dimensional structure is stabilized by a Ca2+ binding loop [12,13] (Figure 1).

formation of the disulfide bonds during protein folding [15].

**3. Calcium-binding properties of α-LA**

partial unfolding and molten globule-like conformation of the protein in MAL [3].

with bovine α-lactalbumin has been called BAMLET.

**2. Structure of α-LA**

Alpha-lactalbumin folding occurs in the lumen of the endoplasmatic reticulum and the protein is subsequently transported to the membrane surface of the Golgi apparatus, where it interacts with galactosyltransferase creating the lactose synthase complex [13,31]. In the absence of α-lactalbumin, the enzyme is a non-specific galactosyltransferase involved in transferring galactosyl groups from UDP-galactose to a range of different substrates [13,31]. Alpha-lactalbumin functions as a substrate modifier, increasing specificity and affinity of the galactosyltransferase for glucose, when catalyzing the final step of lactose production in a lactating mammary gland [7,32].

### **5. Folding of α-LA**

A noteworthy property of α-LA is the ability of the protein to adopt relatively stable partially unfolded intermediates under various conditions. Accordingly, it has been intensely used as a protein folding model in many studies. The classical molten globule of α-LA was defined as the acid denatured compact state of α-LA at pH 2.0 with fluctuating tertiary structure [13,33,34]. However, α-LA can adopt molten globule-like states during Ca2+-free (apo-state) conditions, during high temperatures at 90°C and in the presence of denaturants [14,17,35,36]. Similar conformational states can also be formed by reduction of the disulfide bonds in α-LA [37,38].

The molten globule-like conformational states of α-LA are not significantly populated at physiological conditions [26]. In addition, stable unfolded states of α-LA only persist at low pH or in the absence of metal ions, since the protein instantly returns to the native conformation, if solution conditions are re-normalized [19]. In the molten globule conformation, α-LA has a native-like secondary structure, a less well-defined tertiary structure and a larger stokes radius compared to the native protein [14,34,39].

The apo-state of α-LA, a molten globular-like conformation, is required for the formation of HAMLET. This conformation has high instability and sensitivity towards the ionic strength of the solution, compared to the native state of the protein [19]. However, the apo-state α-LA is more hydrophobic than the native state of the protein, rendering it prone to fatty acid binding [2]. The apo-state of α-LA is thus stabilized by binding to specific fatty acids in the HAMLET complex [6]. The proposed requirement of a special molten globular-like conformation of the proteinaceous component in HAMLET-like complexes has been challenged, as bovine β-lactoglobulin in complex with OA had cytotoxic activity exceeding that of HAMLET without retaining such a structure [40].

### **6.  The release of the Ca2+ ion and subsequent unfolding of the protein triggers α-LA to achieve a new fatty acid binding profile, with a higher affinity for cis-unsaturated fatty acids than for the corresponding trans conformations.

The interaction between apo-state α-LA and distinctive fatty acids has been studied, with the observation that certain fatty acids interact with apo-state α-LA in a stereo-specific manner [6]. Is has been found, that saturated C18 fatty acids, unsaturated C18:1 *trans* fatty acids and fatty acids with longer or shorter carbon chains only form insignificant amounts of complexes with apo-state α-LA [6]. All unsaturated *cis* fatty acids are able to form stable complexes with apo-state α-LA, with oleic acid showing the highest conversion efficiency [6]. It was found, however, that only oleic acid (C18:1 *cis* Δ9 – in HAMLET) and vaccenic acid (C18:1 *cis* Δ11) in complex with apo-state α-LA showed biological activity in apoptosis assay, suggesting that unsaturated C18:1 *cis* fatty acids have the correct stereo-specificity to bind apo-state α-LA in the formation and stabilize the HAMLET complex [6]. Another study has found that vaccenic acid, palmitoleic acid linoleic acid had similar activity as oleic acid while elaidic acid and stearic acid shoved a weaker activity [42]. The fatty acid profile of α-LA thus seems to be rather broad and not as specific as initially anticipated.

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a lactating mammary gland [7,32].

**5. Folding of α-LA**

it interacts with galactosyltransferase creating the lactose synthase complex [13,31]. In the absence of α-lactalbumin, the enzyme is a non-specific galactosyltransferase involved in transferring galactosyl groups from UDP-galactose to a range of different substrates [13,31]. Alpha-lactalbumin functions as a substrate modifier, increasing specificity and affinity of the galactosyltransferase for glucose, when catalyzing the final step of lactose production in

A noteworthy property of α-LA is the ability of the protein to adopt relatively stable partially unfolded intermediates under various conditions. Accordingly, it has been intensely used as a protein folding model in many studies. The classical molten globule of α-LA was defined as the acid denatured compact state of α-LA at pH 2.0 with fluctuating tertiary structure [13,33,34]. However, α-LA can adopt molten globule-like states during Ca2+-free (apo-state) conditions, during high temperatures at 90°C and in the presence of denaturants [14,17,35,36]. Similar conformational states can also be formed by reduction of the disulfide bonds in α-LA [37,38].

The molten globule-like conformational states of α-LA are not significantly populated at physiological conditions [26]. In addition, stable unfolded states of α-LA only persist at low pH or in the absence of metal ions, since the protein instantly returns to the native conformation, if solution conditions are re-normalized [19]. In the molten globule conformation, α-LA has a native-like secondary structure, a less well-defined tertiary

The apo-state of α-LA, a molten globular-like conformation, is required for the formation of HAMLET. This conformation has high instability and sensitivity towards the ionic strength of the solution, compared to the native state of the protein [19]. However, the apo-state α-LA is more hydrophobic than the native state of the protein, rendering it prone to fatty acid binding [2]. The apo-state of α-LA is thus stabilized by binding to specific fatty acids in the HAMLET complex [6]. The proposed requirement of a special molten globular-like conformation of the proteinaceous component in HAMLET-like complexes has been challenged, as bovine β-lactoglobulin in complex with OA had cytotoxic activity exceeding

Alpha-lactalbumin possesses several fatty acid binding sites, and has been shown to bind stearic acid, palmitic acid and oleic acid spin-labelled fatty acid analogs [6,41]. The release of the Ca2+ ion and subsequent unfolding of the protein triggers α-LA to achieve a new fatty acid binding profile, with a higher affinity for cis-unsaturated fatty acids than for the

The interaction between apo-state α-LA and distinctive fatty acids has been studied, with the observation that certain fatty acids interact with apo-state α-LA in a stereo-specific manner [6]. Is has been found, that saturated C18 fatty acids, unsaturated C18:1 *trans* fatty

structure and a larger stokes radius compared to the native protein [14,34,39].

that of HAMLET without retaining such a structure [40].

**6. Fatty acid binding profile of α-LA**

corresponding trans conformations.

Based on the three-dimensional structures of native and apo-state α-LA, two tentative hydrophobic regions and possible fatty acid binding sites within α-LA have been proposed. One is suggested in the interface between the two sub-domains including the C- and D-helix and the β-sheet domain [6,43,44]. The other is anticipated to be formed by residues within the A, B and 310-helices.

The binding of oleic acid to bovine apo-state α-LA has previously been shown to induce changes in the secondary structure of the protein, resulting in a typical molten globule state of the protein [45]. Far-UV CD of oleic acid binding to human apo-state α-LA showed similar results, with an increase in α-helical structure, largely independent of the temperature conditions [30].

Upon Ca2+ release structural changes in α-LA also occur in the cleft between the two domains, with the slight expansion of the Ca2+-binding loop tilting the 310 helix toward the C helix, causing disruption of the aromatic cluster composed of Trp60, Trp104, Phe53 and Tyr103 in the interface between the two domains [11]. X-ray crystallography and NMR spectroscopy studies have revealed that apo-state bovine α-LA contains a largely intact αhelical domain with native side chain packing and an unstructured β-sheet domain [11,46]. Similar findings have been made for the molten globule-like conformation of human apostate α-LA [47]. Therefore, it has been proposed that oleic acid binds in the area between the two domains, thus stabilizing the molten globule-like state of HAMLET and allowing Ca2+ binding without any effect on the tumoricidal activity [2,6]. Nevertheless, it is still unclear exactly how oleic acid binds to apo-state α-LA. Although the apo-state of α-LA is negatively charged, it may be hydrophobic interactions mediating the binding of the oleic acid, considering the increased hydrophobicity of apo-state α-LA compared to native α-LA [11,46,48,49]. Furthermore, even though the apo-state is negatively charged, it may also possess positively charged residues that are able to bind to the negatively charged polar head-groups of the oleic acids through electrostatic interactions [48,49]. Accordingly, it has been suggested that hydrophobic amino acids in the interface between the two domains may bind the fatty acid tail, while positively charged amino acids such as Arg70, Lys94 and Lys99 are plausible coordination residues for the fatty acid head group [2].

In contrast to these above-mentioned suggestions, one of the same studies showed that several proteolytically generated fragments of α-LA were able to incorporate oleic acid,

### 124 Milk Protein

implying that there is not only one single fatty acid binding site in HAMLET [48]. The exact mechanism of fatty acid binding to α-LA remains uncertain.

### **7. Structure of HAMLET**

HAMLET is defined as a conversion complex between apo-state α-LA and a C18:1 *cis* fatty acid co-factor, which has the ability to induce apoptosis selectively in tumor cells [6]. HAMLET has been described as the first discovered example of a protein that exhibits a well-defined function in the native structure, but additionally acquires a potentially beneficial function following partial unfolding [1,5,38].

Studies of the HAMLET structure by tryptophan fluorescence spectroscopy, near-UV CD spectroscopy and far-UV CD spectroscopy have revealed that the complex retains a stable, partially unfolded, molten globule-like conformation even at physiological conditions, in contrast to partially unfolded α-LA, which reverts to the native conformation, if solution conditions are re-normalized [3,5,14,19,29,45]. In this molten globule-like conformation, α-LA in the HAMLET complex showed retention of the secondary structure, near-complete loss of the tertiary structure, a larger stokes radius, and increased exposure of hydrophobic surfaces, compared to native α-LA [3,5,14,34,39,50].

### **8. Characteristics of HAMLET**

The formation, stabilization and cytotoxic activity of the HAMLET complex have been shown to require both partial unfolding of α-LA and the binding of a fatty acid co-factor [5,6,19,51]. The partial unfolding of α-LA, e.g. through metal ion depletion, results in destabilization of the β-sheet domain, while leaving the α-helix domain largely unchanged, paving the way for fatty acid binding and subsequent HAMLET formation [19,50]. In accordance with this, it has been shown that the tumoricidal activity of HAMLET is largely independent of the β-sheet domain and C-terminal portion of human α-LA, since the isolated α-helix domain of α-LA was able to form a cytotoxic complex with OA [52].

Additional changes in the α-LA structure can induce the formation of HAMLET or HAMLET analogs. It has been proposed that the entire 123-residue sequence of α-LA is not required for cytotoxic activity, as fragments of α-LA in a complex with OA were able to induce apoptosis in Jurkat tumor cells [48]. Furthermore, it has been shown that a recombinant variant of human α-LA, where all cysteine residues were substituted with alanine residues, had the ability to form cytotoxic HAMLET complex analogs [38]. In addition, an α-LA mutant where Asp87 was shifted to an alanine with no Ca2+ binding activity was also able to form cytotoxic complexes with oleic acids, implying that a functional Ca2+ site is not required for the conversion of α-LA to the active complex or to cause cell death [19]. This being the case despite the fact that HAMLET has a high Ca2+ affinity, with a Ca2+ association constant of 5.3×106 M-1 at physiological salt conditions [19]. However, the structural changes induced upon Ca2+ binding have no effect on the cytotoxic activity of the complex [19,30].

Interestingly, human α-LA is not the only variant of the protein able to form cytotoxic complexes with OA. Alpha-lactalbumin of bovine, equine, caprine, and porcine origin also have the ability to form complexes with OA, showing HAMLET-like cytotoxicity, while natural HAMLET formation in acid precipitates of casein was unique to human milk [9]. Comparison of highly purified human and bovine α-LA´s ability to form complexes with OA showed that the two proteins behaved very similarly and that the cytotoxic activity was comparable [53].

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implying that there is not only one single fatty acid binding site in HAMLET [48]. The exact

HAMLET is defined as a conversion complex between apo-state α-LA and a C18:1 *cis* fatty acid co-factor, which has the ability to induce apoptosis selectively in tumor cells [6]. HAMLET has been described as the first discovered example of a protein that exhibits a well-defined function in the native structure, but additionally acquires a potentially

Studies of the HAMLET structure by tryptophan fluorescence spectroscopy, near-UV CD spectroscopy and far-UV CD spectroscopy have revealed that the complex retains a stable, partially unfolded, molten globule-like conformation even at physiological conditions, in contrast to partially unfolded α-LA, which reverts to the native conformation, if solution conditions are re-normalized [3,5,14,19,29,45]. In this molten globule-like conformation, α-LA in the HAMLET complex showed retention of the secondary structure, near-complete loss of the tertiary structure, a larger stokes radius, and increased exposure of hydrophobic

The formation, stabilization and cytotoxic activity of the HAMLET complex have been shown to require both partial unfolding of α-LA and the binding of a fatty acid co-factor [5,6,19,51]. The partial unfolding of α-LA, e.g. through metal ion depletion, results in destabilization of the β-sheet domain, while leaving the α-helix domain largely unchanged, paving the way for fatty acid binding and subsequent HAMLET formation [19,50]. In accordance with this, it has been shown that the tumoricidal activity of HAMLET is largely independent of the β-sheet domain and C-terminal portion of human α-LA, since the

isolated α-helix domain of α-LA was able to form a cytotoxic complex with OA [52].

Additional changes in the α-LA structure can induce the formation of HAMLET or HAMLET analogs. It has been proposed that the entire 123-residue sequence of α-LA is not required for cytotoxic activity, as fragments of α-LA in a complex with OA were able to induce apoptosis in Jurkat tumor cells [48]. Furthermore, it has been shown that a recombinant variant of human α-LA, where all cysteine residues were substituted with alanine residues, had the ability to form cytotoxic HAMLET complex analogs [38]. In addition, an α-LA mutant where Asp87 was shifted to an alanine with no Ca2+ binding activity was also able to form cytotoxic complexes with oleic acids, implying that a functional Ca2+ site is not required for the conversion of α-LA to the active complex or to cause cell death [19]. This being the case despite the fact that HAMLET has a high Ca2+ affinity, with a Ca2+ association constant of 5.3×106 M-1 at physiological salt conditions [19]. However, the structural changes induced upon Ca2+ binding have no effect on the cytotoxic

mechanism of fatty acid binding to α-LA remains uncertain.

beneficial function following partial unfolding [1,5,38].

surfaces, compared to native α-LA [3,5,14,34,39,50].

**8. Characteristics of HAMLET**

activity of the complex [19,30].

**7. Structure of HAMLET**

The revelations that neither native-state, apo-state nor otherwise partially unfolded α-LA alone shows significant cytotoxic activity, in addition to the above-mentioned findings, have caused researchers to focus on the possibility that the cytotoxic component of HAMLET may be the fatty acid and not the α-LA protein [3,5,19,50]. As a consequence, it has been suggested that α-LA is merely a carrier or synergist of the tumor-killing activity of the associated oleic acid [48,49]. In agreement with this, studies have found that the cytotoxicity of OA alone is very similar to that of BAMLET, the bovine ortholog of HAMLET, and that of HAMLET-like complexes composed of bovine β-lactoglobulin and pike parvalbumin in complex with OA [40,54]. The cytotoxic activity of the β-lactoglobulin and paralbumin complexes even exceeded that of HAMLET, indicating that the proteinaceous component of the complexes is of less importance than the OA component in relation to the cytotoxic activity [40]. The potent toxicity of free oleic acid against Jurkat and HL-60 tumor cells has been shown, supporting the proposal that the fatty acid could be the cytotoxic component of the HAMLET complex [54,55]. However, in contrast with this proposal, α-LA has previously been found to be cytotoxic in the absence of oleic acid [56-60].

### **9. Stoichiometry of the protein and fatty acid in HAMLET and HAMLETlike complexes**

The stoichiometry of protein and fatty acid in HAMLET or HAMLET-like complexes remains a topic of debate. Initial gas chromatography and mass spectrometry data suggested that the average number of oleic acid molecules bound in the HAMLET complex was 0.9, with some batch variation [6]. The assumption of a 1:1 protein/fatty acid ratio was also reported in the preparation of BAMLET by chromatography [61]. By gas chromatography and mass spectrometry a later study estimated an α-LA to OA ratio of 1:5.4 in a HAMLET complex prepared by the OA-preconditioned anion exchange chromatography method [38]. An α-LA to OA ratio of 1:8.2 in HAMLET determined by LC-MS has also been reported [52]. A ratio of 1:10-1:13 was found for BAMLET and HAMLET complexes formed by a new alternative preparation method, using two consecutive DEAE-Sepharose ion exchange columns for the purification of α-LA and subsequent complex formation, but high variation in stoichiometry was common within different batches [53].

The average number of oleic acid molecules bound per α-LA in LA/OA complexes has been spectrofluorimetrically estimated to be 2.9 for LA/OA formed at 17°C and 9 for LA/OA formed at 45°C [30]. Another study has reported that the bovine LA/OA complex, prepared by direct mixing of the constituents, consists of 4-5 protein molecules with 68-85 bound molecules of OA, thus indicating that every α-LA binds on average 17 OA molecules [49].

A possible explanation for the variation in the observed α-LA to OA ratios might be that the different preparation techniques and experimental conditions (e.g. changes in temperature or altered α-LA to OA ratios) could have a major impact on the observed stoichiometry. In addition, it has been proposed that some of the OA might be present in an unbound form resulting in a higher OA to α-LA ratio [30].

### **10. Methods of HAMLET or HAMLET-analog preparation** *in vitro*

Several different methods have been utilized for the *in vitro* preparation of HAMLET or its orthologs from α-LA and OA. These include OA-conditioned anion exchange chromatography using either a DEAE-Trisacryl M in the conventional method [3,5,6,19] or a DEAE-Sepharose column in an alternative method [53]. Direct mixing under heat denaturation [30,36,52], heat denaturation of α-LA and OA-conditioned anion exchange chromatography [62], direct mixing in solution at room temperature with alternated pH conditions [45,48,49,63], direct mixing followed by anion exchange chromatography [61] and alkaline conditions [64] have also been used.

The conventional method of HAMLET preparation is OA-preconditioned anion exchange chromatography [5]. This method is initiated with the purification of native-state α-LA by hydrophobic interaction chromatography and subsequent EDTA-treatment, resulting in the loss of Ca2+ and partial unfolding of the protein. The apo-state α-LA is subjected to a DEAE-Trisacryl M ion exchange chromatographic column previously exposed to human milk or pre-loaded with fatty acids. The apo-state α-LA interacts with the OA pre-loaded column, forming the HAMLET complex, which elutes at a salt concentration of 1M. The eluted complex is desalted by dialysis and lyophilized to obtain HAMLET dry powder.

Regardless of the preparation method used in the different experiments, it has been shown that the cytotoxicity of the resulting complexes was similar to that of conventionally prepared HAMLET or BAMLET [30,36,48,49,54,61-65].

### **11. Cellular trafficking of HAMLET**

In order to clarify the subcellular localization of HAMLET and the interactions of the complex with different tumor cell compartments, researchers have used biotinylated [5,66], I125 radioactive labeled [66,67] and Alexa Fluor 568-labeled HAMLET complexes in confocal microscopy and subcellular fractionation experiments [67-69]. Site-specific labeling of HAMLET and HAMLET-like complexes with aminooxy-Alexa Fluor 488 or biotin molecules has also been used [52].

It has previously been described that α-LA interacts with cell membranes and lipid bilayers, as well as fatty acids [41,70-74]. In addition, it has been observed that α-LA is able to rapidly insert itself into the lipid bilayer at pH 2 [75]. However, native human α-LA is only inefficiently bound to and internalized by tumor cells, and does not translocate to the nucleus, nor influence tumor cell viability [3,5,68,69,76]. Even partial unfolding of human α-LA has been shown to be insufficient for increased protein internalization into tumor cells [38]. In addition, human α-LA and oleic acid alone were unable to alter membrane structures, further indicating the necessity of interaction between unfolded α-LA and an associated fatty acid for efficient internalization of the HAMLET complex in tumor cells [76].

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resulting in a higher OA to α-LA ratio [30].

alkaline conditions [64] have also been used.

prepared HAMLET or BAMLET [30,36,48,49,54,61-65].

**11. Cellular trafficking of HAMLET**

has also been used [52].

by direct mixing of the constituents, consists of 4-5 protein molecules with 68-85 bound molecules of OA, thus indicating that every α-LA binds on average 17 OA molecules [49].

A possible explanation for the variation in the observed α-LA to OA ratios might be that the different preparation techniques and experimental conditions (e.g. changes in temperature or altered α-LA to OA ratios) could have a major impact on the observed stoichiometry. In addition, it has been proposed that some of the OA might be present in an unbound form

Several different methods have been utilized for the *in vitro* preparation of HAMLET or its orthologs from α-LA and OA. These include OA-conditioned anion exchange chromatography using either a DEAE-Trisacryl M in the conventional method [3,5,6,19] or a DEAE-Sepharose column in an alternative method [53]. Direct mixing under heat denaturation [30,36,52], heat denaturation of α-LA and OA-conditioned anion exchange chromatography [62], direct mixing in solution at room temperature with alternated pH conditions [45,48,49,63], direct mixing followed by anion exchange chromatography [61] and

The conventional method of HAMLET preparation is OA-preconditioned anion exchange chromatography [5]. This method is initiated with the purification of native-state α-LA by hydrophobic interaction chromatography and subsequent EDTA-treatment, resulting in the loss of Ca2+ and partial unfolding of the protein. The apo-state α-LA is subjected to a DEAE-Trisacryl M ion exchange chromatographic column previously exposed to human milk or pre-loaded with fatty acids. The apo-state α-LA interacts with the OA pre-loaded column, forming the HAMLET complex, which elutes at a salt concentration of 1M. The eluted

Regardless of the preparation method used in the different experiments, it has been shown that the cytotoxicity of the resulting complexes was similar to that of conventionally

In order to clarify the subcellular localization of HAMLET and the interactions of the complex with different tumor cell compartments, researchers have used biotinylated [5,66], I125 radioactive labeled [66,67] and Alexa Fluor 568-labeled HAMLET complexes in confocal microscopy and subcellular fractionation experiments [67-69]. Site-specific labeling of HAMLET and HAMLET-like complexes with aminooxy-Alexa Fluor 488 or biotin molecules

It has previously been described that α-LA interacts with cell membranes and lipid bilayers, as well as fatty acids [41,70-74]. In addition, it has been observed that α-LA is able to rapidly insert itself into the lipid bilayer at pH 2 [75]. However, native human α-LA is only

complex is desalted by dialysis and lyophilized to obtain HAMLET dry powder.

**10. Methods of HAMLET or HAMLET-analog preparation** *in vitro*

Even though a number of studies have focused on investigating the mechanism of HAMLET uptake by tumor cells, it still remains unclear. HAMLET has been shown to interact with the cell surface of tumor cells, be internalized and subsequently accumulate in the nucleus [3,5,67,68]. In the case of healthy cells, HAMLET interacted with the cell membrane and was internalized, but did not accumulate in the nuclei of the cells [4,67].

A study of the interaction between HAMLET and natural or artificially generated, negatively charged, model lipid membranes has revealed that the interaction results in loss of cell membrane integrity, leakage of vesicular contents and morphological changes of the membranes [76]. Interestingly, HAMLET disturbs the integrity of the tested membranes, even under physiological conditions, as opposed to native-state α-LA that only disrupts liposomes at acidic pH [77]. Similarly, at physiological conditions HAMLET readily distorts lipid monolayers at low concentrations, while forming pore-like oligomeric structures resembling annular oligomers at higher concentrations [78]. The ability to form oligomers is suggested to be a property of the α-LA polypeptide chain, enhanced by the fatty acid component, which might be important for the cytotoxic activity of HAMLET [78]. It has been suggested that the degree to which HAMLET and other LA-OA-complexes are able to disturb the membrane integrity, depends on the lipid composition and physical characteristics of the membrane [74,76].

HAMLET binds to the cell surface of intact tumor cells in a patchy distribution, indicating the possible targeting (e.g. receptor-binding) of the complex to specific membrane regions [76]. Another study found that HAMLET and an α-LA/OA complex formed at 17**°**C, interacts with and alters trans-membrane integral currents of artificial vesicles and natural plasma membranes of the green alga *C. coralline* [65]. The binding of the OA-complexes caused suppression of Ca2+ current and Ca2+-activated Cl current, as well as increased nonspecific K+ leakage, indicating nonselective permeability of the depolarized plasma membrane [65]. Furthermore, all the OA-bound states of α-LA had a higher affinity for interaction with the membranes compared to α-LA alone, indicating a Ca2+-independent association. Similar results were obtained regarding the bactericidal activity and membrane depolarization effect of HAMLET, bovine β-lactoglobulin and pike parvalbumin in complex with OA on *S. pneumococci* bacteria [40,64]. Accordingly, it has been proposed that once the complex has adsorbed to the plasma membrane, it is most likely internalized by nonspecific pinocytosis, while other mechanisms of endocytosis, such as receptor-mediated endocytosis have not been ruled out [65].

Translocation of HAMLET from the membrane to the cytoplasm has been followed by confocal microscopy, showing the formation of intracellular aggregates in both normal cells

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and in tumor cells, although accumulation of HAMLET was higher in tumor cells [4]. The mechanism of HAMLET redistribution from the cytoplasm to different organelles of the cell, especially the nucleus, remains to be clarified. Initial studies found that the nuclear uptake of α-LA/OA occurred through the nuclear pore complex in a Ca2+-independent manner, and that the accumulation of α-LA in the nucleus resulted in Ca2+-dependent DNA fragmentation in the tumor cells [66]. Later, it was suggested that the interaction between HAMLET and ribosomal proteins could initiate nuclear translocation and targeting of the complex [2].

### **12. Tumor cell death mechanisms of HAMLET**

A key unresolved issue regarding HAMLET is by what biological mechanisms the complex induces tumor cell death. A broad variety of transformed and immature tumor cells, but not healthy differentiated cells, have been found to be affected by the complex and even antibiotic-resistant strains of *S. pneumoniae* bacteria seem to be prone to the cytotoxic activity, showing apoptosis-like morphological and mechanistic changes [4,79,80]. HAMLET is believed to have multiple intracellular targets and has metaphorically been called a "Lernaean Hydra", a mythological creature that uses numerous heads to attack enemies [51]. It has been shown that HAMLET induces at least three major responses, namely an apoptotic pathway, an autophagic pathway and chromatin structure disorder [1,67,81]. All the suggested tumor cell death mechanisms of HAMLET or HAMLET-like complexes are listed in Table 1.

Besides the three major responses, it has been shown that BAMLET activates a caspaseindependent lysosomal cell death pathway mainly in tumor cells, causing lysosomal membrane permeabilization possibly contributing to BAMLET-induced cell death [61]. In a study of HAMLET binding to α-actinin, it has been indicated that the apoptosis-promoting p38 pathway is the top-scoring pathway in HAMLET-induced cell death, with p38 inhibition delaying death of HAMLET-treated tumor cells [82].

An elevated expression level of c-Myc, an oncogene that has the ability to bind a significant fraction of all known gene promoters, has been shown to increase HAMLET-sensitivity of various tumor cells, suggesting that the level of c-Myc expression could be a direct determinant of HAMLET susceptibility [83]. Furthermore, a reduction in the extracellular glucose levels or degree of glycolysis, predominantly the aerobic glycolysis of tumor cells known as the Warburg Effect, of A549 lung carcinoma cells resulted in an enhanced HAMLET-sensitivity [83]. HAMLET was shown to bind and reduce the activity of the glycolytic enzyme hexokinase 1 in the A549 carcinoma cells, which could partly explain the inhibitory effect of HAMLET on glycolysis [83].

HAMLET has been shown to interact directly with 20S proteasome subunits *in vitro* as well as *in vivo*, resulting in structural modifications and partial inhibition of 20S proteasome activity, possibly leading to accumulation of incorrectly folded proteins contributing to cell death [69]. Moreover, HAMLET was found by N-terminal sequencing and MALDI-TOF-MS to bind intact ribosomes, as well as individual ribosomal proteins L4, L6, L15, L13a, L30, L35a, S12 and L21 [2]. The plausible effects of the interaction are protein translational blocking and ribosome-initiated nuclear targeting of HAMLET [2].

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complex [2].

listed in Table 1.

and in tumor cells, although accumulation of HAMLET was higher in tumor cells [4]. The mechanism of HAMLET redistribution from the cytoplasm to different organelles of the cell, especially the nucleus, remains to be clarified. Initial studies found that the nuclear uptake of α-LA/OA occurred through the nuclear pore complex in a Ca2+-independent manner, and that the accumulation of α-LA in the nucleus resulted in Ca2+-dependent DNA fragmentation in the tumor cells [66]. Later, it was suggested that the interaction between HAMLET and ribosomal proteins could initiate nuclear translocation and targeting of the

A key unresolved issue regarding HAMLET is by what biological mechanisms the complex induces tumor cell death. A broad variety of transformed and immature tumor cells, but not healthy differentiated cells, have been found to be affected by the complex and even antibiotic-resistant strains of *S. pneumoniae* bacteria seem to be prone to the cytotoxic activity, showing apoptosis-like morphological and mechanistic changes [4,79,80]. HAMLET is believed to have multiple intracellular targets and has metaphorically been called a "Lernaean Hydra", a mythological creature that uses numerous heads to attack enemies [51]. It has been shown that HAMLET induces at least three major responses, namely an apoptotic pathway, an autophagic pathway and chromatin structure disorder [1,67,81]. All the suggested tumor cell death mechanisms of HAMLET or HAMLET-like complexes are

Besides the three major responses, it has been shown that BAMLET activates a caspaseindependent lysosomal cell death pathway mainly in tumor cells, causing lysosomal membrane permeabilization possibly contributing to BAMLET-induced cell death [61]. In a study of HAMLET binding to α-actinin, it has been indicated that the apoptosis-promoting p38 pathway is the top-scoring pathway in HAMLET-induced cell death, with p38

An elevated expression level of c-Myc, an oncogene that has the ability to bind a significant fraction of all known gene promoters, has been shown to increase HAMLET-sensitivity of various tumor cells, suggesting that the level of c-Myc expression could be a direct determinant of HAMLET susceptibility [83]. Furthermore, a reduction in the extracellular glucose levels or degree of glycolysis, predominantly the aerobic glycolysis of tumor cells known as the Warburg Effect, of A549 lung carcinoma cells resulted in an enhanced HAMLET-sensitivity [83]. HAMLET was shown to bind and reduce the activity of the glycolytic enzyme hexokinase 1 in the A549 carcinoma cells, which could partly explain the

HAMLET has been shown to interact directly with 20S proteasome subunits *in vitro* as well as *in vivo*, resulting in structural modifications and partial inhibition of 20S proteasome activity, possibly leading to accumulation of incorrectly folded proteins contributing to cell death [69]. Moreover, HAMLET was found by N-terminal sequencing and MALDI-TOF-MS to bind intact ribosomes, as well as individual ribosomal proteins L4, L6, L15, L13a, L30,

**12. Tumor cell death mechanisms of HAMLET**

inhibition delaying death of HAMLET-treated tumor cells [82].

inhibitory effect of HAMLET on glycolysis [83].

**Table 1.** The tumor cell death mechanisms of HAMLET and HAMLET-like complexes.

For elaboration of the specific tumor cells prone to the mentioned effect, the reader is encouraged to see the references. HK1, hexokinase 1; IMM, inner mitochondrial membrane; MAL, multimeric α-lactalbumin; MPT, mitochondrial permeability transition.

### **13. HAMLET and apoptosis**

Several studies have focused on clarifying the precise role of apoptosis in the HAMLETinduced cell death of tumor cells. Initially, it was shown by the use of an anti-CD95 antibody that the CD95 receptor-mediated apoptotic pathway had no effect on the apoptosis-inducing activity of MAL [84]. It was also found that MAL co-localized with mitochondria and caused release of cytochrome *c* from the inner mitochondrial membrane space to the cytosol, resulting in activation of caspase-3-like enzymes and, to a lesser degree, caspase-6-like enzymes of the caspase cascade [84]. The activation of cytosolic caspases by apogenetic factors released from the inner mitochondrial membrane (e.g. cytochrome *c*), and subsequent Apaf-1, procaspase-9 and dATP association, resulting in formation of the apoptosome complex, are normal events of the classical apoptotic pathway [85-87]. It was later revealed that MAL induced a loss of the mitochondrial membrane potential (ΔΨm) and abnormal mitochondrial permeability transition (MPT) in isolated mitochondria through opening of the MPT pore, resulting in Ca2+-dependent release of cytochrome *c* to the cytosol [87].

 However, classical apoptosis was not the cause of death, as a pan-caspase inhibitor zVAD-fmk was unable to rescue HAMLET-treated cells from dying [84,90]. This was confirmed by the finding that HAMLET-induced cell death is independent of the anti-apoptotic Bcl-2 and Bcl-xL proteins in Jurkat leukemia, K562 promyelocytic leukaemia and FL5.12 murine prolymphocytic tumor cells, as over-expression of Bcl-2 failed to prevent cell death [90]. In addition, the tumoricidal activity of HAMLET is independent of the tumor suppressor protein p53, as there was no difference in HAMLET susceptibility of tumor cells with wild-type, deleted or mutated p53 gene [2,90,91]. Analyzing the effect of BAMLET on Jurkat and THP1 cells by flow cytometry indicated that the death of Jurkat cells looked more apoptotic than the death of THP1 cells which were more necrotic, showing that the actual mechanism of cell death apparently varies between different types of cells [53].

### **14. HAMLET and macroautophagy**

Autophagy has been a topic of comprehensive debate concerning its function as an alternative caspase-independent type II programmed cell death pathway, in addition to serving as a survival mechanism during cellular stresses [92-95]. Macroautophagy, the only autophagic response included in type II programmed cell death, is an adaptive stress response observed in cells exposed to cellular stresses (e.g. starvation), which triggers the recycle of organelles and long-lived proteins as a nutritional source utilized to prolong cell survival [91,92, 96].

HAMLET induces a macroautophagic response in treated tumor cells, with the appearance of cytoplasmic vacuoles and double-membrane enclosed vesicles [81,91]. Furthermore, HAMLET was shown to alter the staining-pattern of LC3-GFP-transfected cells from uniform (LC3-I) to granular (LC3-II), reflecting the translocation of LC3 to autophagosomes during macroautophagy [81]. HAMLET also induced LC3-II accumulation, detected by a Western Blot, when lysosomal degradation was inhibited, which is a clear indicative of macroautophagy [81].

The inhibition of macroautophagy in HAMLET-treated tumor cells by RNA interference of Beclin-1 synthesis resulted in reduced cell death and inhibition of the increase in granular LC3-GFP staining, suggesting that macroautophagy is an important response pathway in HAMLET-induced cell death [81,91]. In accordance with this finding, it was shown that mTOR, an inhibitor of macroautophagy, is inactivated in tumor cells in response to HAMLET [81,97]. An important note is that the mitochondrial damage observed in the different tumor cells of the mentioned experiments also has the potential to trigger macroautophagy in the cells [81,98,99].

### **15. HAMLET cell nuclei interactions**

130 Milk Protein

[87].

**13. HAMLET and apoptosis**

For elaboration of the specific tumor cells prone to the mentioned effect, the reader is encouraged to see the references. HK1, hexokinase 1; IMM, inner mitochondrial membrane;

Several studies have focused on clarifying the precise role of apoptosis in the HAMLETinduced cell death of tumor cells. Initially, it was shown by the use of an anti-CD95 antibody that the CD95 receptor-mediated apoptotic pathway had no effect on the apoptosis-inducing activity of MAL [84]. It was also found that MAL co-localized with mitochondria and caused release of cytochrome *c* from the inner mitochondrial membrane space to the cytosol, resulting in activation of caspase-3-like enzymes and, to a lesser degree, caspase-6-like enzymes of the caspase cascade [84]. The activation of cytosolic caspases by apogenetic factors released from the inner mitochondrial membrane (e.g. cytochrome *c*), and subsequent Apaf-1, procaspase-9 and dATP association, resulting in formation of the apoptosome complex, are normal events of the classical apoptotic pathway [85-87]. It was later revealed that MAL induced a loss of the mitochondrial membrane potential (ΔΨm) and abnormal mitochondrial permeability transition (MPT) in isolated mitochondria through opening of the MPT pore, resulting in Ca2+-dependent release of cytochrome *c* to the cytosol

HAMLET-treated Jurkat leukemia cells and A549 lung carcinoma tumor cells exhibited classical apoptotic changes, as well as apoptosis-like changes, such as proapoptotic caspase activation, phosphatidyl serine externalization, DNA fragmentation, apoptotic body formation and compacted chromatin condensation [88-90]. However, classical apoptosis was not the cause of death, as a pan-caspase inhibitor zVAD-fmk was unable to rescue HAMLET-treated cells from dying [84,90]. This was confirmed by the finding that HAMLET-induced cell death is independent of the anti-apoptotic Bcl-2 and Bcl-xL proteins in Jurkat leukemia, K562 promyelocytic leukaemia and FL5.12 murine prolymphocytic tumor cells, as over-expression of Bcl-2 failed to prevent cell death [90]. In addition, the tumoricidal activity of HAMLET is independent of the tumor suppressor protein p53, as there was no difference in HAMLET susceptibility of tumor cells with wild-type, deleted or mutated p53 gene [2,90,91]. Analyzing the effect of BAMLET on Jurkat and THP1 cells by flow cytometry indicated that the death of Jurkat cells looked more apoptotic than the death of THP1 cells which were more necrotic, showing that the actual mechanism of cell death

Autophagy has been a topic of comprehensive debate concerning its function as an alternative caspase-independent type II programmed cell death pathway, in addition to serving as a survival mechanism during cellular stresses [92-95]. Macroautophagy, the only autophagic response included in type II programmed cell death, is an adaptive stress response observed in cells exposed to cellular stresses (e.g. starvation), which triggers the

apparently varies between different types of cells [53].

**14. HAMLET and macroautophagy**

MAL, multimeric α-lactalbumin; MPT, mitochondrial permeability transition.

A striking feature of HAMLET is the ability to move through the cytoplasm and accumulate in the nucleus of tumor cells, as initially elucidated by the study of the active human milk fraction [3,66]. Healthy, differentiated cells did not accumulate biotinylated MAL or Alexalabeled HAMLET in the nucleus, although uptake in the cytoplasm was observed [66,67].

Through combination of *in vitro* and *in vivo* experiments, HAMLET was found to co-localize with histones and bind strongly to histone H3, as well as histones H4 and H2B to a lesser degree, resulting in perturbation of the chromatin structure and chromatin assembly in tumor cells, possibly impairing transcription, replication and recombination [67,100]. The binding of HAMLET to the histones was independent of the histone tail, and the binding impairs histone deposition on DNA.

In a different study it has also been shown that monomeric Ca2+-loaded α-LA and apo-state α-LA alone can bind histone H3 *in vitro*, possibly through electrostatic interactions with several α-LA molecules bound per histone protein [101]. However, the finding should have no major implications on HAMLET studies, as native monomeric α-LA is unable to reach the cell nuclei of intact tumor cells [3,5,31].

HAMLET has been shown to act in synergy with histone deacetylase inhibitors, as pretreatment of Jurkat tumor cells with these resulted in enhanced lethal effect of HAMLET and an increased hyperacetylation response [100]. Caspase-independent DNA damage and DNA fragmentation was observed after treatment, suggesting that apoptosis was not the key pathway involved in the combined effect [100]. In addition, HAMLET caused chromatin condensation, as HAMLET-treatment of stably transfected HeLa cells resulted in a decrease of nuclear size. The ability of HAMLET to induce both caspase-dependent and caspaseindependent chromatin condensation pattern was found in another study, indicating that the response to HAMLET involves both classical apoptosis and caspase-independent cell death pathways [90].

### **16. Anti-adhesive properties of HAMLET**

The ability of HAMLET to facilitate tumor cell detachment has been described both in early and recent literature. Initially, MAL was found to possess anti-adhesive properties against bacterial attachment to alveolar type II lung carcinoma cells [1]. Subsequently, an *in vivo* experiment studying the therapeutic effects of HAMLET on bladder cancer showed that HAMLET triggers massive shedding of dead tumor cells into the urine, further indicating the anti-adhesive properties of HAMLET towards tumor cells [102]. Later, *in vitro* studies revealed that HAMLET binds to α-actinin-4, in addition to interacting with α-actinin-1, and causes detachment of A549 lung carcinoma cells [82]. Furthermore, a reduction in β1 integrin staining, as well as in FAK and ERK1/2 phosphorylation was observed, suggesting that HAMLET-treatment induced disruption of integrin-dependent cell adhesion signaling.

### **17. Therapeutic applications of HAMLET**

A major reason for studying the cytotoxic activity of HAMLET is the apparent specificity towards cancer cells. Although this specificity of cytotoxic HAMLET-induced cell death remains unclear, there have been continuous indications in many *in vitro* studies that HAMLET could have a therapeutic potential. As a consequence, researchers started conducting *in vivo* studies of the possible applications of HAMLET [68,102-105]. The proposed therapeutic applications of HAMLET and the observed effects are listed in Table 2. One problem in using HAMLET as a potential cancer agent is its interaction with albumin, which through binding of free fatty acids could neutralize the cytotoxic activity of HAMLET or BAMLET [53,87,102]. As albumin is present in nearly all physiologic fluids, it will in many cases be difficult for HAMLET to reach the target cancer cells in an active form.

The observed effects mentioned are *in vivo* unless otherwise noted. GBM: glioblastoma multiforme.

### **18. HAMLET as a skin papilloma treatment**

Papillomas are characterized as premalignant lesions of the skin and mucosal surfaces, caused by human papillomavirus transformation of keratinocytes [4,104]. The therapeutic treatment options are ineffective and include cryotherapy, immuno modulators, curettage, cautery, salicylic acid, CO2 laser, antimitotic agents, or photo-dynamic theraphy [106,107].

Skin papillomas were the first model selected for the examination of the *in vivo* effects of HAMLET [104]. Patients resistant to conventional treatments were included in this randomized, placebo-controlled, double-blinded study. HAMLET was shown to reduce the papilloma volume in 100% (20/20) of the patients and 96% of their lesions (88/92), compared to 15% in the placebo group patients (3/20) and 20% (15/74) of their lesions. By studying the long-term effects of HAMLET-treatment against skin papillomas, it was found that all lesions had completely resolved in 83% of the patients after 2 years. Furthermore, there were no observed differences between immunocompetent and immunosuppressed patients, indicating the potential of HAMLET-treatment for immunosuppressed skin papilloma patients, instead of the currently used invasive methods [104].

**Table 2.** Possible therapeutic applications of HAMLET.

132 Milk Protein

death pathways [90].

multiforme.

**16. Anti-adhesive properties of HAMLET**

**17. Therapeutic applications of HAMLET**

**18. HAMLET as a skin papilloma treatment**

of nuclear size. The ability of HAMLET to induce both caspase-dependent and caspaseindependent chromatin condensation pattern was found in another study, indicating that the response to HAMLET involves both classical apoptosis and caspase-independent cell

The ability of HAMLET to facilitate tumor cell detachment has been described both in early and recent literature. Initially, MAL was found to possess anti-adhesive properties against bacterial attachment to alveolar type II lung carcinoma cells [1]. Subsequently, an *in vivo* experiment studying the therapeutic effects of HAMLET on bladder cancer showed that HAMLET triggers massive shedding of dead tumor cells into the urine, further indicating the anti-adhesive properties of HAMLET towards tumor cells [102]. Later, *in vitro* studies revealed that HAMLET binds to α-actinin-4, in addition to interacting with α-actinin-1, and causes detachment of A549 lung carcinoma cells [82]. Furthermore, a reduction in β1 integrin staining, as well as in FAK and ERK1/2 phosphorylation was observed, suggesting that HAMLET-treatment induced disruption of integrin-dependent cell adhesion signaling.

A major reason for studying the cytotoxic activity of HAMLET is the apparent specificity towards cancer cells. Although this specificity of cytotoxic HAMLET-induced cell death remains unclear, there have been continuous indications in many *in vitro* studies that HAMLET could have a therapeutic potential. As a consequence, researchers started conducting *in vivo* studies of the possible applications of HAMLET [68,102-105]. The proposed therapeutic applications of HAMLET and the observed effects are listed in Table 2. One problem in using HAMLET as a potential cancer agent is its interaction with albumin, which through binding of free fatty acids could neutralize the cytotoxic activity of HAMLET or BAMLET [53,87,102]. As albumin is present in nearly all physiologic fluids, it will in

many cases be difficult for HAMLET to reach the target cancer cells in an active form.

The observed effects mentioned are *in vivo* unless otherwise noted. GBM: glioblastoma

Papillomas are characterized as premalignant lesions of the skin and mucosal surfaces, caused by human papillomavirus transformation of keratinocytes [4,104]. The therapeutic treatment options are ineffective and include cryotherapy, immuno modulators, curettage, cautery, salicylic acid, CO2 laser, antimitotic agents, or photo-dynamic theraphy [106,107].

Skin papillomas were the first model selected for the examination of the *in vivo* effects of HAMLET [104]. Patients resistant to conventional treatments were included in this randomized, placebo-controlled, double-blinded study. HAMLET was shown to reduce the papilloma volume in 100% (20/20) of the patients and 96% of their lesions (88/92), compared

### **19. HAMLET as a bladder cancer treatment**

The development of bladder cancers is common and remains a massive worldwide challenge, despite continuous advances in the therapeutic options available. The prevalence of bladder cancer is 1 in 4000 people, accounting for about 5% of all cancers, making it the fourth most common malignancy in the United States and the fifth most common in Europe [108]. The current treatments of bladder cancer typically involve intravesical instillation of antitumor agents such as *Bacillus* Calmette-Guerin, thiotepa, epirubicin and mitomycin C [109].

Early *in vitro* studies found that HAMLET induced rapid cell death in numerous cell lines, including bladder carcinoma cells [2]. The direct *in vivo* effect of HAMLET on bladder cancers was initially shown, when 9 male superficial bladder cancer patients received intravesical HAMLET-treatment during the week before scheduled transurethral surgery [102]. HAMLET stimulated a rapid increase in the daily shedding of dead tumor cells into the urine in 8 of 9 patients and an apoptotic response was detected by the TUNEL DNA fragmentation assay in 6 of 9 patients. In addition, a reduction in tumor size or a change in tumor character was observed at surgery in 8 of 9 patients.

### 134 Milk Protein

The therapeutic potential of HAMLET was further studied in a mouse bladder carcinoma model, with MB49 carcinoma cells installed via a catheter into the bladder of anesthetized mice, followed by five intravesical installations of HAMLET [105]. The treatment resulted in significantly decreased tumor areas and delayed the progression of bladder cancer compared to controls. Furthermore, whole body imaging of Alexa Fluor 568-labeled HAMLET revealed that the uptake and retention of the complex were tumor tissue-specific.

### **20. HAMLET in treatment of glioblastoma**

Gliomas are a heterogeneous group of intracranial neoplasms originating from neuroglial cells, which accounts for over 60% of all primary brain tumors and have an unfavorable prognosis [110-113]. Glioblastoma multiforme (GBM), grade IV on the current WHO grading-scale for tumors in the central nervous system, is the most malignant of the gliomas, showing a mean survival time of less than a year [113,114]. The current treatment is only palliative, involving surgery, radiotherapy and chemotherapy [115].

HAMLET has been shown to induce apoptosis in GBM biopsy spheroids *in vitro*, as detected by the TUNEL DNA fragmentation assay [68]. Similarly, *in vivo* studies of the effect of HAMLET-treatment on GBM's, established by xenotransplantation of human glioblastoma biopsy spheroids into nude rat brains, showed that HAMLET reduced the intracranial tumor volume and delayed the onset of pressure symptoms in tumor-xenografted rats [68,116].

### **21. HAMLET and involution of the mammary gland**

The development of the mammary gland through puberty, pregnancy and lactation has been intensively studied and much is known about the factors responsible for regulating the processes. When suckling stops, the gland goes into involution, and despite much research little is known regarding the factors involved in this remodeling of the gland.

Milk synthesis occurs in mammary epithelial organized in small hollow "balls" called alveoli. It is known that the accumulation of milk in the alveoli results in down regulation of protein synthesis as well as reduction in the number of secretory cells by apoptosis [117]. It is also believed that local factors secreted into the milk are responsible for the effect, but molecular details of such compounds are lacking. A protein called FIL (Feedback Inhibitor of Lactation) from milk has been isolated and partly characterized [118], but at present its existence is unconfirmed.

Some sea animals have a special lactation cycle due to long periods of foraging trips away from the offspring. Remarkably, despite the absence of suckling for many days no apoptosis occurs in the mammary gland of e.g. Cape fur seals, and this phenotype has been correlated with lack of α-LA in their milk [119]. The possibility that α-LA either alone or in a complex with fatty acids is involved in the regulation of involution has been suggested as apoptotic cell death in the mammary gland was enhanced by the introducing of HAMLET in the gland of lactating mice [103].

### **22. Conclusion**

134 Milk Protein

[68,116].

existence is unconfirmed.

gland of lactating mice [103].

The therapeutic potential of HAMLET was further studied in a mouse bladder carcinoma model, with MB49 carcinoma cells installed via a catheter into the bladder of anesthetized mice, followed by five intravesical installations of HAMLET [105]. The treatment resulted in significantly decreased tumor areas and delayed the progression of bladder cancer compared to controls. Furthermore, whole body imaging of Alexa Fluor 568-labeled HAMLET revealed that the uptake and retention of the complex were tumor tissue-specific.

Gliomas are a heterogeneous group of intracranial neoplasms originating from neuroglial cells, which accounts for over 60% of all primary brain tumors and have an unfavorable prognosis [110-113]. Glioblastoma multiforme (GBM), grade IV on the current WHO grading-scale for tumors in the central nervous system, is the most malignant of the gliomas, showing a mean survival time of less than a year [113,114]. The current treatment is only

HAMLET has been shown to induce apoptosis in GBM biopsy spheroids *in vitro*, as detected by the TUNEL DNA fragmentation assay [68]. Similarly, *in vivo* studies of the effect of HAMLET-treatment on GBM's, established by xenotransplantation of human glioblastoma biopsy spheroids into nude rat brains, showed that HAMLET reduced the intracranial tumor volume and delayed the onset of pressure symptoms in tumor-xenografted rats

The development of the mammary gland through puberty, pregnancy and lactation has been intensively studied and much is known about the factors responsible for regulating the processes. When suckling stops, the gland goes into involution, and despite much research

Milk synthesis occurs in mammary epithelial organized in small hollow "balls" called alveoli. It is known that the accumulation of milk in the alveoli results in down regulation of protein synthesis as well as reduction in the number of secretory cells by apoptosis [117]. It is also believed that local factors secreted into the milk are responsible for the effect, but molecular details of such compounds are lacking. A protein called FIL (Feedback Inhibitor of Lactation) from milk has been isolated and partly characterized [118], but at present its

Some sea animals have a special lactation cycle due to long periods of foraging trips away from the offspring. Remarkably, despite the absence of suckling for many days no apoptosis occurs in the mammary gland of e.g. Cape fur seals, and this phenotype has been correlated with lack of α-LA in their milk [119]. The possibility that α-LA either alone or in a complex with fatty acids is involved in the regulation of involution has been suggested as apoptotic cell death in the mammary gland was enhanced by the introducing of HAMLET in the

**20. HAMLET in treatment of glioblastoma**

palliative, involving surgery, radiotherapy and chemotherapy [115].

**21. HAMLET and involution of the mammary gland**

little is known regarding the factors involved in this remodeling of the gland.

The milk protein alpha-lactalbumin (α-LA) is one of the most studied proteins with respect to structure and function and has been a model for investigating intermediate folding conformations generally called molten globule states. It has a well-described function as part of the enzyme lactose synthetase, where α-LA is one component of the two-polypeptide enzyme. The other part is a non-specific galactosyl transferase, which in complex with α-LA becomes highly specific for forming lactose from UDP-galactose and glucose.

In the late nineties an entirely different activity of α-LA was discovered. When α-LA is in a partly unfolded conformation it can form a complex with oleic acid (OA) where the complex shows cell-killing activity specific for cancer cells. This partly unfolded conformation of α-LA can be obtained by removing a tightly bound calcium ion, leaving the protein in the apo-state capable of interacting directly with OA and thereby forming the cytotoxic complex. The complex between human α-LA and OA has been called HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) and similar complexes can be formed with α-LA from other species.

In cell culture systems HAMLET shows cytotoxic activity in µM concentrations but the activity varies greatly between cell types. The exact mechanism of cell-killing is not clear, but HAMLET has been shown to interact with numerous cell organelles including the nucleus, lysozymes, mitochondria, proteasomes and ribosomes. Apoptosis, autophagy and chromatin structure disorder are three different cytotoxic pathways described when HAMLET is added to cell cultures. It is therefore likely that different cells will show different death mechanisms dependent on the experimental conditions.

When the cytotoxic activity of α-LA/OA complexes are measured in a dose-response manner, it is characteristic that very steep curves are obtained where as little as a four-fold dilution can result in no activity compared to 100% cell death in the undiluted solution. This underlines the importance of defining the experimental conditions exactly when measuring cytotoxic activity as small changes in concentration might lead to contradictory conclusions. This aspect is especially important when the activity of HAMLET is compared between healthy differentiated normal cells and tumor cells. According to numerous reports HAMLET kills tumor cells as well as undifferentiated cells but leave normal cells untouched. This has recently been questioned as it was found that both normal white blood cells and fully developed erythrocytes were highly sensitive to the cell-killing activity.

The potential of HAMLET as a therapeutic agent has been investigated in three different models. First, skin papillomas were treated in a double-blinded study showing positive results compared to the controls, secondly, patients with bladder cancer indicated reduction in tumor size when the bladder was flushed with a solution of HAMLET, and finally, in a glioblastoma rat model HAMLET treatment resulted in a decrease of the intracranial tumor volume. This suggests that tumor cells in some cases might be more sensitive to HAMLET when compared with the healthy cells from which the tumors originate.

It has been theorized that HAMLET could be naturally formed in the digestive system of breast-fed children, due to low pH conditions of the stomach, serving the function as a natural scavenger and selective cytotoxic killer of cancerous cells in early infancy. The development of normal epithelia cells in the digestive system could also be affected. Obviously, many dairy products contains α-LA in fairly high concentrations and numerous production techniques, such as acid pH and heat treatment, facilitate the formation of partly unfolded α-LA making the protein ready to bind fatty acids and other similar compounds. To what degree such complexes in fact are formed in dairy products remains to be seen.

### **Author details**

Marcel Jøhnke and Torben E. Petersen\* *Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark*

### **23. References**

<sup>\*</sup> Corresponding Author

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136 Milk Protein

**Author details**

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[61] Rammer P, Groth-Pedersen L, Kirkegaard T, Daugaard M, Rytter A, Szyniarowski P, Høyer-Hansen M, Povlsen LK, Nylandsted J, Larsen JE, Jäättelä M (2010) BAMLET activates a lysosomal cell death program in cancer cells. Mol. cancer ther. 9: 24-32. [62] Lišková K, Kelly AL, O'Brien N, Brodkorb A (2010) Effect of denaturation of alphalactalbumin on the formation of BAMLET (bovine α-lactalbumin made lethal to tumor

[63] Zhang M, Yang F Jr, Yang F, Chen J, Zheng CY, Liang Y (2009) Cytotoxic aggregates of α-lactalbumin induced by unsaturated fatty acid induce apoptosis in tumor cells. Chem

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[69] Gustafsson L, Aits S, Onnerfjord P, Trulsson M, Storm P, Svanborg C (2009) Changes in proteasome structure and function caused by HAMLET in tumor cells. PLoS ONE 4:

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bovine milk α-lactalbumin. Biosci. biotechnol. biochem. 69: 1082-1089.

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Conserved features of cancer cells define their sensitivity to HAMLET-induced death; c-

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## **Processing on Milk Proteins**

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**Chapter 5**

## **Maillard Reaction in Milk – Effect of Heat Treatment**

Tomoko Shimamura and Hiroyuki Ukeda

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50079

### **1. Introduction**

Milk is usually subjected to heat treatment to ensure microbiological safety before retail and consumption. There are three types of heat treatment; (1) low temperature long time (LTLT) pasteurization, (2) high temperature short time (HTST) pasteurization, and (3) ultra-high temperature (UHT) treatment. In all types of heat treatment, the Maillard reaction occurs in milk.

The Maillard reaction (nonenzymatic glycation) is a chemical reaction between amino group and carbonyl group; it is the extremely complex reaction that usually takes place during food processing or storage. In the case of milk, lactose reacts with the free amino acid side chains of milk proteins (mainly ε-amino group of lysine residue) to proceed to early, intermediate, and advanced stages of Maillard reaction and forms enormous kinds of Maillard reaction products. The reactions of lactose and milk proteins have been frequently investigated and the formations of various Maillard reaction products in milk during heat treatment have been demonstrated [1]. In the general Maillard reaction, firstly an Amadori product is generated, and it progresses to the 3-deoxyosone or 1-deoxyosone route depending on the reaction pH. In the case of the Maillard reaction of disaccharides such as lactose, there is a third reaction route. It is the 4-deoxyosone route. A main carbohydrate in milk is lactose. Thus, the Maillard reaction in milk progresses via the above described three routes. Finally, the Maillard reaction results in the formation of melanoidins (browning compounds).

### **2. Effect of the Maillard reaction on milk proteins**

The Maillard reaction shows various effects on milk proteins such as bioavailability, solubility, forming property, emulsifying property, and heating stability [1-4]. In addition,

### 148 Milk Protein

the formation of flavor compounds and browning compounds is caused as the consequences of the Maillard reaction between lactose and milk proteins [1, 5].

As for the effect of the Maillard reaction on the bioavailability of milk proteins, various studies were performed. Generally, in the Maillard reaction in milk, lactose mainly reacts with ε-amino group of lysine residue of milk proteins. Thus, the lysine loss by the Maillard reaction increases with a severity of heat treatment. The modified lysine cannot be available as a nutrient any more. For example, steam injection process (direct heating) generated 3.6% (120°C for 400 sec) and 6.8% (130°C for 290 sec) of the blocked lysine in whole milk. The indirect heating at 115°C for 10 to 40 min increased the modified lysine from 11.0 to 13.0% [6]. In addition, it was revealed that the lysine residues in skim milk powder were more susceptible to heating than those in skim milk [7].

Le *et al.* [3] recently suggested that the Maillard reaction was responsible for the solubility loss in milk protein concentrate powder. It was also reported that the glycated βlactoglobulin was more stable at acidic pH and more stable against heating. The glycation of β-lactoglobulin, moreover, could improve its forming and emulsifying properties [4]. These results suggested the usefulness of the Maillard reaction for enabling milk proteins to have different properties.

### **3. Monitoring of the Maillard reaction of milk using XTT assay**

The Maillard reaction has a lot of effects on the function of milk proteins and sensory property of milk and dairy products as described above. Particularly, in the manufacturing of milk, the excess progress of Maillard reaction and the formation of melanoidins are undesired, because a commercial value of milk is drastically decreased by them. Therefore, the detection of the Maillard reaction products is important for the quality control of milk. So far, several heat-induced markers have been proposed to control and check the heat treatment given to milk and dairy products. For example, furosine, hydroxymethylfurfural (HMF), and lactulose concentrations have been recognized to be the most promising indicators, since these concentrations increase with the heat treatment [1]. In Japan, protein reducing substance value (PRS) obtained by a ferricyanide assay is also widely-used conventional indicator [8]. It is based on the detection of reducing substances such as sulfhydryl group which are generated by heating in the fraction of acid-precipitated milk protein. These method, however, are generally time-consuming and complicated.

We proposed an assay method for determining the ability of milk to reduce 3'-[1- [(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzensulfonic acid hydrate (XTT: Figure 1) as a method of evaluating the extent of the Maillard reaction [8-11]. The tetrazolium salt XTT is reduced to water-soluble formazan which is suitable for the spectrophotometric measurement. Taking account into an economical view and a rapidity of assay, we tried to develop a microplate assay. The assay conditions were as follows: XTT concentration, 0.5 mM; reaction pH, 7.0; menadione concentration, saturation level (ca. 0.55 mM); reaction temperature, room temperature; volume of XTT solution, 60 μL; volume of sample solution, 40 μL; detection wavelength, 492 nm; reference wavelength, 600 nm; reaction time, 20 min. The procedure of the XTT assay was as follows: the XTT solution was added into each well in a microplate. Afterward, the sample solution was added to the well. After mixing on a microplate shaker at 500 rpm for 15 sec, a difference in the absorbance between 492 and 600 nm was read on a microplate reader as the absorbance at 0 min. After 20 min, the absorbance difference was read again. An increase in the absorbance for 20 min was recorded as the ability of the sample to reduce XTT (XTT reducibility).

**Figure 1.** Structure of tetrazolium salt XTT.

148 Milk Protein

the formation of flavor compounds and browning compounds is caused as the consequences

As for the effect of the Maillard reaction on the bioavailability of milk proteins, various studies were performed. Generally, in the Maillard reaction in milk, lactose mainly reacts with ε-amino group of lysine residue of milk proteins. Thus, the lysine loss by the Maillard reaction increases with a severity of heat treatment. The modified lysine cannot be available as a nutrient any more. For example, steam injection process (direct heating) generated 3.6% (120°C for 400 sec) and 6.8% (130°C for 290 sec) of the blocked lysine in whole milk. The indirect heating at 115°C for 10 to 40 min increased the modified lysine from 11.0 to 13.0% [6]. In addition, it was revealed that the lysine residues in skim milk powder were more

Le *et al.* [3] recently suggested that the Maillard reaction was responsible for the solubility loss in milk protein concentrate powder. It was also reported that the glycated βlactoglobulin was more stable at acidic pH and more stable against heating. The glycation of β-lactoglobulin, moreover, could improve its forming and emulsifying properties [4]. These results suggested the usefulness of the Maillard reaction for enabling milk proteins to have

The Maillard reaction has a lot of effects on the function of milk proteins and sensory property of milk and dairy products as described above. Particularly, in the manufacturing of milk, the excess progress of Maillard reaction and the formation of melanoidins are undesired, because a commercial value of milk is drastically decreased by them. Therefore, the detection of the Maillard reaction products is important for the quality control of milk. So far, several heat-induced markers have been proposed to control and check the heat treatment given to milk and dairy products. For example, furosine, hydroxymethylfurfural (HMF), and lactulose concentrations have been recognized to be the most promising indicators, since these concentrations increase with the heat treatment [1]. In Japan, protein reducing substance value (PRS) obtained by a ferricyanide assay is also widely-used conventional indicator [8]. It is based on the detection of reducing substances such as sulfhydryl group which are generated by heating in the fraction of acid-precipitated milk

**3. Monitoring of the Maillard reaction of milk using XTT assay**

protein. These method, however, are generally time-consuming and complicated.

We proposed an assay method for determining the ability of milk to reduce 3'-[1- [(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzensulfonic acid hydrate (XTT: Figure 1) as a method of evaluating the extent of the Maillard reaction [8-11]. The tetrazolium salt XTT is reduced to water-soluble formazan which is suitable for the spectrophotometric measurement. Taking account into an economical view and a rapidity of assay, we tried to develop a microplate assay. The assay conditions were as follows: XTT concentration, 0.5 mM; reaction pH, 7.0; menadione concentration, saturation level (ca. 0.55 mM); reaction temperature, room temperature; volume of XTT solution, 60 μL; volume of sample solution, 40 μL; detection wavelength, 492 nm; reference wavelength, 600 nm;

of the Maillard reaction between lactose and milk proteins [1, 5].

susceptible to heating than those in skim milk [7].

different properties.

Using the XTT assay described above, the XTT reducibility of milk was examined (Figure 2). In this test, an LTLT milk (Milk A: 65°C for 30 min) and two kinds of UHT milk (Milk B: 130°C for 2 sec, Milk C: 140°C for 3 sec) were used. When the milk was mixed with XTT solution, intense orange color which was derived from the XTT formazan was recognized. As shown in Figure 2, Milk C showed the highest XTT reducibility and the order of XTT reducibility was Milk C, B, and A. At the same time, the HMF content in Milk B and C was also determined as the conventional indicator of the Maillard reaction, because it was reported that the HMF content clearly increased with the severity of the heating treatment of milk [12, 13]. As a result, the content of HMF in Milk C (12.6 μM) was higher than that of Milk B (8.95 μM). From these results, it was revealed that the XTT assay could estimate the degree of thermal stress delivered to the milk as well as the HMF value.

In addition, the changes in the XTT reducibility during storage of UHT milks (Milk B and C) were investigated. For purpose of comparison, the PRS of Milk B and C were also examined by the ferricyanide assay [8]. The UHT milks were stored at 4°C and 37°C for about 4 weeks (Figure 3, 4). The XTT reducibility of Milk B and C gradually decreased depending on the storage period and the rate of decrease in the XTT reducibility was clearly larger at higher storage temperature (Figure 3). This result strongly suggested that, when the XTT assay is applied to the milk heated under a given condition, the result can serve to estimate not only the heating condition but also the storage period after heat treatment if the storage temperature is known or if storage period is known.

On the other hand, the PRS of Milk B and C were almost constant at all storage conditions (Figure 4). This result showed that the ferricyanide-reducing substances might be stable unlike the XTT-reducing substance. In addition, we found that the HMF value of Milk C did not significantly change during its storage for 60 days at 5°C [11]. It was in accordance with the result of Fink and Kessler about HMF [13]. They also reported that the lactulose concentration of UHT milk was constant throughout the storage period for 70 days at room temperature [13]. It could be concluded, therefore, that the XTT assay is applicable for the estimation of storage conditions which was impossible by the conventional method.

**Figure 2.** Comparison of XTT reducibility of LTLT milk (Milk A) and UHT milks (Milk B and C).

Milks were stored at 4°C (●, Milk B; ■, Milk C) and 37°C (○, Milk B; □, Milk C). **Figure 3.** Changes in XTT reducibility during storage.

Milks were stored at 4°C (●, Milk B; ■, Milk C) and 37°C (○, Milk B; □, Milk C).

**Figure 4.** Changes in PRS during storage.

150 Milk Protein

the result of Fink and Kessler about HMF [13]. They also reported that the lactulose concentration of UHT milk was constant throughout the storage period for 70 days at room temperature [13]. It could be concluded, therefore, that the XTT assay is applicable for the

estimation of storage conditions which was impossible by the conventional method.

**Figure 2.** Comparison of XTT reducibility of LTLT milk (Milk A) and UHT milks (Milk B and C).

Milk B 130°C 2 sec

0 5 10 15 20 25 30

Storage period (days)

Milk C 140°C 3 sec

Milks were stored at 4°C (●, Milk B; ■, Milk C) and 37°C (○, Milk B; □, Milk C).

**Figure 3.** Changes in XTT reducibility during storage.

0

0.1

0.2

0.3

XTT reducibility

0.4

0.5

0

Milk A 65°C 30 min

0.1

0.2

0.3

XTT reducibility

0.4

0.5

### **4. Demonstration of the presence of aminoreductone formed during the Maillard reaction in milk**

### **4.1. In the model system of lactose and butylamine [9]**

In order to clarify the XTT-reducing substance that is formed during the Maillard reaction in milk, we firstly used a model system consisting of lactose and butylamine, and then performed the spectrophotometric analysis of the heated model solution. The model solution of lactose-butylamine heated at 80-100°C for 0-30 min showed a characteristic UV absorption maximum at 320 nm. During the heating at 80-100°C for 30 min, the changes in the absorbance at 320 nm (Figure 5) and the XTT reducibility (Figure 6) were investigated. As a result, both indices increased gradually in accordance with the rise in temperature and heating time. This result indicated that the compound with the absorption maximum at 320 nm was formed by heating. Moreover, the behavior of increase in absorbance at 320 nm was similar to that of the XTT reducibility. Since the similar trend was recognized in the time course of both indices, the XTT reducibility was plotted against the absorbance at 320 nm. In consequence, a significant relationship between them was recognized with a correlation coefficient of 0.967 (n = 19, *p* < 0.001). From these results it was found that the compound with the absorption maximum at 320 nm might be responsible for the reduction of XTT.

The 13C- and 1H-NMR analyses of the compound with the absorption maximum at 320 nm which was extracted from lactose-butylamine model solution heated at 100°C for 15 min were performed. The signals of the 13C- and 1H-NMR could be assigned the compound as the aminoreductone, 1-(butylamino)-1,2-dehydro-1,4-dideoxy 3-hexulose (Figure 7). This compound was reported as the Maillard reaction product formed in the 4-deoxyosone route. It was also reported as the characteristic compound in the Maillard reaction of disaccharides [14]. In addition, we demonstrate a linear relationship between the XTT reducibility and the amount of aminoreductone which was determined more specifically by HPLC [10]. These results strongly indicated that the aminoreductone formed during Maillard reaction of lactose was mainly responsible for the reduction of XTT.

Lactose (262 mM) and butylamine (1.16 M) in 1.28 M phosphate buffer (pH 7.0) were heated at 80°C (●), 90°C (○), and 100°C (■).

**Figure 5.** Effect of heating temperature and time on the absorbance at 320 nm.

Lactose (262 mM) and butylamine (1.16 M) in 1.28 M phosphate buffer (pH 7.0) were heated at 80°C (●), 90°C (○), and 100°C (■).

**Figure 6.** Effect of heating temperature and time on the XTT reducibility.

**Figure 7.** Structure of aminoreductone generated by the Maillard reaction of lactose and butylamine. (R = butyl group)

From these results, we presumed that the aminoreductone is formed by the Maillard reaction between lactose and ε-amino groups of milk proteins, and then it is responsible for the reduction of XTT. However, at that time, there was no report to prove the presence of aminoreductone in milk. Thus, we tried to demonstrate it using model system consisting of lactose and milk proteins and UHT milk.

### **4.2. In the model system of lactose and milk proteins**

As a model system of milk, the solution consisting of lactose (4.6%) and casein (2.6%), αlactalbumin (0.12%), or β-lactoglobulin (0.32%) was used and heated at 130°C for 15 min. After heating, the characteristic absorption maximum or shoulder at 320 nm was recognized. In addition, the changes of the absorbance at 320 nm (Figure 8) and the XTT reducibility (Figure 9) were investigated. In all model systems, the increases in the absorbance at 320 nm and the XTT reducibility depended on the heating time. Because similar tendencies were observed between two indices in all model systems, correlations were examined. Consequently, there were significant linearities as follows: casein (r = 0.993, n = 6, *p* < 0.001), α-lactalbumin (r = 0.996, n = 6, *p* < 0.001), and β-lactoglobulin (r = 0.975, n = 6, *p* < 0.001). From these results, it was suggested that aminoreductone is generated in the Maillard reaction between lactose and milk proteins and it is responsible for the reduction of XTT.

### **4.3. In milk [15]**

152 Milk Protein

100°C (■).

0

0

10

20

30

XTT reducibility

40

50

60

500

1000

Absorbance at 320 nm

1500

2000

100°C (■).

It was also reported as the characteristic compound in the Maillard reaction of disaccharides [14]. In addition, we demonstrate a linear relationship between the XTT reducibility and the amount of aminoreductone which was determined more specifically by HPLC [10]. These results strongly indicated that the aminoreductone formed during Maillard reaction of

Lactose (262 mM) and butylamine (1.16 M) in 1.28 M phosphate buffer (pH 7.0) were heated at 80°C (●), 90°C (○), and

0 5 10 15 20 25 30

Heating time (min)

Lactose (262 mM) and butylamine (1.16 M) in 1.28 M phosphate buffer (pH 7.0) were heated at 80°C (●), 90°C (○), and

0 5 10 15 20 25 30

Heating time (min)

**Figure 5.** Effect of heating temperature and time on the absorbance at 320 nm.

**Figure 6.** Effect of heating temperature and time on the XTT reducibility.

lactose was mainly responsible for the reduction of XTT.

As described above, a possibility of the formation of aminoreductone on the milk proteins during the Maillard reaction with lactose was clearly shown. However, direct demonstration of the presence of aminoreductone in milk had not been accomplished because of a difficulty in isolation of an intact aminoreductone from milk proteins. For instance, aminoreductone is labile and hence not suitable for enzyme hydrolysis and multiple extraction steps. To achieve the practical application of the XTT assay in food industries including dairy products, it was essential to demonstrate the presence of aminoreductone in milk. Because of this background, we attempted to isolate aminoreductone from milk proteins using 2,4-dinitrophenylhydrazine (DNP), a common labeling reagent for the carbonyl group, and Cu2+ [16]. A mechanism of derivatization of aminoreductone in milk is shown in Figure 10. In this derivatization step, Cu2+ plays as an oxidizing agent against aminoreductone, and the oxidized aminoreductone (OAR) has two or three carbonyl groups. Finally, it was assumed that two or three carbonyl groups in OAR are derivatized by DNP (OAR-DNP).

Lactose (4.6%) and casein (■: 2.6%), α-lactalbumin (●: 0.12%), or β-lactoglobulin (▲: 0.32%) in 20 mM phosphate buffer (pH 6.7) were heated at 130°C.

**Figure 8.** Effect of heating time on the absorbance at 320 nm.

Lactose (4.6%) and casein (■: 2.6%), α-lactalbumin (●: 0.12%), or β-lactoglobulin (▲: 0.32%) in 20 mM phosphate buffer (pH 6.7) were heated at 130°C.

**Figure 9.** Effect of heating time on the XTT reducibility.

**Figure 10.** Derivatization mechanism of aminoreductone in milk using DNP and Cu2+.

154 Milk Protein

are derivatized by DNP (OAR-DNP).

(pH 6.7) were heated at 130°C.

0

2

4

6

Absorbance at 320 nm

8

10

(pH 6.7) were heated at 130°C.

0

0.15

XTT reducibility

0.3

0.45

**Figure 9.** Effect of heating time on the XTT reducibility.

**Figure 8.** Effect of heating time on the absorbance at 320 nm.

aminoreductone in milk is shown in Figure 10. In this derivatization step, Cu2+ plays as an oxidizing agent against aminoreductone, and the oxidized aminoreductone (OAR) has two or three carbonyl groups. Finally, it was assumed that two or three carbonyl groups in OAR

Lactose (4.6%) and casein (■: 2.6%), α-lactalbumin (●: 0.12%), or β-lactoglobulin (▲: 0.32%) in 20 mM phosphate buffer

0 3 6 9 12 15

Heating time (min)

Lactose (4.6%) and casein (■: 2.6%), α-lactalbumin (●: 0.12%), or β-lactoglobulin (▲: 0.32%) in 20 mM phosphate buffer

0 3 6 9 12 15

Heating time (min)

The derivatization using DNP and Cu2+ was applied to aminoreductone in UHT milk (140°C, 3 sec). In this study, the UHT milk was reheated at 130°C for 15 min in order to increase the content of aminoreductone, because the original content of aminoreductone in the commercially available UHT milk was not so high. As a result, the reheating of UHT milk could increase the content of DNP derivative by 40 times. The DNP derivative which was thought to be corresponding to OAR-DNP in the reheated UHT milk was purified by preparative normal-phase HPLC and preparative reversed-phase HPLC. Finally, the purified compound (4.2 mg) was obtained from 980 mL of UHT milk and analyzed by 13Cand 1H-NMR. The NMR signals of the DNP derivative from UHT milk could be assigned to the structure of OAR-DNP shown in Figure 10. In addition, the NMR signals of DNP derivative from UHT milk were nearly the same as those of the OAR-DNP from lactosebutylamine model system. These results demonstrated that aminoreductone was formed by the Maillard reaction on the milk proteins and present in milk.

Therefore, considering the above, the principle of the present XTT assay can be concluded as follows (Figure 11): (1) Lactose and ε-amino groups of lysine residue in milk proteins react non-enzymatically to form the Amadori product by the heating process. (2) Aminoreductone structure is formed on the milk proteins after elimination of galactose moiety from lactose through 4-deoxyosone pathway. (3) Aminoreductone is oxidized by XTT, whereas XTT is simultaneously reduced to the corresponding water-soluble formazan.

It would be thought that the above mentioned steps (1) and (2) progresses depending on the time and temperature of heating process in milk production, so the XTT assay can differentiate the extent of heat treatment of milk. Based on the study using model system of lactose and butylamine, the relationship between aminoreductone concentration and XTT reducibility was examined. As a result, there was a good linearity was recognized (r = 0.98) and a regression equation was *y* = 0.606 *x* + 0.046, in which *x* and *y* represented the concentration of aminoreductone (mM) and the XTT reducibility [17]. Based on this equation, the concentration of aminoreductone in UHT milk could be estimated as 0.44 mM.

**Figure 11.** Principle of XTT assay.

As described above, during the course of elucidation of the XTT-reducing substance, Cu2+ was used as the oxidizing agent (Figure 10), because we empirically knew that it was easily decomposed by the addition of Cu2+ [18]. In fact, the aminoreductone extracted from the heated model solution of lactose and butylamine was rapidly oxidized by the addition of Cu2+, and simultaneously the XTT reducibility was also lost. On the other hand, in our previous work, it was revealed that the commercially available UHT milk contains Cu2+ at the concentration of 30 μg/L [18]. Thus, it was easily presumed that the aminoreductone formed by the heating process was gradually oxidized by endogenous Cu2+ in milk during storage period. This was the reason why that the XTT reducibility decreased depending on the storage period (Figure 3). The detailed investigation about the relationship between the aminoreductone concentration, Cu2+ concentration, and storage stability of milk is now in progress.

### **5. Functionality of aminoreductone**

The functionalities of aminoreductone have attracted interest and, so far, some studies were performed. Trang *et al.* [17] reported a protective effect of aminoreductone against riboflavin (vitamin B2) photolysis. It is well known that the milk is important source of riboflavin and its content is 1.5 mg/L. It is stable to heat and oxidation, but is rapidly photo-degraded. In experimental condition at 7000 lux light intensity, the riboflavin (1.5 mg/L) was almost completely degraded for 150 min. On the other hand, the addition of aminoreductone (0.22 mM: half concentration in UHT milk) could extend the half-life period of riboflavin. The protective effect of aminoreductone against riboflavin photolysis was higher than that of ascorbic acid which was famous antioxidant. In addition, the antioxidative activity of aminoreductone was reported [19]. From these results, it was suggested that the aminoreductone formed by the Maillard reaction would contribute to keep the nutritional value and sensory quality of milk.

Furthermore, aminoreductone showed antimicrobial activity against *Helicobacter pylori* (*H. pylori*). *In vitro* it effectively inhibited the growth of 24 kinds of *H. pylori* strains including antibiotic-resistant strains and had a bactericidal activity [20, 21]. The Killing ability was observed even in acidic condition. In addition, aminoreductone also had the antimicrobial activity against methicillin-resistant *Staphylococcus aureus* (MRSA) [21, 22]. These results indicated that foods containing aminoreductone, such as milk and dairy products, have a potential health benefits in medical practice.

### **6. Conclusion**

156 Milk Protein

C OH C O CH2 CHOH CH2OH

Aminoreductone bound

**5. Functionality of aminoreductone**

to milk proteins

**Figure 11.** Principle of XTT assay.

progress.

HC NH

Amadori product

(1)

(2)

NH

C O

Oxidized form of aminoreductone

As described above, during the course of elucidation of the XTT-reducing substance, Cu2+ was used as the oxidizing agent (Figure 10), because we empirically knew that it was easily decomposed by the addition of Cu2+ [18]. In fact, the aminoreductone extracted from the heated model solution of lactose and butylamine was rapidly oxidized by the addition of Cu2+, and simultaneously the XTT reducibility was also lost. On the other hand, in our previous work, it was revealed that the commercially available UHT milk contains Cu2+ at the concentration of 30 μg/L [18]. Thus, it was easily presumed that the aminoreductone formed by the heating process was gradually oxidized by endogenous Cu2+ in milk during storage period. This was the reason why that the XTT reducibility decreased depending on the storage period (Figure 3). The detailed investigation about the relationship between the aminoreductone concentration, Cu2+ concentration, and storage stability of milk is now in

The functionalities of aminoreductone have attracted interest and, so far, some studies were performed. Trang *et al.* [17] reported a protective effect of aminoreductone against riboflavin (vitamin B2) photolysis. It is well known that the milk is important source of riboflavin and its content is 1.5 mg/L. It is stable to heat and oxidation, but is rapidly photo-degraded. In experimental condition at 7000 lux light intensity, the riboflavin (1.5 mg/L) was almost completely degraded for 150 min. On the other hand, the addition of aminoreductone (0.22 mM: half concentration in UHT milk) could extend the half-life period of riboflavin. The protective effect of aminoreductone against riboflavin photolysis was higher than that of ascorbic acid which was famous antioxidant. In addition, the antioxidative activity of

XTT formazan

XTT

(3)

O C

HN

(CH2)4 CH

Lactose + Milk protein (casein etc.)

In this chapter, the demonstration of the presence of aminoreductone formed by the Maillard reaction in milk and the specific assay method for aminoreductone were focused and introduced. Since the novel functionality of aminoreductone have come out one after another, the information of aminoreductone obtained by the XTT assay would gain importance in the quality control in milk and dairy products.

### **Author details**

Tomoko Shimamura\* and Hiroyuki Ukeda *Faculty of Agriculture, Kochi University, Nankoku, Japan*

### **7. References**

<sup>\*</sup> Corresponding Author

**Complexation Between Casein Micelles and Whey Protein by Indirect UHT- Processing of Milk: Influence of Surface Hydrophobicity and Dye-Binding Characteristics of Micelles in Relationship with Their Physico-Functionality**

Bärbel Lieske

158 Milk Protein

(in Japanese) 48: 840-843.

48: 6227-6229.

Chem. 46: 928-931.

Chem. 129: 1088-1092.

Tokyo 4: 258-263.

Agric. Food Chem. 46: 2945-2950.

Agric. Food Chem. 57: 11343-11348.

Food Chemistry 1: 1-12.

Food Chem. 59: 8953-8960.

[8] Shimamura T., Ukeda H., Sawamura M. (2001) Quality evaluation of milk using XTT assay –comparison with ferricyanide assay. Nippon Shokuhin Kagaku Kogaku Kaishi

[9] Shimamura T., Ukeda H., Sawamura M. (2000) Reduction of tetrazolium salt XTT by aminoreductone formed during the Maillard reaction of lactose. J. Agric. Food Chem.

[10] Shimamura T., Ukeda H., Sawamura M. (2004) Relationship between the XTT reducibility and aminoreductone formed during the Maillard reaction of lactose: the

[11] Ukeda H., Goto Y., Sawamura M., Kusunose H., Kamikado H., Kamei T. (1995) Reduction of tetrazolium salt XTT with UHT-treated milk: its relationship with the

[13] Fink R., Kessler H. G. (1988) Comparison of methods for distinguishing UHT treatment

[14] Pischetsrieder M., Schoetter C., Severin T. (1998) Formation of an aminoreductone during the Maillard reaction of lactose and *Nα*-acetyllysine or proteins. J. Agric. Food.

[15] Shimamura T., Kurogi Y., Katsuno S., Kashiwagi T., Ukeda H. (2011) Demonstration of the presence of aminoreductone formed during the Maillard reaction in milk. Food

[16] Shinohara K., Fukumoto Y., Tseng Y.-K., Inoue Y., Omura H. (1974) Action of amino reductones on nucleic acids. Nippon Nogei Kagaku Kaishi (in Japanese) 48: 499-506. [17] Trang V. T., Kurogi Y., Katsuno S., Shimamura T., Ukeda H. (2008) Protective effect of

[18] Ukeda H., Shimamura T., Hosokawa T., Goto Y., Sawamura M. (1998) Monitoring of the Maillard reaction based on the reduction of tetrazolium salt XTT. Food Sci. Technol. Int.

[19] Pischetsrieder M., Rinaldi F., Gross U., Severin T. (1998) Assessment of the antioxidative and prooxidative activities of two aminoreductones formed during the Maillard reaction: effects on the oxidation of *β*-carotene, *Nα*-acetylhistidine, and *cis*-alkenes. J.

[20] Trang V. T., Takeuchi H., Kudo H., Aoki A., Katsuno S., Shimamura T., Sugiura T., Ukeda H. (2009) Antimicrobial activity of aminoreductone against *Helicobacter pylori*. J.

[21] Takeuchi H., Shimamura T., Kudo H., Ukeda H., Sugiura T. (2012) Antimicrobial activity of natural products and food components. Current Research in Agriculture and

[22] Trang V. T., Takeuchi H., Kudo H., Katsuno S., Shimamura T., Kashiwagi T., Son V. H., Sugiura T., Ukeda H. (2011) *In vitro* antimicrobial activity of aminoreductone against the pathogenic bacteria methicillin-resistant *Staphylococcus aureus* (MRSA). J. Agric.

extent of heat treatment and storage conditions. Food Sci. Technol. Int. 1: 52-57. [12] Kessler H. G., Fink R. (1986) Changes in heated milk and stored milk an interpretation

detection of aminoreductone by HPLC. Food Sci. Technol. Res. 10: 6-9.

by reaction kinetics. J. Food Sci. 51: 1105-1111.

and sterilization of milk. Milchwissenschaft 43: 275-280.

on the photo-degradation of riboflavin. Int. Dairy J. 18: 344-348.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/45981

### **1. Introduction**

Milk is nearly always heat treated and the aim of heating was to make the milk safe for human health by killing microorganisms and inactivating the enzymes. Now there has been consistent interest in more novel and sophisticated strategies to improve the rheological properties and stability of acid gels by a more tailored complexation between casein and whey protein *via* SH/SS-exchange. The most frequented approach explored has been (ultra)high-pressure processing. Pressure treatment alone decreases the casein micelle size; however, this effect was less marked when heat and pressure treatments were combined and gels formed on acidification revealed a range of firmness, yield stresses and yield strains which were not to related to whey protein denaturation by oneself (1).Today, the understanding of the effects of high pressure seems quite advanced, e.g.; that the casein micelle properties were changed and that the denaturation of whey protein affected the gel assembly process in cheese and yoghurt (2). Investigations in the effect of high pressure (in the range of 50 to 350 MPa) on constituents of the colloidal phase of milk revealed that nondenatured β-lactoglobulin is left reaching maximum value of 62% in the range of 100 to 350 MPa (7). Other studies addressed the effects of pressure, pH, and temperature on the casein micelle dissociation in skim milk during high-pressure treatment with measurements of light scattering. A relationship between the barostability of casein micelles and pH was established, whereas no effect of temperature was ascertainable, and a mechanism for high pressure-induced disruption of micelle integrity is suggested in which the state of calcium

© 2012 Lieske, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Lieske, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

plays a crucial role in the micelle dissociation process (8). A study in high pressure effects on the structure of casein micelles in milk by cryo-transmission electron microscopy showed that in the range 150 to 300 MPa a large number of small micelles coexisted with a fraction of large micelles, which appeared perfectly spherical with smooth and well-defined surfaces (6).

The manufacture of acid gels from ultra-high pressure homogenization-treated milk in combination with a thermal treatment (90 °C/5min) resulted in a firmer gel quality to be used for improving the rheological properties and stability of yoghurt and, thus decreasing the need for additives (5). This expectation proved to be right as shown in two further experimental studies dealing with acid coagulation properties of set and stirred yoghurt (3, 4).

Thermo-sonification has proved to be an alternative technique to extent the shelf life of whole milk (10) and the combination of preheating (45°C) with thermosonification (24 kHz for 10 min) allowed the preparation of yoghurts with rheological properties superior (9) to those of control yoghurts produced from conventionally heated milk (90°C for 10min) (11). The effects of preheating milk (80°C for 10min) for preparing acid milk gels were studied with reference to the preheating pH by dynamic oscillation measurements resulting in firmer gel qualities at a higher pH (>6,65), probably due to greater involvement of S-S interactions (12). A short-time of preheating in terms of procedure is also an element of ultrahigh temperature processing (UHT) of milk which is well-tried in the dairy practice today. Indirect UHT processing was used to prepare dairy desserts for studying the influence of varying concentrations of additives on the dessert rheology. As a result, a more extensive whey protein denaturation and subsequent complexation with casein micelles is believed to contribute to the improvement of rheological properties of the UHT desserts (13).

Own research into the effects on indirect UHT processing are in agreement with this and confirmed that perfect complexation may result only by the indirect procedure (14). Moreover, perfect complexation and inherent physico-functionality of the protein were to set not only by the technology alone but also by the current colloid-chemical status of the casein micelles in milk. In herd milk, this status may vary considerably in one period of lactation and inherent negative consequences are seen too late in the finished product.

This research was aimed at the investigation into the influence of a varying colloidal status of casein micelles on the resulting micellar complexation with whey protein on indirect UHT processing of skim milk. For this an own colloid-chemical approach is used to explain the structural changes of casein micelles with given examples of four herd milk samples and one dairy-based product for the purpose of confirmation

### **2. Materials and methods**

### **2.1. Raw milk and thermal treatments**

Bulked herd milk was collected from one farm in Schleswig-Holstein for a period of time of 8 month. Both separation of milk and thermal treatment were carried out using certified equipment. The skimmed milk was first regenerative (principle of heat recovery) preheated to 70-85 °C and then heated indirectly (126 °C for 4 s) and after that cooled down to 5 °C.

### **2.2. Spectrophotometric analysis of ß-lactoglobulin (ß-Lg)**

For quantification of β-Lg (15) from the pH 4,6 -filtrate a spectrophotometric method was applied using a molar absorption coefficient of 0.950 for 0, 1 % (w/w) β-Lg at 280 nm. All spectrophotometric results are means of triplicate determinations repeated twice.

### **2.3. Surface hydrophobicity of proteins in milk**

The development of this method is dated back to 1990/91 and was refined further until 2004. The initial interest was to know the reason of interference of the non-ionic detergent Tween 80 in the Bio-Rad Protein Assay with a variety of proteins. The detergent binding turned out to be a measure of hydrophobic surface area of integral proteins and thus constituting the basis for an assay procedure to determine the hydrophobic potential on the surface of proteins. At first, the methodical research was turned to analyze only isolated proteins (16). A methodical description is given below.

### *2.3.1. Reagent and sample preparation*

*Detergent reagent:* 0,25 % Tween ("commercial-grade") is prepared in destilled and/or deionized water, stored at room temperature for max. 3 days.

*Dye reagent*: The dye reagent is purchased as a five-fold concentrate, which must be diluted and filtered through a large-pored filter prior further use.

*Sample preparation:* Proteins are diluted to about 0,05-0,10 % using *aqua dest.* or 0,05M phosphate buffer. As a rule, the colour intensity of 50 μL diluted sample developed with 2,5 mL dye reagent should not exceed A 595nm, 1cm = 0,500 vs. *aqua dest*. or buffer.

### *Additional items required:*

160 Milk Protein

plays a crucial role in the micelle dissociation process (8). A study in high pressure effects on the structure of casein micelles in milk by cryo-transmission electron microscopy showed that in the range 150 to 300 MPa a large number of small micelles coexisted with a fraction of large micelles, which appeared perfectly spherical with smooth and well-defined surfaces (6).

The manufacture of acid gels from ultra-high pressure homogenization-treated milk in combination with a thermal treatment (90 °C/5min) resulted in a firmer gel quality to be used for improving the rheological properties and stability of yoghurt and, thus decreasing the need for additives (5). This expectation proved to be right as shown in two further experimental studies dealing with acid coagulation properties of set and stirred yoghurt (3, 4). Thermo-sonification has proved to be an alternative technique to extent the shelf life of whole milk (10) and the combination of preheating (45°C) with thermosonification (24 kHz for 10 min) allowed the preparation of yoghurts with rheological properties superior (9) to those of control yoghurts produced from conventionally heated milk (90°C for 10min) (11). The effects of preheating milk (80°C for 10min) for preparing acid milk gels were studied with reference to the preheating pH by dynamic oscillation measurements resulting in firmer gel qualities at a higher pH (>6,65), probably due to greater involvement of S-S interactions (12). A short-time of preheating in terms of procedure is also an element of ultrahigh temperature processing (UHT) of milk which is well-tried in the dairy practice today. Indirect UHT processing was used to prepare dairy desserts for studying the influence of varying concentrations of additives on the dessert rheology. As a result, a more extensive whey protein denaturation and subsequent complexation with casein micelles is believed to

contribute to the improvement of rheological properties of the UHT desserts (13).

one dairy-based product for the purpose of confirmation

**2. Materials and methods**

**2.1. Raw milk and thermal treatments**

Own research into the effects on indirect UHT processing are in agreement with this and confirmed that perfect complexation may result only by the indirect procedure (14). Moreover, perfect complexation and inherent physico-functionality of the protein were to set not only by the technology alone but also by the current colloid-chemical status of the casein micelles in milk. In herd milk, this status may vary considerably in one period of lactation and inherent negative consequences are seen too late in the finished product.

This research was aimed at the investigation into the influence of a varying colloidal status of casein micelles on the resulting micellar complexation with whey protein on indirect UHT processing of skim milk. For this an own colloid-chemical approach is used to explain the structural changes of casein micelles with given examples of four herd milk samples and

Bulked herd milk was collected from one farm in Schleswig-Holstein for a period of time of 8 month. Both separation of milk and thermal treatment were carried out using certified equipment. The skimmed milk was first regenerative (principle of heat recovery) preheated to 70-85 °C and then heated indirectly (126 °C for 4 s) and after that cooled down to 5 °C.

### *2.3.2. Assay procedure*

The hydrophobicity of proteins and casein micelles is calculated from two different protein assays, which are developed at the same time. Triplicates for each single measurement are necessary.

**1a)** 50 μL protein (A sample) is placed on the bottom of a dry and clean test tube whereas 50 μL *aqua dest*. or buffer are used as blank (A blank).

**1b)** 50 μL 0,25 % Tween 80 is placed on the bottom of a dry and clean test tube and in addition to 50 μL protein is placed onto the droplet of Tween 80 (B sample) whereas *aqua dest*. or buffer and Tween 80 are prepared as blank (B blank).

**2)** Only the detergent-containing tubes (1b) are shaken for 10 min. (avoid foaming) to complete detergent binding at temperatures between 18-22 °C.

**3)** Add 2,5 mL diluted dye reagent to each test tube, insert mixing spatulas by use of a pincette to avoid any skin contact. Move the spatula several times up and down without foaming.

**4)** Allow standing for 12 min. to develop the colour. The colour intensity of each tube is measured at 595 nm vs. *aqua dest*. Avoid any warming of cuvettes by a prolonged standing inside the cuvette department of the photometer.

**5)** Calculation

Protein Hydrophobicity (PH) is defined as following (Nakai and Li-Chan) (17)

PH = (nonpolar residues)/ (nonpolar residues) – (polar residues)

Using this definition on detergent binding according the proposed method, PH is calculated as

$$\frac{\text{SHP}\left(\%\right) \, = \left(\text{A}\_{\text{sample}} - \text{A}\_{\text{blank}}\right) \, - \left(\text{B}\_{\text{sample}} - \text{B}\_{\text{blank}}\right) \, ^\ast \, 100 \,\, \frac{\text{m}^2}{\text{s}} \,\text{s}^{-1}$$

Analyzing the casein in its natural colloidal status (18) this method was modified to protect the micelle structure from an to early dissociation on dilution with water for a short time. Among some other things, the purity of water is one crucial point for the colloid-chemical analysis requiring a special conditioning procedure in some laboratories and need to do some research into the causes of this. The water quality plays also an important role in the dilution of the detergent for analysis; for this "protein-grade" Tween 80 is to be used. In original packing the detergent is diluted 10 % (v/v) in distilled, deionized and filtrated water and applicable for a limited period of time after opening. At present, all Tween-preparations on the market are plant-based and replaced the preferred high-grade detergent products of animal origin.

The estimation of micellar SHP is carried out pH-depended between pH 6,0 and 7,5. For this, skimmed milk samples were held at 4-5 °C during pH adjustments at pH 6,0, 6,2, 6,4 and 6,6 ca. 8 gL-1 Glucono-delta-lacton (GDL) or NaOH (4 gL-1) was used for adjustments at pH 6,8, 7,0, 7,2 and 7,4, respectively.

The eight samples were sealed and stored at 4-6 °C overnight. Before analysis the samples are equilibrated in a water bath; first, at 36 °C for 30 min while stirring and then 70 min rocking (120 rpm) at room temperature (19-21 °C). The procedure of equilibration is based on measurements of micellar turbidity. The SHP is analyzed from a watery dispersion (100- 200 μL milk in 25 mL highly pure water) at 21 ± 1 °C. The measurements of samples were carried out in triplicate and six fold in case of the protein-free standards (blanks). The experiments were repeated if the spread of readings do not agree with those permitted for the Bio-Rad- method.

### **3. Results and discussion**

162 Milk Protein

foaming.

as

**5)** Calculation

animal origin.

pH 6,8, 7,0, 7,2 and 7,4, respectively.

**1b)** 50 μL 0,25 % Tween 80 is placed on the bottom of a dry and clean test tube and in addition to 50 μL protein is placed onto the droplet of Tween 80 (B sample) whereas *aqua dest*.

**2)** Only the detergent-containing tubes (1b) are shaken for 10 min. (avoid foaming) to

**3)** Add 2,5 mL diluted dye reagent to each test tube, insert mixing spatulas by use of a pincette to avoid any skin contact. Move the spatula several times up and down without

**4)** Allow standing for 12 min. to develop the colour. The colour intensity of each tube is measured at 595 nm vs. *aqua dest*. Avoid any warming of cuvettes by a prolonged standing

Protein Hydrophobicity (PH) is defined as following (Nakai and Li-Chan) (17)

PH = (nonpolar residues)/ (nonpolar residues) – (polar residues) Using this definition on detergent binding according the proposed method, PH is calculated

>

A – A

SHP % A A — B B \* 100

sample blank

Analyzing the casein in its natural colloidal status (18) this method was modified to protect the micelle structure from an to early dissociation on dilution with water for a short time. Among some other things, the purity of water is one crucial point for the colloid-chemical analysis requiring a special conditioning procedure in some laboratories and need to do some research into the causes of this. The water quality plays also an important role in the dilution of the detergent for analysis; for this "protein-grade" Tween 80 is to be used. In original packing the detergent is diluted 10 % (v/v) in distilled, deionized and filtrated water and applicable for a limited period of time after opening. At present, all Tween-preparations on the market are plant-based and replaced the preferred high-grade detergent products of

The estimation of micellar SHP is carried out pH-depended between pH 6,0 and 7,5. For this, skimmed milk samples were held at 4-5 °C during pH adjustments at pH 6,0, 6,2, 6,4 and 6,6 ca. 8 gL-1 Glucono-delta-lacton (GDL) or NaOH (4 gL-1) was used for adjustments at

The eight samples were sealed and stored at 4-6 °C overnight. Before analysis the samples are equilibrated in a water bath; first, at 36 °C for 30 min while stirring and then 70 min rocking (120 rpm) at room temperature (19-21 °C). The procedure of equilibration is based on measurements of micellar turbidity. The SHP is analyzed from a watery dispersion (100- 200 μL milk in 25 mL highly pure water) at 21 ± 1 °C. The measurements of samples were carried out in triplicate and six fold in case of the protein-free standards (blanks). The

sample blank sample blank

or buffer and Tween 80 are prepared as blank (B blank).

inside the cuvette department of the photometer.

complete detergent binding at temperatures between 18-22 °C.

### **3.1. The influence of UHT-processing at 126 °C/ 4s on some major components in three different skim milk samples**

Effects of indirect UHT-processing on some major components in the three skim milk samples are summarized in Tab.1.

**Table 1.** Composition of three bulked raw milk samples before and on indirect UHT-processing at 126 °C for 4 s

The pH-value of samples No. 1 and 2 is between 6,77-6,82 pointing to an increased oxidative potential in both milk samples and thus to natural changes of the SH/SS- status of the proteins (19).

The high pH determined in sample No. 1a compared to No. 1b is explained by fact that this UHT- processing happened at the day of milk production but two days later to meet the demands of sample preparation in the colloid-chemical analysis (see 2.3). Within this period of time the pH of No. 1 increased in parallel with the decline of the inherent oxidative potential. In this course of events casein serves as the major radical scavenger in milk, whereas β-Lactoglobulin (β-Lg) and α-Lactalbumin (α-La) seem to be far less active (20). In agreement with it, the own results of β-Lg analysis in Tab.1 show a nearly constant rate of denaturation in all heat-processed samples and are probably more linked up with definite conditions on heat- processing.

From this point of view, samples No. 3 and No. 3b seem to be without criticism. In this instance the SHP gained about 5,3 % on UHT-processing. It is a definite indication that casein and whey protein became associated (reversible and/or irreversible) in this sample. Moreover, complexation is also verifiable for the other samples, the increase of SHP amounts to 8,5 % and 0,9 % for No. 1b and 2b, respectively. In both milk samples the participation of casein in complexation is varying. The underlying changes of the micelle status in milk are investigated and discussed below.

### **3.2. Research into demands on the colloid-chemical status of natural casein micelles relative to their suitability as a physico-functional food ingredient**

*3.2.1. The influence of oxidative stress on the colloidal-chemical status of casein and on results of thermal complexation by indirect UHT*