (TFA) in HPLC-grade water and filtered through 0.45 mm filters before HPLC injection. Reversed phase (RP)-HPLC analysis was carried out using a Thermo Finnigan HPLC system (Thermo Finnigan, San Jose, California, USA) consisting of an autosampler, temperature control unit for the column (SpectraSystem AS3000), a degasser system (SpectraSystem SCM1000), a quaternary gradient pump (SpectraSystem P4000) and a photodiode array detector (SpectraSystem UV6000LP). A computer with a software package for system control and data acquisition (ChromQuest 4.0) was used for analyses. Separation was achieved on a Phenomenex Jupiter C18 wide-pore analytical column (5 mm, 300 A˚, 250 4.6 mm, Torrance, CA, USA) equipped with a Phenomenex cartridge holder containing a wide-pore C18 guard cartridge (4 3 mm). Separations were conducted at 30 1C using a mobile phase of two solvents at a flow rate of 1 mL min 1. Eluant A was 0.1% (vol/vol) TFA (HPLC grade, J.T. Baker, USA) in deionized water, and eluant B was 0.1% (v/v) TFA in acetonitrile (ultragradient grade; Merck, Darmstadt, Germany), starting with 100% eluant A for 5 min, and continuing with a linear gradient to 50% B over 45 min, holding at 50% B for 5 min, increasing to 60% B over 5 min, and finally holding at 60% B for 5 min. The column was finally eluted with 100% eluant B for 2 min. An aliquot (100 mL) of the filtrate was injected into the HPLC system. The absorbance of the eluate was monitored at 214 nm (Kelly & Foley, 1997). 2.5. Sensory analysis Sensory evaluation of the UHT milk samples was carried out by five experienced panelists during the storage period at 1, 30, 60, 90, 120, 150 and 180 d. The samples were evaluated for appearance, consistency, colour, odour and flavour. Each sensory property was scored on a 4-point scale ranging from extremely unacceptable (1) to extremely acceptable (4). The intensity of bitterness was scored on a scale from 0 (not bitter) to 4 (very strongly bitter). Gelation
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The effect of storage period, heating temperature and raw milk quality on the chemical composition and the level of proteolysis was assessed using analysis of variance (ANOVA) by the general linear model procedure of the SPSS 10.0 statistical package programme. Significance level was established at p o0.05. 3. Results and discussion The properties of the raw milk samples are given in Table 1. The samples were categorised according to their SCC, proteinase, plasmin activity and total bacteria count as A (low quality), B (medium quality) and C (high quality). After standardization and heat treatment, the compositions of all UHT milk samples were not significantly different. Means of total solids (%), protein (%) and fat (%) content were 12.59, 2.99 and 3.42, respectively. Proteinase and plasmin activity were not detected in the UHT milk samples since the spectrophotometric assays were not sensitive enough to detect low levels of activity. The pH values of all samples produced at 145 and 150 1C fluctuated between 6.59–6.72 and 6.62–6.79 during storage, respectively. The changes of pH in all samples were not statistically significant. Total solids, protein and fat contents of all samples were almost constant during the storage period. The effects of raw milk quality, storage period and heating temperature on chemical composition were not significant. The changes of soluble nitrogen values are given in Figs. 1 and 2. The pH 4.6-SN and TCA–SN in all UHT milk samples increased during storage (po0.05). The pH 4.6-SN of UHT-A1, UHT-B1 and UHT-C1 samples increased by 74.7%, 53.8% and 33.8%, respectively at 180 d compared with 1 d of storage. The high-SCC milk had higher levels of proteolysis during storage than the low-SCC milk due to both bacterial proteinase and plasmin Table 1 Properties of raw milk used in UHT milk production
A B C a
SCCa (mL 1)
TBCb (cfu mL 1)
PBCc (cfu mL 1)
Proteinased Plasmine activity ( 10 4) activity
621,000 315,000 212,000
2.2 106 1.8 106 3.5 105
4872 5100 2121
7.1 5.2 4.2
SCC, Somatic cell count. TBC, Total bacteria count. c PBC, Psychrotrophic bacteria count. d Changes in absorbance units h 1 mL 1of milk. e mmol p-nitroaniline h 1L 1 of milk. b
45.6 31.9 23.8
pH 4.6-SN (%TN)
2.6. Statistical analysis
30
20
10
0 0
50
100 Storage time (days)
150
200
0
50
100 Storage time (days)
150
200
(a) 30
pH 4.6-SN (%TN)
was defined as a custard-like gel in the lower portion of the container, which was weak and easily broken. Sedimentation was defined as dense particles settling to the bottom of the package.
635
20
10
0 (b)
Fig. 1. Changes in pH 4.6-soluble nitrogen of UHT milk samples during storage at room temperature, expressed as percentage of total nitrogen (%TN): (a) direct heating at 145 1C for 4 s and (b) direct heating at 150 1C for 4 s. UHT milk was produced from low-quality (~), medium-quality (’) or high-quality (m) raw milk. Results shown are means of duplicate analyses and two replications.
activity (po0.05). The results are in agreement with the findings of Kelly and Foley (1997) and Auldist et al. (1996). The pH 4.6-SN of UHT-A2, UHT-B2 and UHT-C2 samples produced at the higher temperature (150 1C for 4 s) increased by 57.9%, 31.3% and 17.1%, respectively at 180 d compared with 1 d of storage. The levels of pH 4.6SN and TCA–SN in UHT milk samples produced at 150 o C were low due to greater inactivation of proteolytic enzymes. The effect of sterilization temperature on pH 4.6SN and TCA–SN level was significant (po0.05). RP-HPLC chromatograms of the pH 4.6-SN fraction of UHT milk samples after 180 d of storage at room temperature are given in Fig. 3. The retention time of tryptophan was used to define the hydrophobic zone. The hydrophobic peptide portion consisted of the peptides that eluted after tryptophan (from 24 min). The peptides that eluted between 45 and 52 min were a-lactalbumin and blactoglobulin, respectively. The retention times of tryptophan and serum proteins were identified by comparison with those of standard solutions injected separately under the same conditions (Gomez, Garde, Gaya, Medina, & Nunez, 1997). The peptide profiles of the pH 4.6-SN fractions were quite different both qualitatively and quantitatively at
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636
6
14
UHT-A1
10
4 mVolts
TCA-SN (%TN)
12
2
8 6
UHT-B1
4 2 UHT-C1
0
0 0
50
(a)
100 Storage time (days)
150
200
0
5
(a)
6
10 15 20 25 30 35 40 45 50 55 60 Minutes
14 UHT-A2
10
4 mVolts
TCA-SN (%TN)
12
8 6
UHT-B2
2 4 2 0
(b)
UHT-C2
0 0
50
100 Storage time (days)
150
200
0 (b)
5
10 15 20 25 30 35 40 45 50 55 60 Minutes
Fig. 2. Changes in 12% trichloroacetic acid–soluble nitrogen (TCA–SN) of UHT milk samples during storage at room temperature expressed as percentage of total nitrogen (%TN): (a) direct heating at 145 1C for 4 s and (b) direct heating at 150 1C for 4 s. UHT milk was produced from lowquality (~), medium-quality (’) or high-quality (m) raw milk. Results shown are means of duplicate analyses and two replications.
Fig. 3. RP-HPLC chromatograms of pH 4.6-soluble nitrogen fraction of UHT milk samples after storage for 180 d at room temperature: (a) direct heating at 145 1C for 4 s and (b) direct heating at 150 1C for 4 s. UHT milk was produced from low-quality (UHT-A), medium-quality (UHT-B) or high-quality (UHT-C) raw milk.
180 d of storage. The number and corresponding concentration of peptides were low in UHT milk produced at 150 1C (Fig. 3b). There was also little proteolysis in the UHT-C1 milk sample produced with high-quality raw milk at 145 1C (Fig. 3a). The hydrophobic peptide level is highly related with hydrolysis of b-casein (Lemieux & Simard, 1991; Marcos & Esteban, 1999) and the extent of hydrolysis of b-casein is positively correlated with the intensity of bitterness (Lemieux & Simard, 1991). Undesirable bitter flavours in UHT milk have been associated with late-eluted, hydrophobic peptides (Lemieux & Simard, 1992). In the present study, an extremely bitter flavour was detected in UHT-A1 milk sample at 180 d storage (Table 2). The casein degradation level can be attributed to the activity of heat-stable proteinases of native and/or bacterial origin (Lopez-Fandino, Olano, San Jose, & Ramos, 1993). The pH 4.6-SN fraction shows peptide peaks originating from bacterial proteinases and plasmin. The peptides produced by bacterial proteinases are less hydrophobic and elute early in the RP-HPLC profile, while the peptides produced by plasmin are more hydrophobic and elute later (Datta & Deeth, 2003). The pH 4.6 extract of UHT-C1
milk exhibited only minor peaks up to 24 min but large peaks between 24 and 45 min. However, the chromatogram of UHT-A1 contained peaks in both the hydrophilic and hydrophobic regions, indicating activities of both bacterial proteinase and plasmin (Fig. 3a). The sensory evaluation results are shown in Table 2. Colour and odour changes in milk samples sterilized at 145 1C during storage were limited. UHT-A1 samples showed a tendency to bitterness at 60 d which increased considerably during the storage period. Gelation was observed in the UHT-A1 and UHT-B1 samples at 150 and 180 d, respectively. A high level of sedimentation was observed in the UHT-A1 sample during storage. Sensory changes were not detected in the UHT-C1 sample. Changes in appearance and consistency scores of UHT milk samples sterilized at 150 1C during the storage period were small. Colour scores of the same samples were lower than those of the UHT milk samples sterilized at 145 1C. UHT-A2, UHT-B2 and UHT-C2 samples showed cooked odour and flavour at 1 d, which generally decreased on storage. However, gelation was not observed in the UHT-B2 and UHT-C2 samples at 180 d. It has been reported that more severe heat treatment delays gelation (Datta & Deeth,
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637
Table 2 Sensory scoresa of UHT milk samplesb during storage at room temperature
Overall acceptancec (d) 1 60 120 180
UHT-A1
UHT-B1
UHT-C1
UHT-A2
UHT-B2
UHT-C2
19.5 19.1 15.7 12.9
19.5 19.5 17.7 13.6
19.8 20.0 18.1 17.6
17.2 19.0 18.0 16.4
17.3 18.9 18.9 17.4
17.6 19.2 19.3 18.6
0.0 0.5 1.5 3.0
0.0 0.0 1.0 1.8
0.0 0.0 0.7 1.2
0.0 0.0 1.0 1.5
0.0 0.0 0.2 0.5
0.0 0.0 0.1 0.0
Bitterness (d) 1 60 120 180
UHT-A2, UHT-B2, and UHT-C2: UHT milk samples produced with low-, medium- and high-quality raw milk, respectively, and subjected to direct heating at 150 1C for 4 s. a Values are means of duplicate analyses of two replications allocated by 5 panelists. b UHT-A1, UHT-B1, and UHT-C1: UHT milk samples produced with low-, medium- and high-quality raw milk, respectively, and subjected to direct heating at 145 1C for 4 s. c Calculated by sum of appearance, consistency, colour, odour and flavour scores.
2001). Bitterness was clearly identified in the UHT-A2 sample at 180 d. Sedimentation levels were generally low in the milk samples treated at 150 1C. 4. Conclusion UHT milks produced in a direct system from low-quality raw milk with high SCC and psychrotroph counts showed high levels of proteolysis during storage at room temperature. This caused bitterness, gelation and sedimentation which reduced the shelf-life of the milk. The proteolysis appeared to be due to both bacterial proteinase and plasmin. The proteolysis and defects were reduced by processing the milk at a higher temperature (150 rather than 145 1C). This caused a more intense cooked flavour in the UHT milk but the overall acceptability was not affected. Acknowledgements This study was supported by the research fund of Hacettepe University (Project no: 04.01.602.004) and Pınar Food Inc. The authors wish to thank Pınar Food Inc. staff for their help. References Anonymous (2001). Sekizinci Bes Yillik Kalkinma Plani Gida Sanayi Ozel Ihtisas Komisyonu Raporu Sut ve Sut Urunleri Sanayi Alt Komisyon Raporu. The progress planning of eighth five years. No: DPT: 2636OIK: 644, Ankara, Turkey. AOAC (1990). Official methods of analysis (15th ed.). Washington, DC, USA: Association of Official Analysis Chemists. Auldist, M. J., Coats, S. J., Sutherland, B. J., Hardham, J. F., McDowell, G. H., & Rogers, G. L. (1996). Effect of somatic cell count and stage of
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