Monitoring the changes in UHT whole milk during storage under dynamic and constant temperature profiles

Abstract

The objective of the study was to monitor some physical and chemical changes of UHT whole milk stored under constant and dynamic temperature. UHT milk samples were stored at constant room temperature (25 °C) and at constant abuse temperature (41.5 °C). Dynamic condition was attained by switching the samples between 25 and 41.5 °C with 24 h intervals up to 158 days. Physical and chemical analyses revealed that most dramatic changes obtained in samples stored at 41.5 °C followed by dynamic temperature and 25 °C. The changes in the samples stored dynamically were in between but closer to those of stored at 25 °C. The pH levels reduced, and acidity, electrical conductivity and viscosity increased at the end of the storage period (158 days). Brown color was apparent for the samples stored at 41.5 °C and samples stored dynamically had also darker color than that of the samples stored at 25 °C whereas color of freshly UHT processed milks was indistinguishable from the samples stored at 25 °C. Our results indicate that shelf life studies based on isothermal conditions should be reconsidered for UHT milk since it is almost impossible to attain constant temperature during shipment or storage.

Introduction

Milk can be heat treated to reduce or eliminate bacterial count and to enhance the shelf life. Milk can be pasteurized or sterilized and extended shelf life (ESL) milk is somewhere between these two treatments (Deeth and Lewis 2017). It was shown that ultra-high temperature (UHT) technology, which uses direct or indirect heating at 135–141 °C for 3–6 s, effectively inactivates a variety of mesophilic and thermophilic spores, including those of Clostridium perfringens, C. botulinum and Bacillus cereus. Commercially sterile products with a prolonged shelf life at room temperature are produced by UHT technology (Rauh and Xiao 2022). Although an acceptable “best before” time has been generally accepted as six months, there is increasing pressure to extend this to up to 12 months and even longer (Deeth and Lewis 2017). Therefore “prolonged shelf life” is generally up to one year at room temperature for UHT milk.

There are several studies on the changes of UHT milk during storage at different temperatures. Karlsson et al. (2019) showed that UHT milk stored at 4 and 20 °C had the longest shelf-life of 34–36 weeks, limited by sediment formation, and storage at 30 and 37 °C considerably decreased the shelf-life of UHT milk to 16–20 weeks, whereby changes in sediment formation, taste and color were the limiting factors. Malmgren et al. (2017) observed sedimentation in directly heated UHT milk at all storage temperatures (5, 22, 30 and 40 °C); however, the same amount of sediment as formed after 3 months at 30 and 40 °C was seen first after 6 months at 5 °C. Richards et al. (2014) employed a multivariate accelerated shelf life test to low fat UHT milk samples and determined storage at 35 and 45 °C reduced the shelf life by a factor of 2.9 and 7.8, respectively when compared with 25 °C.

UHT milk undergoes significant chemical and physical changes during storage even at room or ambient temperature and these may have an impact on the end product’s shelf life and consumer acceptance. Temperature is obviously the most important factor in the shelf life of UHT milk because the ambient temperature can vary from 0 to 50 °C in certain areas and some countries have extremely hot summers and bitterly cold winters. Moreover, UHT milks are shipped without refrigeration and it may take more than a month to arrive their destinations. Loggers have recorded temperatures up to 60 °C during these shipments (Deeth and Lewis 2017).

Generally, higher temperatures (≥ 30 °C) cause more and faster changes in UHT milk in contrast to refrigeration and room temperatures. Moreover, studies on the shelf life of UHT milk performed generally at constant temperature conditions. However, during shipment or storage dynamic temperature conditions should not come as a surprise as mentioned above. Two notable examples where changing temperatures used were the works of Sunds (2016) and Sunds et al. (2018). Storage temperatures for UHT milk samples were 10, 20, 30, 40 and 50 °C and three temperature cycles were applied: cycle 1 was exposed to 10 to 30 °C, cycle 2 to 20 to 40 °C and cycle 3 to 30 to 50 °C. Temperatures were switched between the low and high temperature levels with two weeks intervals to simulate the real life conditions during shipping and export of UHT milk (Sunds 2016; Sunds et al. 2018). The temperature cycles included in the chemical analyses (peptide analysis, initial, intermediate and late Maillard reactions, lipid oxidation, and protein composition) increased with a higher rate than at the average temperature of which they were exposed to whereas the temperature cycles included in the physical analyses (physical destabilization, protein and fat content, and fat globule size distribution) followed the same development as at the average temperature of which they were exposed to (Sunds 2016).

Temperature profile is not only important during shipment of UHT milk but also during storage at the market shelves. The works of Sunds (2016) and Sunds et al. (2018) gave insights to the changes in UHT milk under dynamic temperature conditions; however, temperature changed from low to high or high to low levels at every two weeks and this corresponds to a long period of time for the market environment. Therefore, the aim of this study was to investigate the changes of UHT milk during storage under dynamic temperature profile that intends to mimic market conditions and to compare these changes with constant temperature conditions.

Material and methods

Milk samples

UHT whole milk samples in 200 mL Tetra Brik Aseptic packages were obtained from a local producer in Konya, Türkiye. UHT treatment was performed in a commercial scale UHT plant which was equipped with both steam injection and steam infusion modes. Milk was heated by steam injection or steam infusion to a final UHT temperature of 140 °C for 6 s. Two different batches produced at different days were taken and there were three days between the production of these batches. Analyses results of the UHT milk is given in Table 1. The protein content of the UHT milk was 2.5% and this was lower than the expected value which is generally between 3.0 and 3.5%. This might be due to various reasons such as breed, age, stage of lactation, season of milking and nutrition (Linn 1988).

Table 1.

Results of the analyses of UHT milk ± standard deviation (n = 6)

Parameter	Value
pH	6.82 ± 0.02
Conductivity (mS/cm)	5.05 ± 0.02
Acidity (o>SH)	5.0 ± 0.1
oBrix	11.3 ± 0.1
Salt (%)	0.26 ± 0.03
Total dry matter (%)	10.78 ± 0.13
Total dry matter non-fat (%)	7.76 ± 0.04
Protein (%)	2.45 ± 0.16
Density (g/mL)	1.0285 ± 0.0005
Fat (%)	3.23 ± 0.11
Freezing point (oC)	−0.50 ± 0.005
Lactic acid (%)	0.1125 ± 0.00225
Viscosity (cP)	1.47 ± 0.03
L*	95.40 ± 0.34
a*	–1.87 ± 0.02
b*	6.23 ± 0.06

Storage conditions

UHT milk samples were divided into three and stored at either (i) a steady temperature of 25 °C (named as 25C samples) or (ii) a steady temperature of 41.5 °C (named as 41.5C samples) and (iii) samples were switched between 25 and 41.5 °C with 24 h intervals (named as dynamic samples) up to 158 days in temperature-controlled incubators with 55 L of volume (Nüve EN55, Ankara, Türkiye). At most 48 samples were placed in one incubator. The average temperature in Konya region during the months of July and August (highest among other months) are 24.1 and 24.0 °C, respectively and the maximum temperature for these months are 40.6 and 39 °C, respectively (Turkish Sate Meteorological Service 2024). Temperatures used in this study were based on these average and maximum values. Temperature of the samples were checked regularly during storage by use of a lab-scale thermometer (TFA, Wertheim, Germany).

Chemical and physical analyses

pH was measured by a pH-meter (WTW Inolab pH 7310, Weilheim, Germany). EC meter (WTW Cond 3110, Weilheim, Germany) was used to determine electrical conductivity (EC) of the samples. Viscosity was measured by a viscometer (Brookfield DV-E, Middleborough, USA). Acidity expressed as Soxhlet-Henkel degree (°SH) was measured by using AOAC method described by Fabro et al. (2006). Color of the UHT samples were determined by using SL400 spectrocolorimeter (SERLAB İstanbul, Türkiye). L^*, a^*, and b^* values were measured according to the CIELAB color range. The coordinates L^*, a^*, and b^* represent lightness (white: 100, black: 0), redness (red: + , green: −), and yellowness (yellow: + , blue: −) ranges, respectively. The color measurements were repeated six times. Whiteness index, browning index, yellowness index and total color difference (ΔE) were calculated as follows (Buzrul et al. 2022; Milovanovic, et al. 2020):

$$\text{WI}=100-\sqrt{\left(100-L_{}^{\ast }\right)_{}^2+a_{}^{\ast 2}+b_{}^{\ast 2}}$$ 1

$$\text{BI}=\frac{100·\left(\frac{1.75·L_{}^{\ast }+a_{}^{\ast }}{5.645·L_{}^{\ast }+a_{}^{\ast }-3.012·b_{}^{\ast }}-0.31\right)}{0.17}$$ 2

$$\text{YI}=\frac{142.86·b_{}^{\ast }}{L_{}^{\ast }}$$ 3

$$\mathrm{\Delta }E=\sqrt{\left(L_0-L_{}^{\ast }\right)_{}^2+\left(a_0-a_{}^{\ast }\right)_{}^2+\left(b_0-b_{}^{\ast }\right)_{}^2}$$ 4

where L₀, a₀ and b₀ are the color parameters of the freshly produced UHT milk.

Hydroxymethylfurfural (HMF) was determined by the method of Elliot et al. (2003). The steps before HPLC analysis were briefly as follows: 5 mL of milk was mixed with 2.5 mL of 0.3 M oxalic acid solution for 1 h at 100 °C. After cooling, 2.5 mL of trichloroacetic acid solution (%40 w/v) was added and stirred, and the mixture was filtered through a filter paper.

Microbiological analyses

Microbiological analyses in triplicate were determined up to 4 months (120 days) due to limited number of UHT samples. All analyses were conducted according to standard methods (AOAC 2005). All media were obtained from Merck (Darmstadt, Germany). Plate count agar (PCA) was used for total aerobic bacteria, and violet red bile agar (VRBA) was used to detect Escherichia coli/coliforms, plates were incubated at 37 °C for 24 h. Yeast extract glucose chloramphenicol agar (YEGCA) was used for yeasts and molds, plates were incubated at 25 °C for 96 h.

Bacillus cereus selective agar base was used for enumeration of B. cereus. Plates were incubated at 37 °C for 24 h. PCA was also used for the enumeration of spore formers. Heated samples at 80 °C for 10 min were spread onto PCA plates. All plates were incubated in an inverted position for 72 h at 55 °C.

Statistical analyses

All measurements except color were done in triplicate and means ± standard deviations for two different batches of UHT milks were calculated. ANOVA was performed to assess significant (p ≤ 0.05) differences among the samples followed by Tukey’s test by using SPSS (Version 22, Chicago, IL, USA).

Results and discussion

Microbiological results revealed that there were no bacteria in UHT milk samples up to 120 days indicating heat treatment (140 °C for 6 s) was effective to inactivate all vegetative cells and spores (results not shown). The pH values of all samples were decreased with respect to time; however, most drastic reduction (from 6.82 to 6.29) was in 41.5C samples (Fig. 1A). It is known that pH of UHT milk decreases with time even storage at refrigeration temperature (4 °C), but as temperature increases (22 °C or 37 °C) the reduction in pH is more pronounced (Aldubhany et al. 2014). The decrease in pH of UHT milk is explained by calcium phosphate precipitation, casein dephosphorylation, lactose breakdown and proteolysis (Gausher et al. 2008). The pH change during storage are also associated with the advance of the Maillard reaction especially at high temperatures (> 30 °C). Isomerization and degradation of lactose creates high amounts of formic acid, and this is mostly responsible for the storage-induced decline in milk pH besides the formation of brown pigments (Karlsson et al. 2019; van Boekel 1998). If we consider pH 6.5 as the boundary for the shelf life of UHT milk, the shelf life at 41.5 °C was only 74 days (pH = 6.49) according to our results (Fig. 1A) and pH values at 25 °C were always higher than 6.5 (the lowest pH was 6.56 during storage) indicating that shelf life of UHT milk was longer than 158 days at 25 °C. Under dynamic temperature shelf life was 137 days (pH = 6.50) revealing that shelf life of UHT milk stored dynamically would be shorter than that stored at the low temperature, but much higher (about two times) than the ones stored at the high temperature.

Fig. 1.

Changes in pH (A), lactic acid (B), conductivity (C), °SH (D) and viscosity (E) of UHT milk samples stored at different temperatures. Error bars represent standard deviation (n = 6)

Contrary to pH, acidity increased with respect to storage time (Fig. 1B, D). Similar to acidity, EC (Fig. 1C) increased for all samples, but again most dramatic increase was observed in 41.5C samples. Viscosity (Fig. 1E) on the other hand, initially decreased (from 1.49 cP to 1.29 cP for 25C samples), stayed at that level up to 74 days and then increased. Acidity increase (Fig. 1B, D) in UHT milk during storage is attributed to the formation of acid anhydrides and formic acid over time (Malmgren et al. 2017). It was also reported that increase in acidity of UHT milk stored at room temperature is higher than in those stored at refrigeration temperature (Rerkrai et al. 1997). EC measurement was proposed to evaluate the freshness of cow milk by Hwang et al. (2022). These authors found that EC values increased from 5.05 to 6.10 mS/cm during 42 days for the refrigerated milk (5 °C), whereas it was promptly increased from 7.08 to 11.95 mS/cm during 30 days for the milk stored at room temperature (20 °C). Similarly, we obtained that as the storage temperature increased EC values increased (Fig. 1C). Our results on pH and acidity were also in agreement with González et al. (2020) and Ranvir et al. (2020). The initial decrease of the viscosity (Fig. 1E) is due to a thinning effect (de Koning et al. 1985) and increase in viscosity (Fig. 1E) causes age gelation during the storage of UHT milk (Deeth and Lewis 2017). Although the storage temperature may affect the viscosity, it may also be correlated with the heat treatment during processing, percentage of fat and presence of extracellular proteolytic enzymes.

The changes in the color of UHT milk samples are shown in Fig. 2. The lowest L^* values belonged to the 41.5C samples at the end of 158 days. Although significant difference (p ≤ 0.05) was observed between the 25C and dynamic samples eventually, L^* values of these samples were close to each other and they were insignificant (p > 0.05) up to 4.5 months (Fig. 2A). There was a slight change between the initial and final values of L^* values of the 25C samples (only 0.2% decrease); however, about 8% decrease was observed for 41.5C samples. The a^* and b^* values increased dramatically for 41.5C samples (281% and 173% increases, respectively) whereas 25C samples had less changes (16% increase and 6% decrease, respectively) in the end. The dynamic samples were in between but closer to the 25C samples. Moreover, the rate of increase in a^* and b^* values was much higher in 41.5C samples than those of 25C and dynamic samples (Fig. 2C, E). In general, WI decreased while BI and YI increased for all samples, again the dynamic samples were closer to the 25C samples (Fig. 2B, D, F). Our findings on color were similar to Ansari and Sahoo (2018) and Popov-Raljic et al. (2008) who stored UHT milk samples at 20 ± 5 °C. Maillard reaction rate increases with increasing temperature (Al-Saadi and Deeth 2008) and this might be the reason of obtaining different color changes at different storage temperatures (Karlsson et al. 2019).

Fig. 2.

Variation in color (L^*, a^* and b.^*) and whiteness index (WI), browning index (BI) and yellowness index of UHT milk samples stored at different temperatures. Error bars represent standard deviation (n = 6)

Total color difference (ΔE) of UHT samples are shown in Fig. 3. ΔE values of 25C and dynamic samples were always less than 4 and 5, respectively; however, values of 41.5C samples were > 6 during the whole storage period (Fig. 3). ΔE values higher than 3 are generally perceptible to the human eye (Milovanovic et al. 2020) and support for this statement comes from the photo of the samples at the end of 158 days shown in Fig. 4. The 25C samples had almost the same color with freshly produced UHT milk. On the other hand, dynamic samples had slightly browner color and 41.5C samples had the darkest color of all (Fig. 4).

Fig. 3.

Variation in total color differences (ΔE) of UHT milk samples stored at different temperatures

Fig. 4.

Changes in color of UHT milk samples. Samples from left to right: (i) Freshly processed UHT milk, (ii & iii) UHT milk stored at 25 °C for 158 days, (iv & v) UHT milk stored under dynamic temperature for 158 days, (vi & vii) UHT milk stored at 41.5 °C for 158 days

Formation of HMF in UHT milk samples at the end of storage period is shown in Fig. 5. Freshly processed UHT milk had the lowest (1.97 mg/L) and 41.5C samples had the highest (2.40 mg/L) HMF. This corresponded to 22% increase in HMF. The rises was only 6% for 25C samples and it was 14% for the dynamic samples. HMF values of all samples were significantly different (p ≤ 0.05). Jimenez‐Perez et al. (1992) stored UHT milks at different temperatures in the range of 6–50 °C up to 90 days and found an increase in HMF in the temperature ≥ 40 °C. The formation of HMF is possible even at low temperatures; however, as the temperature of the heat treatment as well as the storage temperature increase, HMF concentration increase enormously (Capuano and Fogliano 2011).

Fig. 5.

HMF formation in control sample (freshly processed UHT milk) and UHT milk samples stored at different temperatures at the end of storage period (158 days). Error bars represent standard deviation (n = 6) and letters indicate statistically significant differences among the samples (α = 0.05)

Conclusion

Storage temperature has a major role in physical and chemical changes and therefore the shelf life of UHT milk, but unfortunately it is not easy to control this parameter. In this study, UHT milk stored at constant room (25 °C) and at constant abuse temperature (41.5 °C) as well as under dynamic temperature condition. All physical and chemical analyses showed that dynamic condition results were in between low and high temperature, generally closer to the results of 25 °C indicating that shelf life of UHT milk subjected to dynamic temperature during storage would have shorter shelf life than that of UHT milk stored at room temperature. Although our study was a proof, new studies should be conducted by monitoring the markets’ temperature changes with loggers and performing deeper analyses such as age gelation, sedimentation and flavor to better understand the effects of dynamic conditions on shelf life of UHT milk.

Author contributions

RBZ: Methodology, investigation, data analysis, SB: Conceptualization, methodology, writing-original draft, data analysis, supervision, reviewing and editing, GB: Conceptualization, reviewing and editing, supervision.

Funding

This research did not receive any specific grant.

Data availability

Data are available from the corresponding author on request.

Code availability

Not applicable.

Declarations

Conflict of interest

The authors declare no conflict of interest.

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.