Mining Milk For Valuable Ingredients Biology Essay

Milk serves as a critical dietetic demand in supplying foods and peculiarly, protein to human for growing. Milk is a complex liquid constituent that is excreted from the mammary secretory organs of mammals such as human, cow, American bison, caprine animal, sheep and others. This essay discussed entirely on the bovine milk. Water is the primary components of milk which comprised about 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 ) . However, the variableness of these constituents in milk alterations across species, strains and between single mammals, phase of lactation, provender composing, clime and other factors. In fact, besides of these major constituents, the being of the milk ‘s minor components must non be neglected as they possessed valuable belongingss to be commercial utilised in nutrient industry, excessively. Horton ( 1995 ) brought up the question on the definition of ‘minor constituent ‘ which is neither based on the entire weight footing nor per centum of solids. It is, nevertheless, an single molecule or a series of similar compound that make up the major constituents. Table 1 shows the major constituents of milk and some of the common minor constituents.

Table 1. Milk Components [ Beginnings: Otter and Benjamin ( 2003 ) and Horton ( 1995 ) ]

Major Components

Minor Components

Milk sugar ( lactose )

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Milk fat

-Mono- , di- and triacylglycerols

-Sterols and steroid alcohol esters

-Unesterified fatty acids



Milk protein….Caseins




Milk protein….Whey



-Bovine serum albumen




Milk protein… . Non protein N ( NPN )


-Glyco- and macropeptides

Mineral salts

Calcium, chloride, Fe, Mg, Na, K and phosphate

The minor constituent of milk whey proteins, i.e. ?-lactalbumin ( ?-La ) will be discussed comprehensively in this essay. ?-La made up of 123 aminic acids with a concentration scope of 0.6-1.7g per liter of milk is one of the major whey proteins. The approximative sum of proteins present in whey comprised of 50 % ?-lactoglobulin ( ?-Lg ) , 25 % ?-La, and 25 % of other proteins such as bovine serum albumen and Igs ( Zadow and Benjamin 2003 ) . In comparing with casein, whey proteins are known as the soluble milk protein with ball-shaped construction and will stay in native signifier even at their isoelectric point and when subjected to high temperature. However, whey ( constituted 17-20 % w/w of entire protein in milk ) is ever regarded as the byproduct of casein and milk plasma that remains after the milk is curdled and strained during cheese doing which was non valued before for its important importance as nutrient ingredient, nutraceuticals and antimicrobic maps. Harmonizing to Zadow and Benjamin ( 2003 ) , before the whey proteins gain regard in dairy industry, they were frequently treated as waste merchandises to be used either as carnal provender or allow flow to the river watercourse as wastewaters which so imposed injury on the environment. Finally, nutrient scientists and engineers recognised a great potency of whey proteins and ?-lactalbumin for commercial use.

In fact, ?-lactalbumin in bovine milk is besides the primary protein in human milk ( Otter and Benjamin 2003 ) which contains considerable sum of indispensable amino acids tryptophan, cysteine, methionine, threonine and isoleucine, but present in a lower proportion than in human milk. Interestingly, ?-La is a Ca metalloprotein which is capable to be in two signifiers ; the native signifier ( ?-La-Ca2+ ) that bind strongly to calcium ion and the calcium-free signifier ( apo-?-La ) . This belongings stabilises the third ball-shaped construction of ?-L and hence, increase its heat stableness ( Otter and Benjamin 2003 ; Eugenia Lucena and others 2007 ) . ?-La besides consists of four disulphide bonds with an isoelectric point ( pi ) of 4.2 and the molecular weight is about 14.2kDa.

Since ?-Lg ( MW of ?-Lg = 18.0kDa, pi = 5.2 ) and ?-La have small difference between their molecular weight and isoelectric point, the extraction and purification method that separate ?-La from other whey proteins in bovine milk is instead disputing. The ideal ?-La extraction and purification method to be employed must foremost, achieve high pureness of ?-La and generate greater per centum of concluding recovery output of soluble ?-La ; secondly, free from contaminations such as ?-lactoglobulin, bovid serum albumen, Igs and other substances ; thirdly, retain the innate physical and functional belongingss of ?-La itself ; and fourthly, employ the techniques into commercial capacity for greater productiveness of ?-La as an utile nutrient ingredient. However, it is impractical to carry through the last end at the commercial graduated table due to the comparatively high cost and clip consuming processs in order to carry through the first three purposes stated above. Studies were done by several writers on pull outing and sublimating ?-La methods for both commercial and laboratory degrees as reviewed by Kamau and others ( 2010 ) . These include enzyme hydrolysis, selective precipitation, membrane separation ( ultralfitration or diafiltration ) and ion-exchange chromatography.

After measuring all the techniques, the most possible technique to be applied for commercial degree 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 get downing stuff of the extraction method in order to obtain the concluding ?-La enriched solution is whey protein dressed ore ( WPC ) , ideally with moderate degree of protein content on dry footing ( Eugenia Lucena et Al. 2007 ) , low ?-Lg concentration ( Eugenia Lucena and others 2006 ) and defatted WPC ( Bramaud and others 1997 ) . Previous work was done by Eugenia Lucena and others ( 2007 ) utilizing liquid solutions with changing concentrations of ?-La: liquid whey ( 0.7g/L ?-La ) , WPC 35 ( 5.6g/L ?-La ) , WPC 65 ( 12.0g/L ?-La ) and whey protein isolates with 15g/L ?-La. From the survey, they reported that the initial ?-La concentration will finally hold an impact on the ?-La percipitation output and pureness. Hence, WPC with 65 % protein content on dry footing ( WPC 65 ) with 12.0g/L ?-La concentration was selected as the perfect get downing stuff for the extraction precedure.

In pattern, WPC is isolated from whey utilizing either ultrafiltration or electrodialysis and lactose crystallision method ( Walstra et al. 1999 ) . These readying methods produce higher protein content and has lesser sum of fat and milk sugar. Alternatively, ultralfiltration besides served as the ?-La purification measure to divide whey based on size differences through membrane pore accommodation to bring forth the mark protein with minumum sum of bovine serum albumen ( BSA ) and immunoglobulins so as to heighten the ?-La/?-Lg ratio. Arabelle and others ( 2003 ) reported that the pureness of ?-La after ultrafiltration was carried out was 0.65 but gave dissatisfactory output of 0.15 due to low transmittal of ?-La. The principle elucidated by the research workers was the failure of membrane pore to know apart between ?-La and ?-Lg owing to the close propinquity of their molecular weights to be separate out expeditiously. Nonetheless, Millesime and others ( 1995 ; cited in Eugenia Lucena et Al. 2007 ) noticed the potency of doing use the electrostatic interaction between proteins and the filter membrane surface by changing the pH and ionic strength. Lucas and the squad ( 1998 ) besides took advantage of this logic, they chemically modified the filter membrane by surfacing a bed of polyethyleneimine which is positively charged. Therefore, ?-La was selectively separated out at pH 7 at immediate ionic strength 0.2 mol l-1 whereby the ?-Lg was strongly attracted to that peculiar chemically modified inorganic membrane. The transmittal of ?-La reported by them was 10 % while ?-Lg rentention at the membrane was about 100 % .

Ultrafiltration entirely is non perfect plenty to obtain the preferable ?-La pureness and recovery output. The get downing stuff, WPC 65, is fed into the extractor and maintained at 4oC before transporting out the precipitation. The optimal operating conditions for precipitation must be selected carefully based on the old plants by several writers. Since the stabilization and destabilization of ?-La and ?-Lg is controlled by Ca ions concentration, there is a demand to add calcium ions complexing agents to WPC 65 in order to cut down the concentration of free Ca, meanwhile, promote the formation of Ca free ?-La, i.e. apo-?-La, which is hydrophobic in nature for ?-La to precipitate at its isoelectric point ( Bramaud et al. 1997 ) . Apparently, ?-Lg showed an increase in stableness when the Ca ions concentration is low ( Eugenia Lucena et Al. 2006 ) . Example of Ca ions complexing agents are lactic acid, citric acid and hydrochloric acid, merely to call a few. Eugenia Lucena et Al. ( 2007 ) made a comparing between these three different acids to look into which acid gave a promising pureness value from the obtained precipitated fractions. Both the organic acids ( citric acid and lactic acid ) have the capableness to organize composites with Ca ions. Although add-on of hydrochloric acid will do protein precipitation, due to its strong sourness, all ?-La were denatured irreversibly ; losing their secondary and third construction. Furthermore, when they carried out the experiment under the pH scope between 3.5 and 4.5, which is near to the ?-La isoelectric point ( pi ) , the precipitation output showed important quantitative sum for organic acids than hydrochloric acid. At pH highly higher or lower than the pi, the precipitation output showed a drastic decline. Besides, since the isoelectric point of ?-Lg is at pH 5.2 which is non under the suggested optimal pH scope, ?-Lg will non precipitate and therefore stay in the soluble stage. For the precipitation runing temperature wise, irreversible thermic denaturation is an issue to avoid as this masked the true consequence of precipitation output. Lactic acid precipitated all the proteins ( include BSA and Ig ) , excepting ?-Lg at mild temperature, peculiarly at temperature higher than 50oC, in which the highest ?-La precipitation output was achieved ( Eugenia Lucena et Al. 2006 ) . This can be deduced that the use of lactic acid as complexant is a temperature dependent measure. Conversely, the consequence of Ca ions complexation owing to citric acerb add-on was at maximal at lower temperature scope. After all, the plants of Eugenia Lucena et Al. ( 2006 and 2007 ) concluded that lactic acid is a better complexant than citric acid ; hence, the optimal operating conditions for the selective precipitation for ?-La are utilizing 0.75M lactic acid at temperature 55oC, pH 4.0 and modulate the Ca concentration of lactic acid/Ca2+ grinder ratio higher than 9.

The terminal merchandise of precipitation is the precipitate which contained ?-La, BSA and Ig while ?-Lg proteins remain in solution. The principle that explained presence of ?-Lg proteins in solution is because they are able to accomplish stabilization upon low Ca ions concentration. However, the precipitate might be tainted with a little sum of ?-Lg, hence, the precipitate is centrifuged as a safeguard measure and ?-Lg is separated out as the supernatant ( Eugenia Lucena et Al. 2006 ) . Supernatant will be tested for ?-Lg pureness before discarded. Eugenia Lucena et Al. ( 2006 ) found out that the supernatant obtained gave 85 % of ?-Lg pureness owing to the presence of other constituents such as lactose, mineral and caseinomacropeptide ( CMP ) . Further safeguard measure in order to take ?-Lg fractions finally from the precipitate is carried out through lavation, at least twice, utilizing acidulated fresh H2O. Throughout the washing stairss, the pH maintained at 4.0, which is about at the isoelectric point of ?-La, and performed under the temperature between 15 to 60oC. The results of the rinsing stairss caused partial solubilisation of ?-La and caused decrease of the recovery with respect to ?-La initial concentration in WPC 65, but BSA and Igs will still stay in the precipitate as contaminations. In fact, ?-Lg showed a greater betterment in recovery up to 100 % at temperature 55oC ( Eugenia Lucena et Al. 2006 ) . Bramaud et Al. ( 1997 ) suggested that the solubilisation of ?-La during the washing measure can be avoided by utilizing 0.1M sodium chloride at pH 3.9 to rinse the precipitate, but it will do irreversible ?-La precipitation.

The ultimate end of the ?-La extraction is to bring forth a nutrient ingredient with functional belongings and exists in native signifier. This can be exemplified by the ability of ?-La to solubilise and non subjected to any denaturation during the extraction and purification processs such as thermic denaturation, chemicals taint and in peculiarly, the add-on of acids during the selective precipitation measure. Since the precipitate experienced a alteration in pH, Ca ion concentration and temperature fluctuation, the solubilisation of precipitate can be justified through the ability to retrieve the two signifiers of ?-La: apo-?-La and ?-La-Ca2+ ( native signifier ) under the status of pH 7.5 and temperature 45oC ( Eugenia Lucena et Al. 2006 ) . Bramaud et Al. ( 1997 ) had the same determination that the ?-La-contained precipitate that undergo rinsing measure via Na chloride solution appeared to be milky and turbid will go a clear solution at pH 7.5 at 45oC, in which all the proteins are wholly solubilised. At pH 7.5, the Igs that co-precipitate with ?-La remain indissoluble and no hint of ?-Lg detected in the soluble stage, and therefore, the ?-La pureness gives a promising consequence ( Bramaud et al. 1997 ) . The ability to retrieve the two signifiers of ?-La relied upon the nature of ions in the solubilisation dissolver, either Na+ or Ca2+ ( Arabelle and others 2003 ) . Ca2+ ( utilizing Ca chloride ) was reported by Bramaud et Al. ( 1997 ) was a better solubilisation dissolver since the lone leftovers contained inside the concluding obtained fraction were ?-lactalbumin and bovid serum albumen proteins. Further separation measure of either ultrafiltration or chromatography could be carried out to take BSA for the intent to accomplish higher ?-La pureness. Overall, the solubilisation measure verified that the initial precipitation measure utilizing the Ca ions complexing agent of 0.75M lactic acid at pH 4.0 is a reversible reaction.

The flow chart of the proposed technique to pull out ?-La from WPC 65 in order to obtain the ?-La enriched solution is illustrated in Figure 1.

Figure 1. Flow Chart of the ?-La Extraction

[ Beginnings: Eugenia Lucena et Al. ( 2006 ) and Bramaud et Al. ( 1997 ) ]

Eugenia Lucena et Al. ( 2006 ) carried out the high public presentation liquid chromatography ( HPLC ) and gel cataphoresis to analyze the composing of the fractions obtained at the terminal of each measure and the consequence is presented in Table 2. The concluding recovery of the soluble ?-La in its native signifier from their work was 85 % and a pureness of 75 % with the presence of ?-Lg around 5 % . The ?-La/?-Lg concentration ratio was about 14 and about 99 % of the ?-Lg was removed from the initial natural stuff, WPC 65. The cardinal success of this extraction and purification technique is by and large relied on the initial protein concentration of the get downing natural stuff, precipitation pH, the operating temperature and the figure of precipitate lavation for the purpose to accomplish high ?-La pureness and high ?-La/?-Lg concentration ratio ( Kamau and others 2010 ) .

Table 2. Composition of the Obtained Fraction at Each Measure

[ Beginning: Eugenia Lucena et Al. ( 2006 ) ]


Composition ( % )

?-La pureness

?-La recovery

?-Lg pureness

?-La/?-Lg ratio

Before transporting out precipitation



Centrifugation 2




Centrifugation 2 ( supernatant )






Centrifugation 3

( supernatant )


?-La solubilisation




Alpha-lactalbumin is good known for its functional belongings of supplying wellness benefits due to its high quality and complete protein profile. For case, ?-La is abundant in cysteine amino acid which is the edifice block of antioxidant glutathione to supply unsusceptibility for the organic structure. Another indispensable amino acid of tryptophan in ?-La aid to modulate neurobehavioural effects in order to better the sleeping quality and excite good temper. After all, ?-La is a valuable nutrient ingredient of nutraceutical map that provides both wellness and nutritionary benefits as a mean to forestall physiological unwellness ( Alm & A ; eacute ; cija and others 2007 ) . In add-on, the applications of ?-La are wide, runing from infant pulverization and athlete preparation preparation, nutrition bars, protein munition in drinks to nutrient addendum for nutritionary and dietetic intents, merely to call a few. It is notable to appreciate the invention of Maase and Johannes ( 2002 ) that employ ?-La as a functional nutrient ingredient ; nutrient additive ; and nutrient addendum to heighten its prebiotic consequence in dairy merchandise. ?-La was demonstrated to heighten the good enteric microbic burden so as to forestall stomach flu via encapsulating with appropriate bearer and dilutants. Furthermore, Tome ( 2009 ) patented the thought of utilizing ?-La to help ordinance of glucose degree in blood through betterment in cellular soaking up of glucose, in this manner, prevent the happening of diabetes Type II. The methodological analysis is to utilize ?-La as a nutrient additive to be incorporated into dairy-based of ready-to-eat nutrient merchandises such as yoghurt and milk ; and besides developed as a tablets or gel capsules as dietetic addendums to heighten insulin sensitiveness. In add-on, ?-La promotes whippability in meringue-like sweets to give better sensory experience ( Bhattacharjee and others 2006 ) . Besides of playing of import function in nutrient industry, ?-La aids household planning as a prophylactic agent that binds strongly to glycosylated receptors on surface of oocytes and antherozoids ( Bhattacharjee et al. 2006 ) .

The precipitate obtained from the extraction and purification method is the ?-La enriched fraction which is a perfect get downing stuff to fix infant expression owing to the low sum of ?-Lg, therefore bettering the protein quality of the concluding merchandise. In general, freshly born babies need to be breast fed by the female parent with foremilks in order to beef up the immune system. Then, there is a demand to develop protein enriched infant expression in order to supplement the baby with human milk when chest eating is sometimes impossible for the female parent and the limited measure of foremilks. The research workers took the advantage of the bovid ?-La protein that is every bit indistinguishable to the human ?-La to be exploited in the infant expression fabrication industry. However, researches showed that ?-Lg in bovine milk triggered allergic reaction towards infant if present in infant expression ( Eugenia Lucena et Al. 2006 ) . This explained the importance of extraction and purification method to insulate ?-La single proteins merely so as to guarantee ?-Lg concentration in the concluding obtained fraction is at its minimum possible degree. However, the drawback of retaining 0 % of ?-Lg in the bovine milk to bring forth infant expression is the change of nutritionary and functional belongingss of the proteins present in the milk, for case the hapless fade outing ability of infant pulverization in H2O. By and large, the extremely purified beginning of ?-La with about negligible sum of ?-Lg is used as the nutrient ingredient to fabricate infant expression is rich in indispensable amino acids is an first-class nutritionary beginning for freshly born baby. The protein content in the infant expression enriched with ?-La is found to be higher than that of the regular baby expression in the market ( Kuhlman 2003 ) . Furthermore, ?-La is an outstanding beginning to fabricate lactose-free baby pulverization to provide the baby with lactose intolerance.

The downside of utilizing ?-La as a nutrient ingredient is apparent peculiarly during the extraction and purification process upon cognizing the fact that ?-La is a protein that is easy influenced by the pH and temperature factors. At this point, there is a necessity to execute farther researches to suggest methods that could understate the protein denaturation due to harsh operating conditions during extraction and purification prior using the stray ?-La as nutrient ingredient. The uncertainness of pull outing 100 % pure ?-La constituent from bovine milk still exists, non to advert the capableness of the method to retain the extracted ?-La in their native province, therefore, proficient non feasible. Besides, the method is excessively expensive to transport out in commercial graduated table, in footings of high engineering equipments and chemicals used and when sing the pricing of ?-La protein related merchandises in the current market, therefore, fiscal impracticable. The remotion of lactose, BSA and Ig involved extremely contaminated waste disposal which is hard to be treated and will present an environmental issue.

In decision, dairy-based nutrient merchandises served as an of import beginning of dietetic and nutrition to human, some of the dairy merchandises even confer wellness benefits. In the long tally, the dairy industry continuously develop new engineerings to get the better of the troubles arise from the bing milk composing analysis and milk constituents separation techniques to cover with the altering demands of consumers. Therefore, an apprehension of ?-La constituents in bovid milk in footings of their chemical, physical and functional belongingss so as to pull out them in their purest signifier is decidedly important. This enables the consumers across assorted demographic group addition entree to this valuable nutrient ingredient through most of the dairy merchandises range.