- Robert Adi Nugraha
The phenomenon known as “age thickening” refers to the event of viscosity rise just before the formation of gel and loss of fluidity. It is described by Snoeren et al (1982) as “structural build-up through weak interactions between casein micelles” which could be disrupted through mechanical shear. This effect is observed more on concentrated milk than the single strength milk. (Datta & Deeth, 2001) In addition, the type of milk may have a part as well in determining the susceptibility to age thickening. It was found that skim milk samples were more susceptible to age thickening than regular milk. This is because fat had a protective effect against age thickening, perhaps through the interaction with casein micelles. (Harwalker et al, 1983) Age thickening is also a major limiting factor of shelf life for ultra high temperature (UHT) milk. (Datta & Deeth, 2001)
According to Datta & Deeth (2001), there are 4 stages of age thickening. The initial step is the thinning of the product. There is not much change in viscosity observed in second step. The third step should display the sudden change of viscosity and gel formation. The last step is where the separation begins and gel started to appear. This gel that formed has been characterised by the whey proteins (in particular β-Lactoglobulin) interacting with casein (mainly κ-casein) of the casein particle and forming a three dimensional matrix of protein. Thus, this resulted in the formation of β-Lactoglobulin-κ-casein complexes during the heat treatment process involved in production of UHT milk products. Moreover, further changes occur during storage which involve the β-Lactoglobulin-κ-casein complexes being released from casein micelles and cross linking proteins interactions. This resulted in the formation of three dimensional protein network and can be observed as the milk thicken and then gel. (Datta & Deeth, 2001)
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There are several factors that affect age thickening in milk which are: mode and severity of heat treatment, proteolysis, microbiology quality factor, storage temperatures, and fat content. These factors are related to the degree of the three processes which lead to age thickening or gelation which are 1) β-Lactoglobulin and κ-casein interaction, 2) the β-Lactoglobulin-κ-casein complex release from the casein particle, and 3) the cross linking of the β-Lactoglobulin-κ-casein complexes and proteins. (Datta & Deeth, 2001) There are several additional factors suggested by Walstra et al (2005) that involve the effect of age thickening in condensed milk. The factors added are the stage at which sugar is added (the latter in the process, the less the age thickening) and the concentration factor (the higher the concentration, the more the age thickening).
The effect of heat treatment on preventing age-thickening in cold-stored evaporated milk was looked at by Harwalker et al (1983) and showed that heat treatment was not effective at all. The idea behind their research was because of some changes in casein micelles which resulted from cold storage could be reversed from heating. Thus, the same concept was looked at for application in similar way, but they could not find evidence that the treatment worked. On the other hand, several researches have found that the introduction of UHT heating whether that direct or indirect differentiate the susceptibility of the milk samples to age thickening effect.
McKellar et al (1984) found that an increase in viscosity was observed between 6-10 weeks at 20°C of directly heated UHT milk compared to the no apparent rise in viscosity during 30 weeks storage at the same temperature for the indirectly heated UHT milk. They suggested that the different heating severity where the indirect heating have higher heat load was the crucial factor that caused this result to be seen. Similar researches were carried out by increasing the temperature or time of heating and the same effect could be observed where the milk samples exposed to higher temperature or longer heating time tend to last longer without age thickening effect observed. (Samuelson & Holm, 1966; Zadow & Chituta, 1975) The reasoning behind this was suggested through the research of Manji & Kakuda (1988) where they proposed that the resistance to age thickening effect observed in more severely heat treated milk samples was due to the increased level of denatured whey protein. The result from this research showed that start of the age thickening effect is factored by formation of denatured whey proteins and casein complex.
Some studies have investigated the role of proteolysis of caseins in age thickening of milk. This was accredited to natural milk proteinase (plasmin) and heat stable proteinase produced by contaminants psychrotrophic bacterial. (Datta & Deeth, 2001) During storage, the quantity of plasmin in milk may increase because of the endogenous plasminogen activators which convert plasminogen into plasmin and may cause gelation. Plasminogen is more heat stable than plasmin and thus if proteolysis and subsequently age thickening need to be controlled, denaturation of plasminogen must be targeted. In a study specifically designed to look at the effect of proteolysis in age thickening, serine proteinase inhibitors were added to UHT milk to inhibit plasmin and the result after storage for 9 months at 20°C was that no proteolysis and gelation occurred. (de Koning et al, 1985) As for the heat stable proteinase produced by psychrothropic bacteria, a Pseudomonas fluorescens strain was isolated from raw milk. The presence of this bacteria led to age thickening gelation over time with the time dependent on the bacteria growth prior to heat processing. (Law et al, 1985) Therefore, both bacterial proteinases and plasmin displayed their abilities to initiate proteolysis and thus age thickening effect in UHT milk.
Harwalker et al (1983) tested the microbial quality of the raw milk used for preparing the evaporated milk, but they did not find any significant relation between the age thickening effect and this microbial quality. They proceeded to look at the microbial growth during storage to see if there was a relationship or not. In this part of their research, they did not find evidence to support relationship between an increase in standard plate count of microorganism with age thickening effect. The samples they were testing had an increase in standard plate count to millions, but the viscosity change was less than the samples which had much lower increases in standard plate count. The age thickened samples also showed no microorganisms which are capable of growth with the conditions. (Harwalker et al, 1983)
The addition of additives to milk, such as sodium phosphate and sodium citrate, may speed up the age thickening effect, whereas the addition of polyphospates (e.g. sodium hexametaphosphate) could delay this effect. (Datta & Deeth, 2001) The age gelation protection provided by polyphosphates increases with chain length and concentration with the most effective one being at 4.8 phosphorus atoms per chain. (Leviton et al, 1963) Furthermore, cyclic phosphates are more effective than the corresponding linear polymers in delaying the effect of age thickening. This is because cyclic phosphates are stable against hydrolysis and thus unable to form complexes with calcium ions which also posses anti-gelation activity as well compared to the linear polyphosphates which are converted slowly into orthophosphate that accelerates age thickening effect. (Leviton et al, 1962)
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Oxidising conditions such as aeration and peroxide treatments accelerated age thickening, but reducing conditions such as antioxidant treatments tended to delay but not prevent this phenomenon. (Harwalkar et al, 1983) This research also found that age thickening was also accelerated when evaporated skim milk was cold-stored before sterilisation. In relation to commercial practice, the age thickening problem happens to be a seasonal problem and it is more frequent in the early spring. (Hardham, 1996) Storage temperature is an important factor in age thickening effect as well. As noted by Datta & Deeth (2001), age thickening takes place most easily at room temperatures (20-25°C) compared to the low (4°C) or high temperatures (35-40°C). Moreover, Harwalker et al (1983) concluded that age thickening may not pose a huge problem if cold storage of concentrate milk before sterilisation could be avoided. However, they noted that it is simply not possible in practical term because of the increasing volume of milk processed in combination with shorter working weeks.
Datta & Deeth (2001) summarised ways of controlling age gelation in their paper which will be discussed here. These methods are based on minimising proteolytic activity, delaying dissociation of β-Lactoglobulin-κ-casein complex from casein micelle, and inhibition of crosslinking or protein network formation.
The first and foremost important way is by using raw milk of high quality in combination with low temperature storage for the least amount of time. In this way, the growth of psychrotrophic bacteria and the proteinases generated from bacteria in milk is minimised before thermal processing. (Datta & Deeth, 2001) The second method proposed is heat treatment during preheating and sterilisation. This method needs to achieve the denaturation of most β-Lactoglobulin and complex formation of the aforementioned denatured whey proteins with casein. In addition, the heat treatment would also inactivate plasmin. Indirect heating is more encouraged rather than direct heating to produce gelation stable milk. (McKellar et al, 1984) The drawback from this method is that it may give cooked flavour which would be a negative attribute for consumers. (Datta & Deeth, 2001)
The third method suggested in the literature was reported by Barach et al (1976) which is the low temperature inactivation of heat stable enzymes in milk using T=55 °C and extended holding time of 30-60 minutes. It was suggested that the proteinase undergoes a conformational change, and then the altered proteinase aggregates with casein, and thus an enzyme-casein complex was formed which inactivate the enzyme. However, this method is flawed in the way that the effect of this low temperature inactivation treatment varies between different milk batches and also some proteinases showed resistance to the temperature-time combination treatment proposed above. (Datta & Deeth, 2001) Last method proposed is the addition of additives such as sodium hexametaphosphate to retard age thickening effect. This method was based on Kocak & Zadow (1985) experiment which suggests that polyphosphates (sodium hexametaphosphate in particular) stopped the second phase of age thickening gelation which involves coagulation of protein. The drawback is that the consumer reaction to additives being added to milk product may be unfavourable.
In addition, ultrasound have emerged as a possible method to manage the milk age thickening phenomenon. (Zisu et al, 2012) In the study, they used high intensity ultrasound with frequency of 20 kHz to control the rate of age thickening and reduce the viscosity of concentrated skim milk. (Zisu et al, 2012) This study found that the ageing effect could be reversed which was observed by the reduction of viscosity to similar values that of the starting material. However, they did also note that the ultrasound technique could not prevent age thickening once the process was started (especially if it is already at the advanced thickening stage), they rather delayed the thickening rate. (Zisu et al, 2012) The study concluded that if the ultrasound was to be utilised during the whole evaporation process, the effect on the early stage thickening stage could be prevented. (Zisu et al, 2012)
In conclusion, milk age thickening poses a problem for the dairy industry as it could limit the shelf life of products. There is much to be learned of this phenomenon in order to solve this problem. Novel methods such as the ultrasound technique mentioned last are much needed and may arise as more research being focused on the causes of this age thickening effect. The expectation is that more ways would be suggested and researched to prevent this effect from happening and finally resolving the problem. In the mean time, there are few alternatives methods already available or suggested to minimise or delay the age thickening effect.
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