Milk serves as a vital dietary requirement in providing nutrients and particularly, protein to human for growth. Milk is a complex liquid component that is excreted from the mammary glands of mammals such as human, cow, buffalo, goat, sheep and others. This essay discussed solely on the bovine milk. Water is the primary constituents of milk which comprised approximately 88% w/w, milk sugar 4.8%, milk fat 4%, milk proteins 3.3%, and mineral salts 0.7% (Walstra and others 1999; Otter and Benjamin 2003; Horton 1995). Nevertheless, the variability of these components in milk changes across species, breeds and between individual mammals, stage of lactation, feed composition, climate and other factors. In fact, besides of these major components, the existence of the milk's minor constituents must not be neglected as they possessed valuable properties to be commercial utilised in food industry, too. Horton (1995) brought up the query on the definition of 'minor component' which is neither based on the total weight basis nor percentage of solids. It is, however, an individual molecule or a series of similar compound that make up the major components. Table 1 shows the major components of milk and some of the common minor components.
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Table 1. Milk Components [Sources: Otter and Benjamin (2003) and Horton (1995)]
Milk sugar (lactose)
-Mono-, di- and triacylglycerols
-Sterols and sterol esters
-Unesterified fatty acids
-Bovine serum albumin
Milk protein…. Non protein nitrogen (NPN)
-Glyco- and macropeptides
Calcium, chloride, iron, magnesium, sodium, potassium and phosphate
The minor component of milk whey proteins, i.e. α-lactalbumin (α-La) will be discussed comprehensively in this essay. α-La made up of 123 amino acids with a concentration range of 0.6-1.7g per litre of milk is one of the major whey proteins. The approximate amount of proteins present in whey comprised of 50% β-lactoglobulin (β-Lg), 25% α-La, and 25% of other proteins such as bovine serum albumin and immunoglobulins (Zadow and Benjamin 2003). In comparison with casein, whey proteins are known as the soluble milk protein with globular structure and will remain in native form even at their isoelectric point and when subjected to high temperature. However, whey (constituted 17-20% w/w of total protein in milk) is always regarded as the by-product of casein and milk plasma that remains after the milk is curdled and strained during cheese making which was not valued before for its significant importance as food ingredient, nutraceuticals and antimicrobial functions. According to Zadow and Benjamin (2003), before the whey proteins gain respect in dairy industry, they were often treated as waste products to be used either as animal feed or let flow to the river stream as effluents which then imposed harm on the environment. Eventually, food scientists and technologists recognised a great potential of whey proteins and α-lactalbumin for commercial utilisation.
In fact, α-lactalbumin in bovine milk is also the primary protein in human milk (Otter and Benjamin 2003) which contains considerable amount of essential amino acids tryptophan, cysteine, methionine, threonine and isoleucine, but present in a lower proportion than in human milk. Interestingly, α-La is a calcium metalloprotein which is capable to exist in two forms; the native form (α-La-Ca2+) that bind strongly to calcium ion and the calcium-free form (apo-α-La). This property stabilises the tertiary globular structure of α-L and hence, increase its heat stability (Otter and Benjamin 2003; Eugenia Lucena and others 2007). α-La also consists of four disulphide bonds with an isoelectric point (pI) of 4.2 and the molecular weight is about 14.2kDa.
Since β-Lg (MW of β-Lg = 18.0kDa, pI = 5.2) and α-La have little difference between their molecular weight and isoelectric point, the extraction and purification method that separate α-La from other whey proteins in bovine milk is rather challenging. The ideal α-La extraction and purification method to be employed must firstly, achieve high purity of α-La and generate greater percentage of final recovery yield of soluble α-La; secondly, free from contaminants such as β-lactoglobulin, bovine serum albumin, immunoglobulins and other substances; thirdly, retain the innate physical and functional properties of α-La itself; and fourthly, employ the techniques into commercial capacity for greater productivity of α-La as an useful food ingredient. However, it is impractical to accomplish the last goal at the commercial scale due to the relatively high cost and time consuming procedures in order to accomplish the first three aims stated above. Studies were done by several authors on extracting and purifying α-La methods for both commercial and laboratory levels as reviewed by Kamau and others (2010). These include enzyme hydrolysis, selective precipitation, membrane separation (ultralfitration or diafiltration) and ion-exchange chromatography.
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After assessing all the techniques, the most potential technique to be applied for commercial level is the combination of separation-precipitation-solubilisation as proposed by Eugenia Lucena and others (2006), Bramaud and others (1997), Arabelle and others (2003), and Eugenia Lucena et al. (2007). The starting material of the extraction method in order to obtain the final α-La enriched solution is whey protein concentrate (WPC), ideally with moderate level of protein content on dry basis (Eugenia Lucena et al. 2007), low β-Lg concentration (Eugenia Lucena and others 2006) and defatted WPC (Bramaud and others 1997). Previous work was done by Eugenia Lucena and others (2007) using liquid solutions with varying concentrations of α-La: liquid whey (0.7g/L α-La), WPC 35 (5.6g/L α-La), WPC 65 (12.0g/L α-La) and whey protein isolates with 15g/L α-La. From the study, they reported that the initial α-La concentration will eventually have an impact on the α-La percipitation yield and purity. Hence, WPC with 65% protein content on dry basis (WPC 65) with 12.0g/L α-La concentration was selected as the perfect starting material for the extraction precedure.
In practice, WPC is isolated from whey using either ultrafiltration or electrodialysis and lactose crystallision method (Walstra et al. 1999). These preparation methods produce higher protein content and has lesser amount of fat and lactose. Alternatively, ultralfiltration also served as the α-La purification step to separate whey based on size differences through membrane pore adjustment to produce the target protein with minumum amount of bovine serum albumin (BSA) and immunoglobulins so as to enhance the α-La/β-Lg ratio. Arabelle and others (2003) reported that the purity of α-La after ultrafiltration was carried out was 0.65 but gave disappointing yield of 0.15 due to low transmission of α-La. The rationale elucidated by the researchers was the failure of membrane pore to discriminate between α-La and β-Lg owing to the close proximity of their molecular weights to be separate out efficiently. Nonetheless, Millesime and others (1995; cited in Eugenia Lucena et al. 2007) noticed the potential of making use the electrostatic interaction between proteins and the filter membrane surface by altering the pH and ionic strength. Lucas and the team (1998) also took advantage of this logic, they chemically modified the filter membrane by coating a layer of polyethyleneimine which is positively charged. Therefore, α-La was selectively separated out at pH 7 at immediate ionic strength 0.2 mol l-1 whereby the β-Lg was strongly attracted to that particular chemically modified inorganic membrane. The transmission of α-La reported by them was 10% while β-Lg rentention at the membrane was almost 100%.
Ultrafiltration alone is not perfect enough to obtain the preferred α-La purity and recovery yield. The starting material, WPC 65, is fed into the centrifuge and maintained at 4oC before carrying out the precipitation. The optimum operating conditions for precipitation must be selected carefully based on the previous works by several authors. Since the stabilisation and destabilisation of α-La and β-Lg is controlled by calcium ions concentration, there is a need to add calcium ions complexing agents to WPC 65 in order to reduce the concentration of free calcium, meanwhile, promote the formation of calcium free α-La, i.e. apo-α-La, which is hydrophobic in nature for α-La to precipitate at its isoelectric point (Bramaud et al. 1997). Apparently, β-Lg showed an increment in stability when the calcium ions concentration is low (Eugenia Lucena et al. 2006). Example of calcium ions complexing agents are lactic acid, citric acid and hydrochloric acid, just to name a few. Eugenia Lucena et al. (2007) made a comparison between these three different acids to investigate which acid gave a promising purity value from the obtained precipitated fractions. Both the organic acids (citric acid and lactic acid) have the capability to form complexes with calcium ions. Although addition of hydrochloric acid will cause protein precipitation, due to its strong acidity, all α-La were denatured irreversibly; losing their secondary and tertiary structure. Furthermore, when they carried out the experiment under the pH range between 3.5 and 4.5, which is near to the α-La isoelectric point (pI), the precipitation yield showed significant quantitative amount for organic acids than hydrochloric acid. At pH extremely higher or lower than the pI, the precipitation yield showed a drastic declination. Besides, since the isoelectric point of β-Lg is at pH 5.2 which is not under the suggested optimum pH range, β-Lg will not precipitate and thus remain in the soluble phase. For the precipitation operating temperature wise, irreversible thermal denaturation is an issue to avoid as this masked the true result of precipitation yield. Lactic acid precipitated all the proteins (include BSA and Ig), excluding β-Lg at mild temperature, particularly at temperature higher than 50oC, in which the highest α-La precipitation yield was achieved (Eugenia Lucena et al. 2006). This can be deduced that the usage of lactic acid as complexant is a temperature dependent step. Conversely, the effect of calcium ions complexation owing to citric acid addition was at maximal at lower temperature range. After all, the works of Eugenia Lucena et al. (2006 and 2007) concluded that lactic acid is a better complexant than citric acid; hence, the optimum operating conditions for the selective precipitation for α-La are using 0.75M lactic acid at temperature 55oC, pH 4.0 and regulate the calcium concentration of lactic acid/Ca2+ molar ratio higher than 9.
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The end product of precipitation is the precipitate which contained α-La, BSA and Ig while β-Lg proteins remain in solution. The rationale that explained presence of β-Lg proteins in solution is because they are able to achieve stabilisation upon low calcium ions concentration. However, the precipitate might be tainted with a small amount of β-Lg, hence, the precipitate is centrifuged as a precaution step and β-Lg is separated out as the supernatant (Eugenia Lucena et al. 2006). Supernatant will be tested for β-Lg purity before discarded. Eugenia Lucena et al. (2006) found out that the supernatant obtained gave 85% of β-Lg purity owing to the presence of other components such as lactose, mineral and caseinomacropeptide (CMP). Further precaution step in order to remove β-Lg fractions ultimately from the precipitate is carried out through washing, at least twice, using acidulated fresh water. Throughout the washing steps, the pH maintained at 4.0, which is approximately at the isoelectric point of α-La, and performed under the temperature between 15 to 60oC. The outcomes of the washing steps caused partial solubilisation of α-La and caused reduction of the recovery with regard to α-La initial concentration in WPC 65, but BSA and immunoglobulins will still remain in the precipitate as contaminants. In fact, β-Lg showed a greater improvement in recovery up to 100% at temperature 55oC (Eugenia Lucena et al. 2006). Bramaud et al. (1997) suggested that the solubilisation of α-La during the washing step can be avoided by using 0.1M sodium chloride at pH 3.9 to wash the precipitate, but it will cause irreversible α-La precipitation.
The ultimate goal of the α-La extraction is to produce a food ingredient with functional property and exists in native form. This can be exemplified by the ability of α-La to solubilise and not subjected to any denaturation during the extraction and purification procedures such as thermal denaturation, chemicals contamination and in particularly, the addition of acids during the selective precipitation step. Since the precipitate experienced a change in pH, calcium ion concentration and temperature fluctuation, the solubilisation of precipitate can be justified through the ability to recover the two forms of α-La: apo-α-La and α-La-Ca2+ (native form) under the condition of pH 7.5 and temperature 45oC (Eugenia Lucena et al. 2006). Bramaud et al. (1997) had the same finding that the α-La-contained precipitate that undergo washing step via sodium chloride solution appeared to be whitish and turbid will become a clear solution at pH 7.5 at 45oC, in which all the proteins are completely solubilised. At pH 7.5, the immunoglobulins that co-precipitate with α-La remain insoluble and no trace of β-Lg detected in the soluble phase, and thus, the α-La purity gives a promising result (Bramaud et al. 1997). The ability to recover the two forms of α-La relied upon the nature of ions in the solubilisation solvent, either Na+ or Ca2+ (Arabelle and others 2003). Ca2+ (using calcium chloride) was reported by Bramaud et al. (1997) was a better solubilisation solvent since the only remnants contained inside the final obtained fraction were α-lactalbumin and bovine serum albumin proteins. Further separation step of either ultrafiltration or chromatography could be carried out to remove BSA for the purpose to achieve higher α-La purity. Overall, the solubilisation step verified that the initial precipitation step using the calcium ions complexing agent of 0.75M lactic acid at pH 4.0 is a reversible reaction.
The flowchart of the proposed technique to extract α-La from WPC 65 in order to obtain the α-La enriched solution is illustrated in Figure 1.
Figure 1. Flow Chart of the α-La Extraction
[Sources: Eugenia Lucena et al. (2006) and Bramaud et al. (1997)]
Eugenia Lucena et al. (2006) carried out the high performance liquid chromatography (HPLC) and gel electrophoresis to analyse the composition of the fractions obtained at the end of each step and the result is presented in Table 2. The final recovery of the soluble α-La in its native form from their work was 85% and a purity of 75% with the presence of β-Lg around 5%. The α-La/β-Lg concentration ratio was approximately 14 and almost 99% of the β-Lg was removed from the initial raw material, WPC 65. The key success of this extraction and purification technique is generally relied on the initial protein concentration of the starting raw material, precipitation pH, the operating temperature and the number of precipitate washing for the aim to achieve high α-La purity and high α-La/β-Lg concentration ratio (Kamau and others 2010).
Table 2. Composition of the Obtained Fraction at Each Step
[Source: Eugenia Lucena et al. (2006)]
Before carrying out precipitation
Centrifugation 2 (supernatant)
Alpha-lactalbumin is well known for its functional property of providing health benefits due to its high quality and complete protein profile. For instance, α-La is abundant in cysteine amino acid which is the building block of antioxidant glutathione to provide immunity for the body. Another essential amino acid of tryptophan in α-La helps to regulate neurobehavioural effects in order to improve the sleeping quality and stimulate good mood. After all, α-La is a valuable food ingredient of nutraceutical function that provides both health and nutritional benefits as a mean to prevent physiological illness (Almécija and others 2007). In addition, the applications of α-La are broad, ranging from infant powder and athlete training formulation, nutrition bars, protein fortification in beverages to food supplement for nutritional and dietary purposes, just to name a few. It is noteworthy to appreciate the innovation of Maase and Johannes (2002) that employ α-La as a functional food ingredient; food additive; and food supplement to enhance its prebiotic effect in dairy product. α-La was demonstrated to enhance the beneficial intestinal microbial load so as to prevent gastroenteritis via encapsulating with appropriate carrier and diluents. Moreover, Tome (2009) patented the idea of using α-La to assist regulation of glucose level in blood through improvement in cellular absorption of glucose, in this way, prevent the occurrence of diabetes Type II. The methodology is to use α-La as a food additive to be incorporated into dairy-based of ready-to-eat food products such as yogurt and milk; and also developed as a tablets or gel capsules as dietary supplements to enhance insulin sensitivity. In addition, α-La promotes whippability in meringue-like desserts to give better sensory experience (Bhattacharjee and others 2006). Besides of playing important role in food industry, α-La assists family planning as a contraceptive agent that binds strongly to glycosylated receptors on surface of oocytes and spermatozoids (Bhattacharjee et al. 2006).
The precipitate obtained from the extraction and purification method is the α-La enriched fraction which is a perfect starting material to prepare infant formula owing to the low amount of β-Lg, thus improving the protein quality of the final product. In general, newly born infants need to be breast fed by the mother with colostrums in order to strengthen the immune system. Then, there is a demand to develop protein enriched infant formula in order to supplement the infant with human milk when breast feeding is sometimes impossible for the mother and the limited quantity of colostrums. The researchers took the advantage of the bovine α-La protein that is equally identical to the human α-La to be exploited in the infant formula manufacturing industry. However, researches showed that β-Lg in bovine milk triggered allergic reaction towards infant if present in infant formula (Eugenia Lucena et al. 2006). This explained the importance of extraction and purification method to isolate α-La individual proteins only so as to ensure β-Lg concentration in the final obtained fraction is at its minimal possible level. However, the drawback of retaining 0% of β-Lg in the bovine milk to produce infant formula is the alteration of nutritional and functional properties of the proteins present in the milk, for instance the poor dissolving ability of infant powder in water. Generally, the highly purified source of α-La with almost negligible amount of β-Lg is used as the food ingredient to manufacture infant formula is rich in essential amino acids is an excellent nutritional source for newly born infant. The protein content in the infant formula enriched with α-La is found to be higher than that of the regular infant formula in the market (Kuhlman 2003). Furthermore, α-La is an outstanding source to manufacture lactose-free infant powder to cater the infant with lactose intolerance.
The downside of using α-La as a food ingredient is evident particularly during the extraction and purification procedure upon knowing the fact that α-La is a protein that is easily influenced by the pH and temperature factors. At this point, there is a necessity to perform further researches to propose methods that could minimise the protein denaturation due to harsh operating conditions during extraction and purification prior utilising the isolated α-La as food ingredient. The uncertainty of extracting 100% pure α-La component from bovine milk still exists, not to mention the capability of the method to retain the extracted α-La in their native state, thus, technical not viable. Besides, the method is too expensive to carry out in commercial scale, in terms of high technology equipments and chemicals used and when considering the pricing of α-La protein related products in the current market, hence, financial unfeasible. The removal of lactose, BSA and Ig involved highly contaminated waste disposal which is difficult to be treated and will pose an environmental issue.
In conclusion, dairy-based food products served as an important source of dietary and nutrition to human, some of the dairy products even confer health benefits. In the long run, the dairy industry continuously develop new technologies to overcome the difficulties arise from the existing milk composition analysis and milk components separation techniques to deal with the changing needs of consumers. Therefore, an understanding of α-La components in bovine milk in terms of their chemical, physical and functional properties so as to extract them in their purest form is definitely crucial. This enables the consumers across various demographic group gain access to this valuable food ingredient through most of the dairy products range.