Diagnosing the cause of proteolysis in UHT milk

Diagnosing the cause of proteolysis in UHT milk

Lebensm.-Wiss. U.-Technol. 36 (2003) 173–182 Diagnosing the cause of proteolysis in UHT milk Nivedita Datta*, Hilton C. Deeth Dairy Industry Centre f...

201KB Sizes 0 Downloads 73 Views

Lebensm.-Wiss. U.-Technol. 36 (2003) 173–182

Diagnosing the cause of proteolysis in UHT milk Nivedita Datta*, Hilton C. Deeth Dairy Industry Centre for UHT Processing, School of Land and Food Sciences, The University of Queensland, Gatton, Qld 4343, Australia Received 10 October 2001; accepted 25 February 2002

Abstract Proteolysis of UHT milk during storage at room temperature is a major factor limiting its shelf-life through changes in its flavour and texture. The latter is characterised by increases in viscosity leading in some cases to gel formation. 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 the milk prior to heat processing. These proteinases react differently with the milk proteins and produce different peptides in the UHT milk. In order to differentiate these peptide products, reversed-phase HPLC and the fluorescamine method were used to analyse the peptides soluble in 12% trichloroacetic acid (TCA) and those soluble at pH 4.6. The TCA filtrate showed substantial peptide peaks only if the milk was contaminated by bacterial proteinase, while the pH 4.6 filtrate showed peptide peaks when either or both bacterial and native milk proteinases caused the proteolysis. Results from the fluorescamine test were in accordance with the HPLC results whereby the TCA filtrate exhibited significant proteolysis values only when bacterial proteinases were present, but the pH 4.6 filtrates showed significant values when the milk contained either or both types of proteinase. A procedure based on these analyses is proposed as a diagnostic test for determining which type of proteinase—milk plasmin, bacterial proteinase, or both—is responsible for proteolysis in UHT milk. r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved. Keywords: Plasmin; Bacterial proteinase; Proteolysis; Fluorescamine; RP-HPLC

1. Introduction Age gelation of UHT milk is a major concern of the dairy industry since it limits the shelf-life and market potential of the milk. One of the causes of age gelation is proteolysis of caseins by either native milk proteinase or bacterial proteinase, or both (Harwalkar, 1992; Datta & Deeth, 2001). Proteolysis of UHT milk causes the development of bitter flavours and leads to increases in viscosity, with eventual formation of a gel. These changes are caused, or at least accelerated, by hydrolysis of caseins, releasing the b-lactoglobulin–k-casein complex (bk-complex), formed during heat treatment, from the micelle. The released complex subsequently aggregates and forms a three-dimensional network of cross-linked proteins, which causes the milk to gel (McMahon, 1996). Any processing or storage conditions of UHT milk that accelerate (or delay) release of the bk-complex from the casein micelle will ultimately accelerate (or delay) age gelation of UHT milk. *Corresponding author.

Proteolysis of UHT milk has been attributed to the natural milk alkaline serine proteinase, known as plasmin, and to proteinases produced by psychrotropic bacterial contaminants of raw milk. Milk plasmin is associated with the casein micelle and the milk fat globule membrane in milk, and is highly heat-resistant (Visser, 1981). It exists in milk in both its active form as well as its enzymatically inactive precursor form, plasminogen. Its activity in milk is controlled by a system of enzyme activators and inhibitors (Richardson, 1983). Plasminogen is converted to plasmin by plasminogen activators (PAs) during storage of UHT milk. The activity of the plasminogen activators increases after heat treatment due to heat inactivation of their inhibitors (Richardson, 1983). Plasmin, plasminogen and the PAs are more heat-stable than the inhibitors. The plasminogen activators are only slightly inactivated by UHT processing conditions. Plasminogen is more heat-stable than plasmin, which some researchers have found to be almost completely inactivated by (indirect) UHT processing (Cauvin, Sacchi, Rasero, & Turi, 1999). The milk proteins most susceptible to attack by milk plasmin are b-casein (3 times more susceptible than

0023-6438/03/$30.00 r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0023-6438(02)00214-1

174

N. Datta, H.C. Deeth / Lebensm.-Wiss. U.-Technol. 36 (2003) 173–182

as1-casein) and as2-casein and, to a lesser extent, as1casein, while extracellular proteinases of microbial origin attack predominantly k-casein with the formation of para-k-casein (Snoeren & Riel, 1979), followed by extensive nonspecific hydrolysis (Law, Andrews, & Sharpe, 1977). Both plasmin and bacterial proteinase can accelerate age gelation. They do not hydrolyse the bk-complex, but they hydrolyse the proteins that attach the bk-complex to the micelles, thus facilitating release of the bkcomplex from the micelles. When the bk-complex is primarily attached to as1-casein, any enzymatic reaction of as1-casein promotes the release of the complex and subsequent gelation (McMahon, 1996). It is evident that only trace levels of proteinase are required to cause gelation of UHT milk during storage. For example, Richardson and Newstead (1979) reported that UHT milk containing as little as 1 ng bacterial proteinase/mL may have a shelf-life of only 3 months. In order to address a problem of proteolysis in UHT milk, it is necessary to determine the nature, and hence origin, of the enzyme(s) responsible. This paper discusses the use of reversed-phase (RP)-HPLC and the fluorescamine method for analysing the peptides soluble in trichloroacetic acid (TCA) and at pH 4.6, and how these results enable differentiation between the products of plasmin and bacterial proteinase action on milk proteins.

2. Methods and materials 2.1. Proteinases Bacterial proteinase was obtained by culturing Pseudomonas aureofaciens (API 20NE V5.1 Code 1057547; Deeth, Khusniati, Datta, & Wallace, 2002) in UHT skim milk at 301C for 3 d and centrifuging at 1000g for 30 min. The supernatant containing the extracellular proteinase was used as the Pseudomonas proteinase. Plasmin was from Sigma Chemical Company (Cat. No. P-7911). 2.2. UHT milk samples 2.2.1. Fresh UHT milk Raw milk, with total bacterial count less than 5  103 cfu/mL, was collected from the University of Queensland dairy herd within 24 h of milking and processed on an APV UHT pilot plant, using indirect (tubular) heating at 1461C for 4 s, and homogenised downstream using a 2-stage aseptic homogeniser operating at 35 and 5 MPa in the first and second stages, respectively. The UHT-processed milks were filled aseptically into pre-sterilised bottles in a laminar flow cabinet. The samples were stored at 28–301C. At

4-weekly intervals, they were assessed for viscosity, proteolysis and visual evidence of clotting or gelling. The samples were analysed in duplicate and average results recorded. 2.2.2. Gelled UHT milks Three types of gelled milks were used: (1) Universityprocessed milk stored for 12 months; (2) gelled milks obtained from commercial processors; and (3) University-processed fresh UHT milk, containing 0.2% sodium azide to inhibit bacterial growth, into which Pseudomonas proteinase (10 mL/L) was injected and incubated at 401C for 24 h to produce a gel. Bacterial gel was produced in duplicate and analysed for viscosity and proteolysis by fluorescamine, each in duplicate. The results presented for viscosity and proteolysis by fluorescamine are mean values for the four replicates. 2.2.3. Proteolysed milks Fresh commercial UHT skim milks were obtained directly from the processor or purchased from the local supermarket. They were hydrolysed with the above proteinases at 401C for 3 h. The reactions were stopped by heating at 1001C for 10 min. The levels of enzymes used were: Pseudomonas proteinase (200 mL/L) and plasmin (100 mg/L). A UHT skim milk with no added proteinase was used as a control. The experiments were performed in duplicate. To investigate the effect of extensive proteolysis by plasmin, it was added to freshly processed UHT whole milk at concentrations of 100, 500, 1000, 2000 mg/L and incubated for 3 h at 401C. The reactions were terminated by heating at 1001C for 10 min. Proteolysis was determined in TCA (12%), and pH 4.6 filtrates as described below. A milk sample without added plasmin acted as a control. The experiments were performed in duplicate. 2.3. Preparation of milk extracts 2.3.1. TCA filtrates TCA (12%)-soluble extracts of milks were prepared by adding 24% TCA to an equal volume of milk, mixing thoroughly by vortexing and allowing to stand at room temperature for 1 h. The mixtures were centrifuged at 24,000  g for 15 min and the supernatants filtered through glass fibre filter paper (Whatman G541) and a 0.45 mm Millipore filter. TCA filtrates (4%) were prepared by mixing 8% TCA with an equal volume of milk and following the procedure described above. 2.3.2. pH 4.6 filtrates The fraction soluble at pH 4.6 was prepared by adjusting milk samples to pH 4.6 with 10% acetic acid, while stirring with a magnetic stirrer, and allowing the mixtures to stand for 1 h at room temperature. The

N. Datta, H.C. Deeth / Lebensm.-Wiss. U.-Technol. 36 (2003) 173–182

mixtures were then centrifuged and processed as for the preparation of the TCA (12%)-soluble extract described above to yield the pH 4.6 filtrates. 2.4. Analysis of peptides by RP-HPLC HPLC analyses were performed on a Shimadzu ClassVP chromatograph with a UV/VIS photodiode array detector using a 150  4.5 mm reversed-phase column (SGE 150 GL4-C-18-P-8/5) at 401C and a binary solvent gradient system at a flow rate of 1 mL/min and detection at 210 nm. Solvent A was 0.1% (v/v) trifluoroacetic acid (TFA) in water and solvent B was 0.1% (v/v) TFA in HPLC-grade acetonitrile. The proportion of solvent B was increased from 20% to 35% during the first 20 min and after 5 min raised to 65% in 20 min and finally to 100% in 5 min. Injections of 50 mL of filtrates were made by autoinjector. The column was washed between samples by increasing solvent B to 65% over 15 min, holding for 15 min and returning to 100% solvent A. All samples were analysed in duplicate. 2.5. Analysis of peptides by the fluorescamine method Peptides in TCA filtrates and pH 4.6 filtrates of UHT milks were analysed by the fluorescamine method of Beeby (1980) as modified by Kocak and Zadow (1985). The fluorescence, in relative fluorescence intensity values, was measured on a Turner fluorometer at an emission wavelength of 480 nm and excitation wavelength of 400 nm using fluorescein as the standard. A calibration curve was constructed using the dipeptide, lleucyl-l-leucine (Sigma L-2752) to convert relative fluorescence intensity values to concentration of a-amino groups. The proteolysis was expressed as mmol of l-leucyl-l-leucine released per litre of milk. The variability in the fluorescamine results was low with standard deviations for replicates being less than 0.45. 2.6. Viscosity The viscosity of UHT milks and the apparent viscosity of gelled UHT milks were estimated at 201C using a Brookfield Synchro-Lectric LVT viscometer. A UL adapter (ULA) was used for the determination of viscosity of milk and spindle no. 1 was used for gelled milk at a spindle speed of 60 rpm. Readings were taken after the spindle had been rotating for 30 s, and the mean of three readings was recorded. The viscosity meter was calibrated with Brookfield calibration liquids. The viscosity was expressed in milli Pascal second obtained by multiplying the relevant factor with the mean viscometer reading. The variability in the results was low with standard deviations on replicates of less than 0.74.

175

2.7. Appearance of milks Any changes in the appearance of the milks, such as clotting or gelling were observed by gently tilting the clear container. The onset of gelation was characterised by the development of a curd or custard-like gel in the lower portion of the containers.

3. Results and discussion 3.1. Proteolysis of UHT milk with added Pseudomonas proteinase and plasmin 3.1.1. Appearance During incubation at 401C for 3 h, Pseudomonas proteinase caused the formation of a hard gel, while plasmin caused partial digestion of the casein as evidenced by turbidity in the digested sample. The clarified samples indicated extensive breakdown of casein while the gelled samples indicated limited proteolysis. The hard gel is similar to that caused by rennet, which, like Pseudomonas proteinase, preferentially hydrolyses the hydrophilic glycomacropeptide from k-casein on the outside of the micelle. This leaves the casein micelle largely intact and also reduces steric repulsion between micelles, allowing the formation of a more compact gel. By contrast, plasmin attacks b-casein located inside the micelle, thereby disrupting the micelle and inhibiting the formation of a strong gel. At the concentration of 100 mg/L, plasmin exhibited less proteolysis than the Pseudomonas proteinase under the conditions used in this trial. Extensive proteolysis of skim milk by plasmin completely clarifies skim milk (Visser, 1981). 3.1.2. Proteolysis by RP-HPLC Fig. 1 shows the RP-HPLC profiles of the pH 4.6 filtrates of freshly processed UHT milk proteolysed by plasmin and Pseudomonas proteinases (3 h incubation). Two different groups of peaks can be distinguished from the chromatograms, the first group eluting before 20 min and a second group eluting between 20 and 40 min. The chromatograms of the filtrates from milk hydrolysed by the Pseudomonas proteinase contained only the first group of peaks, while the second group was evident only in the plasmin-hydrolysed sample. Fig. 2 shows the RP-HPLC profile of the corresponding 12% TCA filtrates of the two hydrolysed milk samples depicted in Fig. 1. The plasmin-hydrolysed sample exhibits virtually no peaks in the chromatogram, as the peptide fragments generated by plasmin action on b-casein are precipitated by 12% TCA. In contrast, the Pseudomonas proteinase digest showed substantial peaks eluting before 20 min.

176

N. Datta, H.C. Deeth / Lebensm.-Wiss. U.-Technol. 36 (2003) 173–182

Fig. 1. Peptide patterns of pH 4.6 filtrates of proteolysed UHT milks (Pseudomonas proteinase, 200 mL/L; plasmin, 100 mg/L; 401C for 3 h).

Fig. 2. Peptide patterns of TCA filtrates of proteolysed UHT milks (Pseudomonas proteinase, 200 mL/L; plasmin, 100 mg/L; 401C for 3 h).

3.1.3. Proteolysis by the fluorescamine method Table 1 (rows 1–3) shows the levels of proteolysis products determined by the fluorescamine method in the control and proteolysed milk samples discussed in the previous section. The results show high levels of proteolysis in both the pH 4.6 and 12% TCA filtrates of the milks digested with Pseudomonas proteinases and in the pH 4.6 filtrate of the plasmin-proteolysed milk. By contrast, the TCA filtrate of the plasmin-proteolysed milk showed virtually no fluorescamine-reactive proteolysis products.

3.2. Proteolysis of gelled UHT milk Fig. 3 shows the elution patterns of pH 4.6 filtrates of fresh UHT milk, plasmin-mediated gelled milk and the same UHT milk inoculated with bacterial proteinases. This clearly illustrates the difference between the patterns of the fresh and gelled milks as well as the difference between the milks in which gelation was initiated by the different proteinases. The HPLC results are reinforced by the levels of proteolysis, as measured by the fluorescamine method, shown in Table 1 (rows 4

N. Datta, H.C. Deeth / Lebensm.-Wiss. U.-Technol. 36 (2003) 173–182

177

Table 1 Effects of proteolysis by bacterial proteinase and plasmin on UHT milk Sample

1. Control (fresh UHT milk) 2. Proteolysed milk (due to Pseudomonas proteinase added to 1 at 200 mL/L) 3. Proteolysed milk (due to plasmin added to 1 at 100 mg/L) 4. Gelled UHT milk (due to endogenous plasmin only) 5. Gelled UHT milk (due to added bacterial proteinase at 10 mL/L) a

State of milk

Viscosity (mPa s)

Normal Bitter and gelled Bitter and turbid Dispersed gel Thick gel

3.0

110 100

Proteolysisa by florescamine 12% TCA filtrate

pH 4.6 filtrate

0 11.2

1.0 11.5

0.09 0.1 5.5

3.4 2.8 6.1

As concentration of a-amino groups in the milk (mmol/L).

Fig. 3. Peptide patterns of pH 4.6 filtrates of fresh and gelled UHT milks (Pseudomonas proteinase added in sample 2 at 10 mL/L; gel in sample 3 due to endogenous plasmin only).

and 5). These show high levels in the pH 4.6 filtrates for both gelled milks but only the bacterially gelled milk showed a high level in the 12% TCA filtrate. The fresh milk showed zero and low levels in the TCA and pH 4.6 filtrates, respectively. The low but measurable level of proteolysis products in the pH 4.6 filtrate or fresh milk is attributable to the normal content of noncasein nitrogenous compounds that occur in all milks, even when fresh. The HPLC patterns were similar to those reported by Lopez-Fandino, Olano, San Jose, and Ramos (1993). They compared the patterns for peptides soluble in 4% TCA and at pH 4.6, which resulted from proteolysis of casein by plasmin and P. fluorescens proteinase. In their chromatograms, most peptides from the bacterial proteolysis eluted before 24 min, while those from plasmin eluted after this time. Morales and Perez (1998) reported similar results for the RP-HPLC chromatograms of peptides soluble in 4% TCA fraction and at pH 4.6 of heat-induced proteolysed milk. They

also reported that there was very little difference in the chromatograms for 4% and 12% filtrates, a finding confirmed in this investigation for gelled UHT milks (results not shown). The explanation for the fluorescamine results is that only very small peptides and amino acids are soluble in 12% TCA, while some larger peptides, such as those formed by plasmin action, are soluble at pH 4.6 but insoluble in 12% TCA. Chen (1971) calculated that 90% of the hydrolysates of b-casein were precipitated by 2% TCA. Since the solubility of protein hydrolysates increases with decreasing concentration of TCA (Yvon, Chabnet, & Pelissier, 1989), more than 90% of the hydrolysates of b-casein will remain insoluble in 12% TCA. This is consistent with the findings of Driessen (1981) who showed that bacterial contamination of UHT milk resulted in high nonprotein nitrogen (NPN) values in 12% TCA filtrates, while plasmin-digested milk showed a high value of noncasein nitrogen (NCN), which was

178

N. Datta, H.C. Deeth / Lebensm.-Wiss. U.-Technol. 36 (2003) 173–182

measured in pH 4.6 filtrates. Driessen (1981) also demonstrated that milk alkaline proteinase splits b-casein into larger fragments, which are precipitated by 12% TCA and do not contribute to the NPN value. The large peptides in the filtrates from the plasmin digestion are probably proteose peptone components arising from b-casein as these peptides are soluble at pH 4.6 but are precipitated by 12% TCA (Walstra & Jenness, 1984; Mulvihill & Donovan, 1987). It can be concluded that the first group of HPLC peaks in Fig. 1 (eluted in the first 20 min) represents not only the least hydrophobic peptides but also the smallest and most acid-soluble, while the second group (eluted after 20 min) represent the largest and least acid-soluble peptides as well as the most hydrophobic. On this basis, the Pseudomonas proteinase produced mostly small, acid-soluble peptides, while plasmin produced predominantly large peptides soluble at pH 4.6 but not in 12% TCA. The viscosity data in Table 1 (rows 1, 4 and 5) are typical of normal and freshly gelled milks. After milks gel, they slowly separate into ‘curds and whey’ and the apparent viscosity decreases from the level of the freshly gelled milks (results not shown). The plasmin-mediated gel was less dense and more easily broken when stirred compared to the gel caused by the bacterial proteinase. Aroonkamonsri (1996) showed that casein micelles treated with plasmin formed gels that were not as intact as gels formed by treatment with bacterial proteinase. The plasmin-treated samples appeared to contain partially disintegrated casein micelles and their linkages. This was also confirmed by other researchers (Visser 1981; de Koning, Kaper, Roellama, & Driessen, 1985); the bacterial gels appeared to have more intact casein micelles, casein micelle aggregates and their linkages. 3.3. Proteolysis of UHT milk with high concentration of plasmin The RP-HPLC chromatogram of the 12% TCA filtrates of milks proteolysed with a very high concentration of plasmin (2 g/L) (Fig. 4) showed some similar-

ity in the earlier part of the chromatogram to those from milks digested with bacterial proteinase. However, it still exhibited long-retention-time peaks, which are absent from chromatograms of bacterial proteinase digests. Consistent with this result are the fluorescamine proteolysis results that show a significant amount of proteolysis in the 12% TCA filtrate of the sample treated with 2 g/L of plasmin (Table 2). However, the corresponding results for the lower levels of plasmin, (less than or equal to 1 g/L) showed negligible proteolysis levels. All samples showed proteolysis in the pH 4.6 filtrates. This indicates that plasmin at a very high concentration produces amino acids and small peptides of low hydrophobicity, which are soluble in 12% TCA. Such a level of plasmin proteolysis is much higher than is likely to be encountered in commercial bulk milk. The only possible exception to this is mastitic (high somatic cell count) milk with raised plasmin and/or PA levels (Auldist et al., 1996). However, commercial UHT milk should not be produced from such poor quality milk. Therefore, it is highly unlikely, under normal circumstances, that proteolysis detected by fluorescamine in 12% TCA filtrates of UHT milk could be due to plasmin action. 3.4. Proposed methodology for distinguishing between proteolysis in milk caused by milk plasmin and bacterial proteinases The above results suggest a useful approach to determining the cause(s) of proteolysis in UHT milk,

Table 2 Proteolysis of UHT milk by various levels of plasmin Plasmin concentration (mg/L)

100 500 1000 2000 a

Proteolysisa by fluorescamine 12% TCA filtrate

pH 4.6 filtrate

0.09 0.12 0.16 5.0

3.4 4.1 5.8 11.6

As concentration of a-amino groups in the milk (mmol/L).

Fig. 4. Peptide patterns of TCA filtrate of UHT milk gelled by plasmin at high concentration (2 g/L).

N. Datta, H.C. Deeth / Lebensm.-Wiss. U.-Technol. 36 (2003) 173–182

which can lead to age gelation, by analysis of the peptides in 12% TCA and pH 4.6 extracts. The action of Pseudomonas proteinases is indicated by a high level of fluorescamine-reactive proteolysis products in the 12% TCA extract or a chromatogram of the peptides soluble at either pH 4.6 or in 12% TCA containing many peaks within 20 min and virtually none after this time indicated.

*

*

This observation is most likely the result of milk having poor bacteriological quality at the time of processing. On the other hand, the action of the native milk proteinase, plasmin, is indicated by a high level of fluorescamine-reactive proteolysis products soluble at pH 4.6 but not in 12% TCA or major peaks from 20 to 40 min in the RP-HPLC chromatogram of the pH 4.6 extract and virtually no peaks from the 12% TCA extract in the same region of the chromatogram.

*

*

This may arise from residual activity of the plasmin after UHT treatment and/or plasmin generated during storage by the action of a PA, either endogenous or exogenous, on the precursor plasminogen. These outcomes and their interpretation are summarised in Table 3 together with the outcomes expected

179

for unproteolysed milk and milk proteolysed by both plasmin and bacterial proteinase. 3.5. Proteolysis in gelled commercial UHT milks In order to apply the above methodology to elucidating the cause of gelling in commercial UHT milks, proteolysis in gelled commercial UHT milk samples (0.1–3.5% fat) was analysed by the fluorescamine method (Table 4) and RP-HPLC (representative chromatograms appear in Figs. 5 and 6). Table 4 clearly indicates two distinct types of proteolysis; samples 1–4 show negligible amounts of TCA-soluble proteolysis products but substantial levels in pH 4.6 filtrates while samples 5–9 show high levels in both the TCA and pH 4.6 filtrates. This suggests that samples 1–4 were affected by plasmin only whereas samples 4–9 suffered proteolysis by both plasmin and bacterial proteinase. This is consistent with the RP-HPLC elution profiles shown in Fig. 5 (sample 1) and Fig. 6 (sample 5). The pH 4.6 extract of sample 1 (Fig. 5a) exhibits only minor peaks up to 20 min but a large and broad peak at 35– 40 min; the corresponding chromatogram for the TCA filtrate (Fig. 5b) shows virtually no peaks, even at the lower attenuation shown. In contrast, both chromatograms in Fig. 6 (sample 5) contain peaks before 20 min indicating bacterial proteolysis. The chromatogram for

Table 3 Interpretation of peptide analyses by fluorescamine and HPLC for diagnosing the cause of proteolysis in UHT milk RP-HPLCa

Proteolysis by fluorescamine

Interpretation

TCA filtrate

pH 4.6 filtrate

TCA filtrate

pH 4.6 filtrate

Few, if any, peaks Few, if any, peaks

Few, if any, peaks Mostly late peaks (>20 min), few early peaks Mostly early peaks (o20 min) Early and late peaks

Close to zero Close to zero

Low Significant

Good quality milk Plasmin action only

Significant Significant

Significant Significant

Bacterial proteinase action only Both bacterial proteinase and plasmin action

Early peaks (o20 min) only Early peaks (o20 min) only a

HPLC retention data apply to the HPLC conditions given in the methodology section.

Table 4 Proteolysis and viscosity data for gelled commercial UHT milks and diagnosis of the cause of proteolysis Samples

Viscosity (mPa s)

Proteolysisa by fluorescamine 12% TCA filtrate

pH 4.6 filtrate

1 2 3 4

20 4.0 (completely clarified) 129 125

0.1 0.1 0.1 0.1

4.0 9.0 2.8 3.0

5 6 7 8 9

50 40 50 111 48

3.7 3.4 4.3 7.1 3.6

8.4 7.4 9.0 12.0 8.0

a

As concentration of a-amino groups in the milk (mmol/L).

Diagnosis

Plasmin-mediated gel

Plasmin- and bacterial proteinase-mediated gel

180

N. Datta, H.C. Deeth / Lebensm.-Wiss. U.-Technol. 36 (2003) 173–182

Fig. 5. Peptide patterns of plasmin-mediated, gelled commercial UHT milk (sample 1 in Table 4): (a) pH 4.6 filtrate; (b) TCA filtrate.

Fig. 6. Peptide patterns of plasmin- and bacterial proteinase-mediated, gelled commercial UHT milk (sample 5 in Table 4): (a) pH 4.6 filtrate; (b) TCA filtrate.

the pH 4.6 filtrate (Fig. 6a) also shows late-eluting broad peaks attributable to peptides from plasmin hydrolysis. The viscosities of the gelled milk (Table 4) were high, but there was no correlation between viscosity

and proteolysis in these milks. This is easily explained since the viscosity is high when the milk is approaching, and at the point of, gelation, but decreases thereafter when the gel breaks up or is clarified by

N. Datta, H.C. Deeth / Lebensm.-Wiss. U.-Technol. 36 (2003) 173–182

further proteolysis. Proteolysis continues and reaches the highest levels when the milk’s viscosity returns to a low level. 3.6. Practical significance Analysis of milk in the manner proposed can enable the UHT milk manufacturer to determine if proteolysis is occurring, or has occurred in the milk and if so, whether it is caused by milk plasmin, bacterial proteinase, or both. If it is caused by plasmin, it is likely that the UHT processing conditions are too mild. This causes less denaturation of plasmin and also less denaturation of whey proteins, which results in less whey protein–casein interaction and less inhibition of plasmin action on the casein. This situation is most commonly encountered in the direct UHT processes, steam infusion or injection (Manji, Kakuda, & Arnott, 1986; Manji & Kakuda, 1988). If the proteolysis is caused by bacterial proteinases, the quality of the raw milk is implicated. The most common cause is high levels of psychrotrophic bacteria in the raw milk. Inadequately cleaned equipment that supports bacterial growth and production of proteinases can also be a cause (Driessen, 1983).

4. Conclusion The peptides produced by bacterial proteinase are less hydrophobic and elute early in the RP-HPLC, while the peptides produced by the native milk plasmin are more hydrophobic and elute later. Acid precipitation to pH 4.6 and with 12% TCA allows the peptides to be fractionated in a similar manner. Determination of the peptides in the fraction soluble at pH 4.6, using the fluorescamine method, provides a measure of the total amount of proteolysis from both plasmin and bacterial proteinases, while the peptides soluble in 12% TCA represent proteolysis by bacterial proteinases only. Therefore, this paper presents two methods of distinguishing between proteolysis in UHT milk by the native milk plasmin and by bacterial proteinases: RP-HPLC and fluorescamine analysis of pH 4.6 and TCA filtrates. Knowledge of the cause of proteolysis, which can lead to gelation of UHT milk, facilitates remedial action to prevent or ameliorate proteolysis problems.

Acknowledgements The authors wish to thank Miss Katherine Raymont for her excellent technical assistance, and the Australia Dairy Research and Development Corporation for financial support.

181

References Aroonkamonsri, J. (1996). The role of plasmin and bacterial proteinases on age gelation of UHT milk (Pseudomonas fluorescens, Pseudomonas fragi, Pseudomonas luteola, Vibrio fluvialis). Ph.D. thesis, University of Guelph, Canada. 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. Beeby, R. (1980). The use of fluorescamine at pH 6.0 to follow the action of chymosin on kappa-casein and to estimate this protein in milk. New Zealand Journal of Dairy Science and Technology, 15, 99–108. Cauvin, E., Sacchi, P., Rasero, R., & Turi, R. M. (1999). Proteolytic activity during storage of UHT milk. Industrie Alimentari, 38, 825–829. Chen, J. H. T. (1971). Purification and characterization of milk protease. Ph.D. thesis, Cornell University, p. 69. Datta, N., & Deeth, H. C. (2001). Age gelation of UHT milk. Transactions of the Institute of Chemical Engineers C. Food and Bioproducts Processing, 79, 197–210. Deeth, H. C., Khusniati, T., Datta, N., & Wallace, R. B. (2002). Spoilage of skim and whole milks. Journal of Dairy Research, 68, 228–242. de Koning, P. J., Kaper, J., Roellama, H. S., & Driessen, F. M. (1985). Age thinning and gelation in unconcentrated and concentrated UHT-sterilized skim milk. Effect of native milk proteinase. Netherlands Milk and Dairy Journal, 39, 71–87. Driessen, F. M. (1981). Enzymic proteolysis in UHT milk products: Production of proteinases by bacteria during their growth in milk. Zuivelzicht, 73, 656–658. Driessen, F. M. (1983). Lipases and proteinases in milk: Occurrence, heat inactivation and their importance for the keeping quality of milk products. Ph.D. thesis, Agricultural University, Wageningen, p. 157. Harwalkar, V. R. (1992). Age gelation of sterilized milks. In P. F. Fox (Ed.), Advanced dairy chemistry, Vol. 1: Proteins (pp. 691–734). London: Elsevier Science Publishers Ltd. Kocak, H. R., & Zadow, J. G. (1985). Age gelation of UHT whole milk as influenced by storage temperature. Australian Journal of Dairy Technology, 40, 14–21. Law, B. A., Andrews, A. T., & Sharpe, M. E. (1977). Gelation of ultrahigh-temperature-sterilized milk by proteinases from a strain of Pseudomonas fluorescens isolated from milk. Journal of Dairy Research, 44, 145–148. Lopez-Fandino, R., Olano, A., San Jose, C., & Ramos, M. (1993). Application of reversed-phase HPLC to the study of proteolysis in UHT milk. Journal of Dairy Research, 57, 541–548. Manji, B., & Kakuda, Y. (1988). The role of protein denaturation, extent of proteolysis and storage temperature on the mechanism of age gelation in a model system. Journal of Dairy Science, 71, 1455–1463. Manji, B., Kakuda, Y., & Arnott, D. R. (1986). Effect of storage temperature on age gelation of ultra-high temperature milk processed by direct and indirect heating systems. Journal of Dairy Science, 69, 2994–3001. McMahon, D. J. (1996). Age-gelation of UHT milk: Changes that occur during storage, their effect on shelf life and the mechanism by which age-gelation occurs. Heat treatments and alternative methods (pp. 315–325). International Dairy Federation ref S.I. 9602. Brussels, Belgium, International Dairy Federation. Morales, F. J., & Perez, S. J. (1998). Monitoring of heat-induced proteolysis in milk and milk-resembling systems. Journal of Agricultural and Food Chemistry, 46, 4391–4397.

182

N. Datta, H.C. Deeth / Lebensm.-Wiss. U.-Technol. 36 (2003) 173–182

Mulvihill, D. M., & Donovan, M. (1987). Whey proteins and their thermal denaturation—A review. Irish Journal of Food Science and Technology, 11, 43–75. Richardson, B. C. (1983). Variation of the concentration of plasmin and plasminogen in bovine milk with lactation. 18, 247–252. Richardson, B. C., & Newstead, D. F. (1979). Effect of heat-stable proteinases on the storage life of UHT milk. New Zealand Journal of Dairy Science and Technology, 14, 273–279. Snoeren, T. H. M., & van Riel, J. A. M. (1979). The properties of alphaS2-group casein. Zuivelzicht, 71, 766–768.

Visser, S. (1981). Proteolytic enzymes and their action on milk proteins. A review. Netherlands Milk and Dairy Journal, 35, 65–88. Walstra, P., & Jenness, R. (Eds.), (1984). Dairy chemistry and physics. New York: Wiley. Yvon, M., Chabnet, C., & Pelissier, J. P. (1989). Solubility of peptides in trichloroacetic acid (TCA) solutions. Hypothesis on the precipitation mechanism. International Journal of Peptide and Protein Research, 34, 166–176.