COMPREHENSIVE
`REV EWS
`
`IN FOOD SCIENCE mo FOOD SAFETY
`
`Alpha-
`Lactalbumin: Its
`
`Production
`
`Technologies
`and Bioactive
`
`Pepfides
`
`Samuel Mburu Kamau, Seronei Chelulei Che-ison, Wei Chen,
`Xiao-Ming Liu, and Rang-Rong Lu
`
`ABSTRACT: Alpha-lactalbumin {or-La}, a globular protein found in all mammalian milk, has been used as an in-
`gredient in infant formulas. The protein can be isolated from milk using chromatographytgel filtration, membrane
`separation, enzyme hydrolysis, and precipitationiaggregation technologies. or-La is appreciated as a source of pep-
`tides with antitumor and apoptosis, antiulcerative, immune modulating, antimicrobial, antiviral, antihypertensive,
`opioid, mineral binding, and antioxldative bioactivities, which may be utilized in the production of functional foods.
`Nanotubes formed by the protein could find applications in foods and pharmaceuticals, and understanding its amy~
`loid fibrils is important in drawing strategies for controlling amyloidal diseases. Bioactive peptides in or«I.a are re
`leased during the fermentation or ripening of dairy products by starter and nonstarter microorganisms and during
`digestion by gastric enzymes. Bioactive peptides are also produced by deliberate hydrolysis of ot—I.a using animal,
`microbial, or plant proteases. The occurrence, structure, and production technologies of ot~La and its bioactive pep-
`tides are reviewed.
`
`Introduction: Occurrence and Structural Properties
`of as-La
`
`A|pha—lactalbumin to—Lal is one of the globular proteins found
`in bovine and human milk. Bovine u—La is quantitatively the
`2nd most important protein in whey; it makes up approximately
`20% to 25% of the whey proteins, while human or-La is the
`dominant whey protein and has a 74% conserved amino acid
`sequence homology with bovine r:r—La. Bovine rr—La occurs as an
`acidic, single-chain Ca“ binding protein made up of 123 amino
`acids including essential and branched—chain amino acids. It has
`NI |3—terminal glulamic acid and COO] |—termina|
`leucine, has
`no free thiols and has 4 disuliide bonds. rt-La is relatively small
`with a molecular mass corresponding to 14070 Da in human
`milk and 14178 Da in bovine milk and its content in bovine
`
`whey is approximately 1.2 g/L. The protein is relatively high
`
`MS 20090?28 Submitted ?x3o-2009, Accepted I0;’1‘0x2009. Authors Kamau.
`Chen, Liu. and Lu are with State Key Lab. of Food Science and Technology.
`School of Food Science and Technology. Jiangrtan Univ.. 1800 Lihu Ave., Wuxi.
`Jiangsu 214122, P. Ft. China. Author Kamau is also with Dairy Training Inst..
`P.O. Box 449, Naivasha 201 1?. Kenya. Author Cheison is with ZiEL—Junior Fle-
`search Group: Bioactive Peptides and Protein Technology. Technische Univer-
`sitat lvlunchen, Weihenstephaner Berg 1. D-85354 Freising, Germany. Author
`Cheison is also with School of Public Health and Community Development.
`Maseno Univ_, Private Bag, Kisumu. Kenya. Direct inquiries to author Lu {E-
`mam|un@mangnaneducnL
`
`lysine, and cys-
`tryptophan,
`in essential amino acids, namely,
`teine, which amounts to 4% to 5%, 11%, and 6% moles of to-
`tal amino acids in tr-L3, respectively t/\ppe| and others 1994),
`compared to other milk proteins. However, these amino acids
`can react with oxitli/_ing lipids in Maillard reactions leading to
`their losses.
`lts isoelectric point
`is 4.2 to 4.6 and it
`is highly
`soluble in water and chloride salt solutions, a property that has
`been exploited in its selective pre aration (Tolkach and others
`2005). In addition, iv-La is relative y heat-stable when bound to
`calcium compared with other whey proteins and may be gly-
`cosylated with mannose {Man}, galactose (Gal), fucose (Fucl,
`glucose (Clc), and lactose {Lac} {Barman 1970). In bovine or-
`La, approximately 10% is lactosylatetl, whereas the human milk
`protein is unmodified (Lonnerdal and Lien 2003). Lactosylated
`ar—La may prevent infection b
`inhibiting binding of pathogens
`lo the intestinal epithelial cel luminal surface due to the ab-
`sence of lactosamine, which is required for their adherence. it
`has also been reported to bind to E. coli heat—labile enterotoxin
`at either asparagine amino acid position at 45 or 74 (Barman
`19701.
`
`u—La plays a fundamental physiological role during milk lac-
`tose synthesis. u—La forms the regulatory subunit of lactose syn-
`lhase complex {EC 2.4.1.22). When (Y-La binds to galaclosyl
`translerase (GT),
`it promotes the conversion of galactose into
`N—acetylglucosamine and results in the efficient synthesis of lac-
`tose lrom uridine diphosphate tUDPl galactose and glucose (Xue
`
`2010 Institute of Food Technologists‘-T57’
`
`Vol. 9. 2010 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY
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`197
`
`Amgen Exhibit 2009
`Apotex Inc. et al. V. Amgen Inc. et al., IPRZO16-01542
`Page 1
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`Comprehensive Reviews in Food Science and Food Safety
`
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`
`Figure 1 — Human or-La. The oz-helical domain contains the
`4 oz-helices and 2 disulfide bonds (f6-120) and (f28-111).
`The )3-sheet domain contains 2 short )3-strands and sev-
`eral loop structures and disulfide bond (f61-77). A single
`calcium ion (black ball) binds to the calcium-binding loop,
`comprised of residues f78-89. An inter-domain disulfide
`bond exists at (f73-91), with the cysteines in the oz-helical
`domain replaced by alanines (Wu and others 1996).
`
`and Others 200l l‘
`
`Galactosyl — transferase
`oz—La
`metal ions
`
`UDP — D — ga/actose + D — glucose4 D
`
`— lactose + UDP
`
`at-La contains 2 major domains: the oz-domain, which contributes
`4 oz-helices, and the )3-domain, which contains a )3-sheet and loop
`regions (Figure 1) (Wu and others 1996). A noteworthy feature of
`oz-La structure is the acidic conformational transition occurring
`between pH 3 to 4, which is accounted for,
`in part, by a com-
`petition between calcium ions and protons for the carboxyl side
`chains comprising the calcium binding site (Permyakov and oth-
`ers 1985). The folding of oz-La in acid pH is considered as a
`prototype of the molten globule (MG) state. MG state is defined
`as a collapsed state of the whole polypeptide retaining a substan-
`tial amount of secondary structure, but lacking the fixed packed
`interactions of the native protein. The MC of oz-La attains an even
`more flexible conformational state during the early phases of the
`aggregation process at acidic pH, as deduced from the enhance-
`ment of its susceptibility to proteolysis by pepsin (Laureto and
`others 2005).
`Bovine oz-La MGs have been produced through hydrolysis with
`porcine pepsin (EC 4.3.3.1) and proteinase K from Tritirachium
`album (EC 34.21.64) at temperatures of 20 to 22 °C and pH of
`2 and 8.3, respectively, under depleted protein-bound calcium
`ions (De-Laureto and others 2002). The oz-La MG state can also be
`generated upon exposing the protein to thermal stress or mild de-
`naturants, removal of bound calcium by use of calcium chelators
`as well as reduction of disulfide bonds (Chowdrury and Raleigh
`2005). Human oz-La forms one ofthe most stable molten globules,
`while bovine oz-La forms a less stable molten globule. The human
`oz-La is a 2-domain Ca“-binding protein that partially unfolds
`at low pH to form a molten globule (Ramboarina and Redfield
`2008).
`An interesting property of at-La is its ability to interact with
`hydrophobic substances such as hydrophobic peptides, model
`lipid membranes, hydrophobic chromatographic supports, and
`fatty acids (Barbana and others 2006). These properties are vital
`in purification processes (Conrado and others 2005; Tolkach and
`
`Kulozik 2005) and as an important ingredient in certain foods
`where it imparts functional properties.
`Purified oz-La is most readily used in infant formula manufactur-
`ing, as it has the most structurally similar protein profile compared
`to breast milk (Marshall 2004; Kuhlman and others 2005). The ad-
`dition of bovine oz-La to infantformula has been proposed to mod-
`ify the plasma amino acid pattern ofthe recipient infant and lead
`to growth patterns more similar to those of breastfed infants com-
`pared with standard formula-fed infants (Sandstrom and others
`2008). The functionality of at-La is probably owed to being a rich
`source of tryptophan, which has been suggested to be involved
`in satiety via brain serotonin. Serotonin is synthesized from tryp-
`tophan and is an important regulator of appetite, macronutrient
`preference, and mood (Beulens and others 2004). In a study on
`whether consumption of a diet enriched with oz-La may increase
`the plasma tryptophan to large neutral amino acids ratio (Trp:
`LNAA ratio), and reduce depressive mood and cortisol concen-
`trations in stress-vulnerable subjects under acute stress, returned
`positive results (Markus and others 2000) signifying the diet in-
`duced increase in tryptophan availability for serotonin synthesis.
`A separate double-blind, placebo-controlled study on evening
`consumption of the tryptophan-rich oz-La diet has shown an in-
`crease of plasma tryptophan availability for uptake into the brain
`and sustained alertness early in the morning after an overnight
`sleep (Markus and others 2005), most likely because of improved
`sleep. Experimental data with rats has also shown that ingestion
`of diets enriched in oz-La as a source of proteins should have ben-
`eficial effects in coping with stress and in anxiety at short term
`(Orosco and others 2004). A suitable source of oz-La is a com-
`mercial product, Alpha-lactalbumin, containing 90% oz-La on a
`protein basis that has lately been produced by Davisco Foods Intl.
`Inc. The product has 4.8 g tryptophan per 100 g of protein. Be-
`sides the numerous benefits of intact oz-La,
`it can also be used to
`produce bioactive peptides with functional significance (Otte and
`others 2007b), that include peptides having improved Trp: LNAA
`ratios.
`
`in food, a suit-
`To determine the quantity of oz-La present
`able technique has been developed. The technique involves
`an automated, label-free biosensor-based immunoassay for oz-La
`in bovine milk utilizing surface plasmon resonance (SPR) de-
`tection (Indyk 2009). The label-free, real-time, and automated
`immunoassay is rapid, sensitive, precise, and accurate, and it
`provides analytical
`information comparable with that from al-
`ternative methods available. at-La content is estimated from the
`
`specific interaction with an antibody immobilized on the sensor
`surface in a direct-binding assay format. The direct biosensor im-
`munoassay has been utilized in the quantification ofoz-La in milk,
`colostrum, WPC, and infant formula.
`The emergence of technologies enabling preparation ofoc-La in
`large quantities and its continued significance in infant formula-
`tions and the bioactive peptides industry justifies a review of the
`protein in the current form. Currently, in spite of the great strides
`made in the manufacture and hydrolysis of pure oz-La, there is
`no comprehensive review on the knowledge base available. This
`review addresses the processing methods available to produce
`kilogram amounts, as well as peptides identified so far derived
`from it that are bioactive.
`
`Isolation of oz-La
`
`oz-Lactalbumin’s primary source is whey, a by-product of
`cheese making where it
`is found with other components:
`)3-
`lactoglobulins ()3-Lg),
`lactose, minerals (mainly calcium, phos-
`phorus, magnesium, and zinc), vitamins, and traces of milk
`fat. There are numerous technologies described to obtain pure
`
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`AIphci—IcictciIbumin—biocictive peptides. .
`
`.
`
`Figure 2—A summary of the available
`methods for the isolation and
`manufacture of or-La from milk.
`
`Bordin and others200l
`
`Whey protein
`
`Diafiltration
`
`Konrad and Kleinschmidt 2008
`
`Chromatography
`
`I
`
`Gel filtration
`
`Precipitation
`/ aggregation
`
`I
`
`Bordin and
`others 2001
`
`Centrifugation
`
`Cheang and
`Zydney 2003
`
`V
`Membrane filtration
`
`V
`
`Tryptic hydrolysis
`
`Neyestani and
`others 2003
`
`Bramaud and
`others l997
`
`I
`(X-
`Lactalbumin
`
`Konrad and Kleinschmidt 2008
`
`or-La fractions from whey protein concentrate (WPC), whey
`protein isolate (WPI), whey hydrolysates (WH), liquid whey, or
`even milk (Figure 2). The choice of production technology to use
`is determined by the quantity of or-La present in the raw mate-
`rial and the required purity levels, as well as production scale;
`and in most cases a combination of techniques is necessary. An-
`other critical consideration is the subsequent processes to be
`undertaken. Chromatographic methods, for instance, are efficient
`in obtaining or-La samples that are pure and with which laboratory
`analysis and/or further isolations are conducted. However, for
`products processing, there is a need to produce large quantities
`of or-La, which inevitably has accompanying by products that re-
`quire further attention. Precipitation/aggregation when combined
`with membrane separation can handle production involving large
`volumes of or-La and )3-Lg as a by-product. Choosing to precipi-
`tate or-La in this process is advantageous in that the proteins or-La
`and )3-Lg retain their properties. If the resulting )3-Lg is to be used
`for the production of hydrolysates, then combining the hydrolysis
`process and the membrane separation would be the best provided
`the enzyme fully hydrolyzes the )3-Lg and does not hydrolyze or-
`La.
`It should be noted that to obtain a desired purity and yield
`one must optimize production conditions for each method used.
`A method for the production of or-La that has more than 85%
`protein purity and is 99% lactose and salt free has been patented
`(Heine and others 1992). The resulting or-La is soluble and almost
`tasteless.
`
`Chromatography and Gel Filtration
`Chromatographic techniques have been used for partitioning
`protein mixtures depending on their affinity to either mobile or
`stationary phases. Gel filtration (a form of chromatography), sep-
`arates proteins based on size as they pass through a gel medium
`in a packed column. Proteins separated by gel filtration do not
`bind to the medium and therefore a buffer does not directly af-
`fect resolution or their biological activity. Chromatography using
`a gel filtration column has been used to isolate or-La from other
`milk proteins (lvlanji and others 1985) and is often combined
`with other separation principles such as differences in acidity,
`basicity, charge, hydrophobic interaction, metal chelating, and
`adsorption affinities. Preparation steps are necessary to extract
`protein fractions from other milk constituents and may involve
`several steps and methods depending on the nature of the raw
`material.
`In most cases whey proteins are first separated from
`casein fractions before being subjected to chromatographic iso-
`lations. Ahmed and others (1998) described a chromatographic
`method where at least 5 different proteins including or-La were
`separated.
`The differences in physicochemical properties of or-La from
`other proteins are utilized in designing fractionation techniques
`or combinations that result in a highly pure product. Neyestani
`and others (2003) separated or-La from bovine serum albumin
`using Sephadex G-50 gel filtration. The 2 proteins or-La and
`bovine serum albumin had co-eluted together in anion-exchange
`
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`chromatography previously used to separate )3-Lg. The method
`was reproducible and showed high purity and well-preserved
`antigenicity of oz-La.
`oz-La has also been isolated from rennet whey using 2 ion-
`exchange chromatographic steps. The 1 st step employed a strong
`cationic exchanger, sulfopropyl-Toyopearl SPP-TP column, fol-
`lowed by elution of lactoferrin and lactoperoxidase. The 2nd
`chromatography step used a strong anion exchanger, quater-
`nary aminoethyl-Toyopearl QAE-TP, and oz-La was separated at
`a NaCl concentration lower than 0.13 M at pH 8.5, while B-
`lactoglobulins were separated at higher NaCl concentration and
`a pH of 6.8 (Ye and others 2000).
`A fast protein liquid chromatography system was similarly used
`to fractionate the protein from sweet and acid wheys that were
`previously adjusted to pH 6.7 (ivianji and others 1985), although
`electrophoresis of the oz-La showed some contamination (pos-
`sibly by immunoglobulins). Results reported by the researchers
`were qualitative only, but reproducibility of peak areas indicated
`that quantitative analysis should be possible (ivianji and others
`1985).
`Expanded-bed chromatography based on Ca“-dependent hy-
`drophobic interactions is another technique that has been used in
`the purification of oz-La from cow and goat milk samples (Noppe
`and others 1999). A procedure for the concentration of oz-La
`from cow milk whey using a high-density, hydrophobic resin
`(Streamline® Phenyl) and an expanded-bed column was even-
`tually developed. The procedure was fast and efficient for the
`purification ofoz-La achieving a purity of 79% after 1 cycle ofthe
`adsorption/elution protocol (Conrado and others 2005). The fun-
`damental aspect of this approach relies on the binding capacity
`ofoz-La to Ca“ ions. oz-La without Ca“ has hydrophobic charac-
`teristics and undergoes a significant conformational change with
`Ca“ and other metal
`ions making it more hydrophilic. Using
`Tris-EDTA buffer in the adsorption step the Ca“ ions are re-
`moved, making the protein more hydrophobic and increasing its
`binding to hydrophobic adsorbents. The elution step using Ca“
`ions permits recovery of oz-La because of the reversible change to
`hydrophilic character. Expanded-bed chromatography offers the
`advantage of reducing the number of steps in this application.
`Separation and quantification of oz-La along other proteins
`without the need to isolate whey protein from caseins has been
`demonstrated as possible by chromatographic techniques (Bordin
`and others 2001). The method used involves ion-pair reversed-
`phase high-performance liquid chromatography (HPLC) with
`photodiode array detection and a C4 column. Identification of
`the protein was by calculation of the peak area ratio (area at 214
`nm/area at 280 nm, A214/A230) generated by the aromatic amino
`acids phenylalanine, tyrosine, and tryptophan. The proportion of
`total aromatic amino acid content allows a rather good distinc-
`tion from other proteins (Bordin and others 2001). Defatting of
`sample is, however, a necessary treatment to avoid saturation of
`the column and can be achieved through centrifugation at 7500
`x g for 10 min at 4 °C (Noppe and others 1999).
`Chromatographic methods are usually expensive and therefore
`restricted to laboratory scale for the production of pure isolates.
`No chromatographic techniques provide 100% yield of active
`material, and overall yield depends on the number of steps in
`the purification protocol. By optimizing each step and combin-
`ing several techniques, one may improve the purity and deliver
`enough quantity of oz-La without compromising its biological
`quality.
`
`Membrane Separation
`Membrane separation techniques are attractive for oz-La isola-
`tion because of their relatively easy scale-up and low processing
`
`costs in comparison to chromatographic techniques. Bottomley
`(1991) described a 2-stage membrane process for obtaining con-
`centrates enriched in oz-La. The separation principle is differences
`in molecular weight of proteins and a likely challenge is lack of
`adequate selectivity for the separation of )3-Lg and oz-La due to
`the very similar molecular weight of these 2 proteins (Cheang and
`Zydney 2003; Muller and others 2003). Efficient membrane sep-
`aration should give low content of contaminants (bovine serum
`albumin,
`immunoglobulins) in or-La and an enhanced oz-La/fi-
`Lg ratio. Ultrafiltration (UF) membranes have been used for the
`separation and concentration of oz-La from WPC with both an
`enhanced purity of 0.35 from initial 0.25 and a satisfactory yield
`of 90% recovery in the permeate (Muller and others 1999). The
`membranes used had 3 channels, were 1.20 m long, 3.6 mm of
`inner hydraulic diameter, and had a filtering area of 0.045 m2.
`The support was oz-alumina with a filtering layer of zirconium
`oxide and had a molecular weight cut-off of 300000 Da corre-
`sponding to a mean pore diameter of about 28.4 nm (Poiseuille’s
`law). The pH and ionic strength of WPC were adjusted to 7 and
`0.2 M NaCl, before separation. UF was performed at 50 °C, at
`tangential flow velocity of 7 m/s and a transmembrane pressure
`of 1 bar. The ceramic membranes used were preferred owing
`to their better resistance against cleaning and disinfection com-
`pared to polymer UF membranes. The purity and yield of oz-La
`were directly related to the initial purity of the feed and transmis-
`sion ratio TR (transmission of or-La/transmission of )3-Lg) of the
`membranes.
`
`To increase purity, a preliminary concentration step of oz-La or
`removal of impurities (bovine serum albumin, immunoglobulins)
`is necessary. This can be achieved through the use of successive
`UF stages, where the 1st filtration is a concentration step. A di-
`afiltration (DF) process can further increase purification of oz-La
`in the permeate. DF has recently been described as part of an
`important method for separating mixtures employing membrane
`cascades (Lightfoot 2006) that have been proposed in separat-
`ing oz-La from protein mixtures. The membranes are combined to
`carry out DF and UF operations in such a manner as to approach
`and /or achieve an ideal cascade. The permeate is further sepa-
`rated by UF to separate the solvent. The properties of solvent used
`(which is usually water) may be manipulated to determine which
`proteins dissolve; at low ionic strength the solubility of )3-Lg is
`limited. Alternatively, precipitation methods to remove impurities
`may be combined with UF.
`UF performance depends on the operation mode adopted.
`These modes include concentration and DF or a combina-
`tion thereof and can be continuous or discontinuous. With the
`concentration modes (continuous concentration CC, discontinu-
`ous concentration DC) oz-La permeate concentration (purity) in-
`creases with time, being faster with CC operation mode compared
`to DC, which can be attributed to concomitant faster resultant
`feed concentration. The oz-La purity of the permeate ranged from
`0.40 to 0.45 with both modes from the initial purity of 0.25. On
`the other hand, the yield of oz-La recovered was higher for the
`CC mode, at 45% against 30% for the DC. In continuous diafil-
`tration CD, oz-La permeate concentration (purity) decreased with
`time from 0.58 to 0.42 due to a decreased oz-La concentration
`in the retentate from 27.6 down to 15.4 g/L, but it gives better
`yield. Purity and yield are antagonistic whatever the operation
`mode: an increase in yield during filtration is accompanied by a
`decrease in purity in the permeate. The most appropriate single
`mode for recovery of oz-La in the permeate is CC (better yield
`than DC and better purity than CD). However, Continuous con-
`centration of up to 11-15 volume reduction ratios is required to
`obtain a fraction with both an enhanced purity of approximately
`0.90 and a satisfactory yield of oz-La in the permeate (Muller and
`others 1999).
`
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`AIphci—IcictciIbumin—biocictive peptides. .
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`
`There have been attempts to increase the purity and yield of
`or-La by increasing TR of the protein by change of membranes,
`transmembrane pressure, and protein concentration, but without
`significant improvement as TR remained low (TR < 0.2) (Muller
`and others 2003). Fortunately, modification of physicochemical
`conditions of membranes can improve their selectivity and hence
`TR. Proper choice of buffer conditions, ultrafiltration membrane,
`and filtration velocity can maximize the overall selectivity of
`the membrane process (Cheang and Zydney 2003). Positively
`charged membranes, obtained by chemical modification of inor-
`ganic membranes with polyethyleneimine coating, appears to be
`an efficient route for improving membrane selectivity owing to
`the strong interactions between the positive charges and )3-Lg. At
`low ionic strength (I < 0.02 mol/L), the transmission of )3-Lg was
`reduced to about 1%, while that of or-La was close to 1 0% (Lucas
`and others 1998), thus allowing effective fractionation.
`The main drawback of membrane separation is decline in TR
`during UF processes as a consequence of fouling, concentra-
`tion polarization, protein adsorption, and protein-protein inter-
`actions. This could be reduced through high cross-flow velocity to
`remove foulants from membrane surfaces or back-pulsing, which
`is an in-place method for cleaning the membrane by forcing per-
`meate back through the membrane to the feed side. This dislodges
`deposited foulants when the transmembrane pressure is reversed
`and the foulants are then carried out of the membrane module
`
`by the tangential flow of retentate. Occurrence of irreversible,
`noncleanable fouling decreases membrane performance and de-
`termines membrane lifetime.
`
`Enzyme Hydrolysis
`Enzymatic hydrolysis combined with membrane filtration can
`be used for isolation and purification of milk proteins. Caseins are
`highly digestible by proteases compared to whey protein (Guo
`and others 1995) and may be selectively digested by proteases
`converting them to low-molecular-weight fractions that can be
`sieved out through membrane filters. However, other convenient
`methods to fractionate casein from whey proteins, such as pre-
`cipitation at their iso-electric point, as it happens during cheese
`making, have been used as alternatives. Selective digestion of
`either or-La or )3-Lg has been attempted in their isolation from
`whey. or-La and )3-Lg are fairly resistant to digestion due to their
`globular structure and have different susceptibility to digestion
`by either trypsin or pepsin. Native )3-Lg that was heat-treated at
`90 to 100 °C for 5 to 10 min underwent changes in structure or
`conformation that rendered it accessible to porcine pepsin (EC
`3.4.23.1) and enhanced the extent of proteolysis by trypsin
`(EC 3.4.21.4)(Guo and others 1995). Conversely, or-La was slowly
`hydrolyzed by trypsin but rapidly by pepsin in either pure form or
`in whole whey. Their hydrolysates had molecular weights from
`about 8 kDa to less than 500 Da with the majority being 3 to 4
`kDa (Pintado and Ivialcata 2000).
`Recently isolation of or-La from sweet whey was achieved
`through a novel approach involving membrane filtrations and
`a tryptic treatment (Figure 3) (Konrad and Kleinschmidt 2008).
`The concentration and fractionation of whey proteins was done
`by UF of sweet whey using membranes with 100 and 150 kDa
`molecular mass and cut-off limits, respectively, at a temperature
`of 45 °C, pressure of 2 bars, and pH of 6.7 followed by tryptic
`hydrolysis of the permeate. The hydrolysis conditions were tem-
`perature 42 °C, pH 7.7, and E/S ratio of 5 mAU/g. At degree of
`hydrolysis (DH) of 10%, all the )3-Lg was digested, while or-La
`was not affected and serum albumin remained in the hydrolysate
`as the only impurity. Further hydrolysis led to partial digestion of
`or-La. The hydrolysis was stopped by changing the pH to 6.0 and
`heating at 65 °C for 10 min. A 2nd UF and DF of the hydrolysate
`
`using a 10 kDa membrane recovered native or-La with a high
`purity of about 93% on the basis of total protein. The method
`produced nearly no waste products and can be easily scaled up.
`The correct termination of the tryptic hydrolysis is critical as
`trypsin attacks or-La immediately after completion of )3-Lg diges-
`tion, while incomplete digestion of )3-Lg leaves it as an impurity
`in or-La. The or-La may also suffer impurities from hydrolysates of
`B-Lg thus affecting its properties.
`
`Precipitation and Aggregation
`This isolation method is based on precipitation of either )3-Lg
`or or-La aggregates under variation of different environmental or
`process parameters. The precipitation is carried out by heat pro-
`cess, addition of ferric chloride, or by use of the limited solubility
`of )3-Lg at low ionic strength, pH 4.65, and high protein con-
`centration (Tolkach and others 2005), which may be achieved by
`concentration using UF. The selective precipitation of or-La is the
`basis for the production of fractions enriched in or-La and )3-Lg
`(Bramaud and others 1997). The or-La is precipitated by acidifica-
`tion of whey or WPC using organic acids, citric and lactic acids
`at 50 °C, pH of 4, and with control of calcium concentration at
`an organic acid/Ca“ molar ratio higher than 9. However, there
`are no conditions under which or-La is the only protein that pre-
`cipitates and subsequent purification procedures are necessary.
`Centrifugation is recommended for the separation of precip-
`itated and soluble fractions, as it
`is more efficient
`than mi-
`crofiltration (Eugenia Lucena and others 2007). The 2 forms
`(apo- and native) of or-La are then recovered after solubilization
`of the precipitate. The apo- is or-La with a calcium-free solvent,
`whereas the native form has calcium solvent. When a solution
`
`of calcium chloride is used, solubilization is a fractionation step
`(increase of 23% in or-La purity), as the immunoglobulins re-
`main insoluble (Bramaud and others 1997). Tolkach and others
`(2005) have also optimized thermal pretreatment conditions for
`the separation of native or-La from WPC by means of selective
`denaturation of )3-Lg. The identified optimal initial composition
`showed 5 to 20 g/L protein content, 0.5 g/L lactose content,
`0.55 g/L calcium content, and pH 7.5. The major advantage of
`this method is that the extracted or-La has a high degree of purity,
`keeps its native structure, and consequently, its properties.
`The reversible precipitation of or-La remains more promising
`provided proper conditions (initial protein concentration, precip-
`itation pH, length of precipitation time, and number of precipitate
`washings) are maintained.
`
`Production and Purification of oz-La Bioactive Peptides
`Bioactive peptides encrypted in intact or-La molecules can be
`generated by the starter and nonstarter bacteria used in the man-
`ufacture of fermented or ripened dairy products as well as by
`digestive and commercial enzymes (Table 1).
`In addition, mi-
`croorganisms together with the microflora extracted from ripened
`dairy products have been reported to fully hydrolyse or-La to
`produce bioactive peptides (Hammea and others 2009). Pep-
`tide fractions with bioactive properties have also been obtained
`through chemical modification of the protein (Oevermann and
`others 2003; Svanborg and others 2003). Commercial enzymes
`capable of hydrolyzing or-La to produce bioactive peptides are
`obtained from plants (Barros and others 2003; Barros and Mal-
`cata 2006), micro-organisms (Ipsen and Otte 2007), and gastric
`juices (Almaas and others 2008).
`These proteolytic enzyme extracts vary in specificity and thus
`give peptides of different characteristics and bioactivities at-
`tributed to size, amino acid sequence, and occurrence of specific
`amino acids at either C-terminal or N-terminal. Low-molecular-
`weight and hydrophobic amino acids have been associated with
`
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`Page 5
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`
`Figure 3—Purification of native or-La
`from sweet whey using a combined
`selective trypsinolysis of
`)3-Iactoglobulin and membrane
`separation (Konrad and Kleinschmidt
`2008).
`
`Comprehensive Reviews in Food Science and Food Safety
`
`Defatted sweet whey
`(pH 6.75)
`
`Ultrafiltration (concentration)
`(150 kDa; VRR 5-20)
`
`Retentate
`di scarded
`
`Tryp sin hydrolysis
`(DH: 9-10%)
`
`Utrafiltration (fractionation)
`(10 kDa; VRR 5)
`
`Hydrolysates
`rernoved
`
`Bioactive
`peptides
`
`Diafiltration (purification)
`
`Lactose, rninerals,
`peptides rernoved
`
`Heat treatment (enzyme
`inactivation/preservation)
`(72 “C; 15 s)
`
`Spray-drying
`
`Pure
`on-lactalbumin
`
`high bioactivities (Hernandez-Ledesma and others 2005; Lopez-
`Fandino and others 2006; Pihlanto 2006). Most of the enzymes
`reported to release bioactive peptides from or-La are serine en-
`dopeptides that are common digestive enzymes.
`Bioactive peptides may be liberated from the protein by gas-
`tric and microbial enzymes during their transit through both small
`and large intestines and they can display bioactivity at the luminal
`side of intestinal tract or, after absorption, in peripheral organs.
`The bioavailability of these peptides is predominantly determined
`by their resistance to further degradation by digestive enzymes
`and rate of intestinal absorption. Short peptides (di- and tripep-
`tides) might be transported across the intestinal barrier and could
`have a physiological effect. Some oligo-peptides may also survive
`degradation under special conditions,
`like naturally permeable
`or leaky intestinal tract allowing their passage into the peripheral
`organs or the blood stream intact. However, the peptides still
`have to contend with possi