Open Access
Issue
Dairy Sci. Technol.
Volume 90, Number 5, September–October 2010
Page(s) 549 - 563
DOI https://doi.org/10.1051/dst/2010018
Published online 07 April 2010

© INRA, EDP Sciences, 2010

1. INTRODUCTION

The main function of milk proteins is to supply the young neonate with amino acids required for development and growth, and to provide a number of biologically active substances thought to protect the newborn from infections. The composition of milk proteins differs between species and breeds, and also lactation stage, to fulfill the needs for development of the growing mammal [8].

Milk proteins from different species vary in concentration and amino acid composition. The protein content in milk from bovine and caprine species is approximately 2.1–3.5%, whereas equine and human milks contain less protein, approximately 2% and 0.8–1.5%, respectively. Bovine and caprine milks have a high casein to whey ratio of 5.0 and 6.0, whereas equine and human milks have a ratio of 1.5 and 0.9, respectively [11, 21, 24].

The casein fraction consists of αs1-, αs2-, β- and κ-casein. The whey fraction consists of mainly β-lactoglobulin (β-lg), α-lactalbumin (α-la), serum albumin (SA), immunoglobulins, lactoferrin (LF) and lysozyme (LZ) [11]. However, αs2-casein and β-lg are not expressed in human milk [22, 24]. Another notable difference between the milks is their content of the two antimicrobial proteins LF [26] and LZ [10]. Human milk has a high content of LF, whereas equine milk contains high levels of LZ as shown in Table I [21, 24].

Table I.

Protein content (g·100 g−1) in bovine, caprine, human and equine milks.

Nearly all caseins in milk are organized into colloidal particles referred to as casein micelles [11]. αs1-, αs2- and β-caseins are mainly located in the interior of the micelle and are bound to calcium phosphate nanoclusters by their phosphoserine domains [8, 23]. The proteins are also linked to each other by calcium bridges and hydrophobic interactions [23]. κ-Casein exists mainly on the surface of the micelle with the hydrophilic C-terminal part protruding into the solvent [5, 23]. κ-Caseins are not evenly distributed on the surface, but are present in clusters that are easily accessible for attack by chymosin (and related enzymes).

The size of the casein micelle is inversely related to their κ-casein content [11]. Equine milk has a very low content of κ-casein (0.25 g·L−1), while milks from bovine and caprine species contain 3.5 and 5 g·L−1, respectively. Human milk contains 1.0 g·L−1 κ-casein [24]. It has been suggested that the high content of β-caseins and the variable degree of phosphorylation of equine milk compensate for the low content of κ-casein in the micelle formation [25].

The high digestibility of ~ 95%, combined with a superior amino acid composition for human beings, makes milk protein a “high quality protein” [4]. In addition to the nutritional role, milk proteins have been shown to have different biological effects. In vitro digestion of milk proteins has revealed promising results related to the production of bioactive peptides. Protein digestion starts in the stomach by the action of pepsins, followed by intestinal digestion by trypsin, chymotrypsin and various carboxy- and aminopeptidases. The extent of milk protein degradation in human beings and the transfer of peptides or intact proteins into the serum are not very well understood. Only a limited number of in vivo studies have been performed on the identification of the protein degradation products. Studies with either milk or purified casein and whey proteins in healthy human beings showed that whey proteins were translocated more rapidly to the upper jejunum than the caseins. A large proportion of intact whey proteins were detected in the jejunum, while only traces of intact casein were detected [19, 20]. It was concluded that the caseins were retained as a clot in the acid stomach environment, while the soluble whey proteins were released to the duodenum. This would increase the time before the caseins reach the intestine compared to the soluble whey proteins, and therefore also the time before further degradation and absorption in the intestine. These results led to the concept of “slow” digested caseins and “fast” digested whey proteins [6, 7, 17].

On the contrary, our earlier studies using human gastric (HGJ) and duodenal (HDJ) juices for the in vitro digestion of bovine and caprine milk proteins revealed that the whey proteins, α-la and β-lg, were very resistant to hydrolysis and that the caseins were degraded fast [2]. There are many studies performed on in vitro digestion using commercial enzymes, such as purified pepsin and trypsin, in an attempt to reveal peptides with physiological activities [15, 28]. However, we observed different degradation patterns when comparing human digestive enzymes and commercial enzymes [3].

The aims of the study were:

  • To compare protein degradation of human, bovine, caprine and equine milks by human gastric and duodenal enzymes;

  • To quantify the protein degradation of milks with high content of casein, represented by bovine and caprine versus milks with low content of casein such as human and equine milks;

  • To study possible differences in the protein degradation between raw and heated milks of different species (bovine, caprine, human and equine).

2. MATERIALS AND METHODS

2.1. Milk

The milk samples used were: commercial bovine milk (pasteurized skim milk from TINE BA), commercial equine milk (raw, frozen milk from Equi Libre AS), human milk (raw, a batch from three lactating women) and caprine milk (raw, represented by one individual dairy goat from the University Farm). The protein content of the respective milks measured by the Kjeldahl method was lying within the range of normal values observed by our earlier studies of individual and batch milk [2], and also reported by others (Tab. I).

Fat was removed by heating the milks to 37 °C for 20 min followed by centrifugation at 3000× g for 20 min. Samples were kept at −20 °C for 10–20 min before fat was removed by a spatula, and the defatted samples were stored at −20 °C until further use.

This study also included a high heating step. The same milk samples, as described above, were heated before digestion. The heat treatment of milk was performed at 95 °C for 1 min. After the heat treatment, the milk was cooled and stored at 4 °C for one day before analysis.

2.2. Human digestive enzymes

To follow up and extend previous work on in vitro digestion of milk and milk proteins [13, 9], the gastric and duodenal juices employed were obtained from the same person. These juices show enzyme activities that lie within the normal range of pepsin and total proteolytic activities observed in 12 individuals (men and women). The gastrointestinal enzymes were obtained in the activated state by collecting HGJ and HDJ according to Holm et al. [12]. In brief, a three-lumen tube (Maxter Catheters, Marseille, France) enabled both simultaneous instillation of stimulation solution in the duodenum, and aspiration of HGJ and HDJ. The stimulation solution (70 g·L−1 sucrose, 1.8 g·L−1 NaCl, 3.2 g·L−1 L-phenylalanine and 2.3 g·L−1 L-valine in H2O) was instilled close to the papilla of Vater (100 mL·h−1) to stimulate the production of pancreatic enzymes, and HDJ was aspirated some 18 cm distally. Aspirates were collected on ice, centrifuged (4500× g for 10 min) to remove mucous and cell debris before it was frozen in aliquots and stored at −20 °C prior to use.

The pepsin activity of HGJ was measured according to Sanchez-Chiang et al. [29] using hemoglobulin (Hb) as a substrate. The total proteolytic activity of HDJ was measured with casein as a substrate according to Krogdahl and Holm [16]. One unit (U) of enzyme activity is defined as the amount of enzyme solution which gives an absorbance of 1.0 at 280 nm after incubation with substrate at 37 °C for 10 min. All enzyme assays were run in triplicate.

2.3. Assay for in vitro digestion

A two-step in vitro assay was used to simulate human digestion in the stomach (step 1) and the duodenum (step 2). A modified protocol described by Almaas et al. [2] was used. Step 1: 10 mL of milk was acidified by dropwise addition of 2 mol·L−1 HCl to a final pH of 2.5. To get a homogeneous milk sample before adding HGJ, the milk was homogenized by Ultraturrax at medium speed. This process has been compared with the earlier non-homogenized milk sample with no effect on the results. Thirty microliters (1.5 U) of HGJ were added to the milk and then incubated for 30 min at 37 °C with constant magnetic stirring. Step 2: pH was adjusted to 8.0 by 2 mol·L−1 NaOH, 280 μL (3.7 U) HDJ was added and the gastric digested milk sample (8 mL) was incubated for 30 min at 37 °C. Aliquots of 0.25–1.0 mL were withdrawn before digestion, after digestion with gastric enzymes and after 5, 15 and 30 min of the subsequent digestion with duodenal enzymes. To stop the enzymatic reaction, the samples were immediately transferred to ice and frozen (−20 °C). The digestion of each milk sample was run in duplicate or more.

2.4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to visualize the protein profile of the samples taken at different hydrolysis times (min) during digestion. Electrophoresis was performed according to standard protocol [18] using homecast 12.5% and 15% acrylamide gels. Gels were stained with Coomassie Brilliant Blue. As a molecular ladder, a low molecular weight standard kit (LMW Calibration Kit, Amersham, GE Healthcare, UK) was used. Samples were diluted 1:2 in 2 X sample buffer and heated at 100 °C for 5 min. To obtain optimal results, different aliquots of sample were applied on the gels; 7 μL of samples of human milk and LMW standard, 3 μL of bovine milk (undiluted and diluted 1:4), 4 μL of caprine milk and 5 μL of equine milk samples. The diluted bovine milk sample was used for quantification of the caseins. More than three SDS gels were run of each milk sample.

2.5. Quantification of protein bands

The protein bands in the gels were quantified by ImageQuant TL 7.0 software (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Image rectangle was applied for background subtraction, and for detection minimum slope was set to 150. Bands of interest were marked in each lane to be able to investigate the degradation pattern of the different proteins. Area of each band was set as close to the band as possible. In order to measure the degradation, the collective number of undigested proteins was set to 100, and the ratio of each band in the first lane was calculated by normalization. The amount of intact proteins remaining in the digested samples was calculated as percentage of its undigested counterpart. Caseins in all milks were considered as one band because of poor resolution of the individual caseins on SDS-PAGE. Standard curves were obtained with different dilutions and were used to ensure linearity between protein content and the intensity of the bands. The bands quantified were inside the linear area of the respective standard curve.

Two gels from each sample were quantified, and the results are given as an average of mean values.

Statistical analysis of raw milk samples was performed by applying a one-way analysis of variance (ANOVA) on caseins in step 1 (after 30 min) and β-lg in step 2 (after 30 min). The results indicated that differences between species should be detectable. Tests for pairwise differences were only carried out using the least different species, as larger differences would be significant if smaller differences were found to be significant. Equine and bovine were compared for caseins, and equine and caprine were compared for the β-lg. To utilize all eight observations of caseins, we set up a hypothesis and used a t test based on a contrast between the equine and bovine levels of the ANOVA as follows: giving a P value of 0.038. For the six observations of β-lg (excluding human), a t value of 3.52 with a corresponding P value of 0.039 was found for the difference between equine and caprine. Both t tests support our claim that equine differs from the other species in the study.

2.6. Casein micelle measurements

Mean size of casein micelles was analyzed by photon correlation spectroscopy (PCS). The measurements were performed using a Zetasizer 3000HS (Malvern Instruments) equipped with a He Ne laser (632.8 nm). The measurements were performed in plastic cuvettes at a scattering angle of 90° at 25 °C. Prior to analysis the milk samples were mixed with 4% glutaraldehyde (GA) in simulated milk ultrafiltrate (SMUF) [13] in the ratio 1:2. The pH of the SMUF was adjusted with 1 mol·L−1 KOH to 6.9 for measurements of equine and human milks, and 6.7 for bovine and caprine milks.

The milk samples mixed with GA were diluted 1:1000 in SMUF, and heated to 25 °C prior to the measurements. To avoid the interference of dust, traces of fat or the former sample, the cuvettes were carefully rinsed in SMUF between each sample, and the diluted samples were filtered through a 0.45 μm filter. All samples were run in triplicate.

3. RESULTS

Milks from different species were digested in vitro by HGJ and HDJ to simulate human digestion. The amount of intact proteins remaining in the digested milk samples visualized by SDS-PAGE is shown in Figure 1. The results revealed that most of the protein degradation occurred after the duodenal digestion. However, it should be noted that the rate of hydrolysis differed among proteins (e.g. caseins versus whey proteins) and between species (bovine β-lg versus equine β-lg) as shown in Figures 14.

thumbnail Figure 1.

Protein profiles of milk from bovine, caprine, human and equine species after digestion in two steps. Step 1: HGJ at pH 2.5 for 30 min/37 °C. Step 2: HDJ at pH 8.0/37 °C for 30 min. 1: Low molecular standard, 2: Undigested milk, 3: HGJ for 30 min, and 4–6: HGJ for 30 min followed by HDJ for 5, 15 and 30 min, respectively. Band within circle is uniquely produced in human milk and seems to be a lower molecular weight fragment of the casein band. α-la = α-lactalbumin, β-lg = β-lactoglobulin, CNs = caseins, HDJ = human duodenal juice, HGJ = human gastric juice, IgHc = immunoglobulin heavy chain, LF = lactoferrin, LZ = lysozyme and SA = serum albumin.

During gastric digestion (pH 2.5) the caseins in bovine and caprine milks were poorly degraded with a remaining casein content of 69% and 82%, respectively. However, in equine milk the caseins were highly degraded and only 30% remained undigested after 30 min. In human milk the degradation of the main casein band resulted in a protein band of lower molecular size (< 20 kg·mol−1) and about 39% undigested casein was observed. The difference in casein degradation between bovine and equine was significant (P = 0.038). The formation of an extra peptide band, most probably originating from the κ-casein, was clearly seen above the α-la band in bovine and caprine milks (Fig. 1). This band was not possible to detect in milks from human and equine species that may be due to the low content of κ-casein in these milks. Further digestion with human duodenal juice (step 2, pH 8.0) resulted in very fast digestion of the caseins in all the species. After 5 min less than 20% were still intact, and after 30 min almost no caseins were left (< 6%) (Figs. 1 and 2, and Tab. II).

thumbnail Figure 2.

In vitro digestion of caseins in raw milks from bovine (♦), caprine (■), human (▲) and equine (●) species by HGJ at pH 2.5/37 °C for 30 min and HDJ at pH 8.0/37 °C for 5, 15 and 30 min. Values are obtained by ImageQuant. Start: Undigested milk, step 1: HGJ pH 2.5/37 °C for 30 min; step 2: HDJ pH 8.0/37 °C for 5, 15 and 30 min. Error bars are omitted for clarity.

Table II.

Remaining protein content (%) in raw milks before digestion (start), after step 1 digested with HGJ at pH 2.5 for 30 min/37 °C and after steps 1 and 2 further digested with HDJ at pH 8.0/37 °C for 30 min. Values are obtained by ImageQuant.

The main whey proteins, β-lg and α-lactalbumin, were very resistant to digestion by human gastric and duodenal enzymes. After gastric digestion, β-lactoglobulin, α-la and LZ remained almost intact (> 99%), while LF, SA and immunoglobulins were degraded to some extent (Tab. II).

Human milk is devoid of β-lg; however, this is one of the major whey proteins in milk from the other species. A notable difference was seen during the duodenal digestion as β-lg was significantly (P = 0.039) more hydrolyzed in equine milk as compared to bovine and caprine milks. Only 25% of the equine β-lg was still intact after 30 min, while ~ 60% remained intact in bovine and caprine milks as shown in Figure 3.

thumbnail Figure 3.

In vitro digestion of β-lactoglobulin (β-lg) in raw milks from bovine (♦), caprine (■) and equine (●) species by HGJ at pH 2.5/37 °C for 30 min and HDJ at pH 8.0/37 °C for 5, 15 and 30 min. Values are obtained by ImageQuant. Start: Undigested milk, step 1: HGJ pH 2.5/37 °C for 30 min; step 2: HDJ pH 8.0/37 °C for 5, 15 and 30 min. Error bars are omitted for clarity.

The most resistant protein to human digestive enzymes in all species was α-la (Fig. 4). Lactoferrin was digested very fast both by gastric and by duodenal juices in all species. Equine milk contained high amount of lysozyme, and interestingly this protein remained mainly intact (65%) after gastric and duodenal digestions.

thumbnail Figure 4.

In vitro digestion of α-lactalbumin (α-la) in raw milks from bovine (♦), caprine (■), human (▲) and equine (●) species by HGJ at pH 2.5/37 °C for 30 min and HDJ at pH 8.0/37 °C for 5, 15 and 30 min. Values are obtained by ImageQuant. Start: Undigested milk, step 1: HGJ pH 2.5/37 °C for 30 min; step 2: HDJ pH 8.0/37 °C for 5, 15 and 30 min. Error bars are omitted for clarity.

High heat treatment of the milk from all the species resulted in very little differences in the protein degradation, only α-la showed a higher degradation, approximately 10–20%, compared to raw milk (Tab. III).

Table III.

Remaining protein content (%) of heated milks (95 °C for 1 min) before digestion (start), after step 1 digested with HGJ at pH 2.5/37 °C for 30 min and after steps 1 and 2 further digested with HDJ at pH 8.0/37 °C for 30 min. Values are obtained by ImageQuant.

The mean size of casein micelles in the milks was measured by PCS. Size of the casein micelles varied between the species as seen in Figure 5. Equine milks showed to have the largest micelles (311.5 nm) and human milk the smallest ones (146.0 nm). The micellar sizes of the caprine and bovine milks were 220.4 and 183.9 nm, respectively.

thumbnail Figure 5.

Mean size (nm) of casein micelles in raw milk. Bovine: 183.9 ± 0.4 nm, caprine: 220.4 ± 2.8 nm, human: 146.0 ± 0.7 nm, equine: 311.5 ± 1.1 nm. Values are an average of three runs (each containing 10 subruns) obtained by PCS.

4. DISCUSSION

Gastric digestion and low pH 1.5–2.5 are the first step in the gastrointestinal degradation of proteins in human beings, followed by stomach emptying and further digestion in the upper part of duodenum by pancreatic and brush border enzymes at pH ~7. In this study, we have used an in vitro digestion model employing HGJ and HDJ, containing crude extracts of enzymes, inhibitors, bile salts, etc. This model developed and published earlier [2] for revealing caprine milk protein degradation by SDS and IEF was used. To compare the digestion of raw and heated milk proteins from species other than caprine, like bovine, human and equine species, only SDS gels are shown.

The equine milk behaved differently from the other species by showing a more rapid gastric digestion of the casein already in step 1, leaving only ~ 30% of the caseins intact after 30 min. Also human milk showed a pronounced casein degradation during the gastric digestion; however, the casein of human milk gave rise to a protein band with only a small reduction in the molecular size than the original casein band (Fig. 1, lane 3). The caseins of the other species, caprine and bovine, showed a significantly lower (P = 0.038 between equine and bovine) gastric digestion. Further digestion by the duodenal juice showed that most of the caseins of all species were degraded after 30 min. Also equine β-lg was digested significantly faster (P = 0.039) compared to bovine and caprine β-lg.

Only a limited number of studies in human beings have been performed. Studies with either milk or purified caseins and whey proteins showed that only traces of intact casein could be detected in the jejunum [6, 7, 19, 20]. The authors concluded that this was due to a delayed emptying of casein in the stomach because of the casein clotting under the gastric pH, which delays the delivery to the gut. Milk soluble proteins like whey proteins, on the other hand, were rapidly evacuated from the stomach as intact proteins and further hydrolyzed by pancreatic enzymes in the duodenum. This causes the whey proteins to be absorbed more distally in the intestine [30].

Our in vitro studies showed, however, that the caseins of all studied species were rapidly digested by the gastrointestinal enzymes and that equine milk showed a very high gastric degradation. Equine casein micelles have a very low content of κ-casein and are larger in size (Fig. 5) than the casein micelles in the milks of all the other species. The low content of κ-casein and the large size of the casein micelles may be the reasons for the high susceptibility to hydrolysis by gastric enzymes. Another factor may be that equine milk contains β-caseins with variable degree of phosphorylation. The less phosphorylated forms of β-caseins can act on the surface of the casein micelles and thereby play the role of the κ-casein [25]. Even though a certain amount of the human casein was degraded in the gastric juice, the degradation pattern was very different from the profile observed for the equine caseins. While the human caseins seemed to be broken down to protein fragments of molecular weight just below 20 kg·mol−1, the equine milk caseins were mainly broken down to smaller peptides that were not seen in the gel (Fig. 1).

With respect to the digestion of whey proteins, Mahé et al. [20] reported that after milk ingestion a large proportion of the whey proteins were detected as intact proteins in the human jejunum. Tomé and Debabbi [30] concluded that the soluble whey proteins were emptied much faster from stomach to the intestine. The proteins detected in an intact form (5–10%) in the upper jejunum were β-lg, α-la, LF and immunoglobulins.

Our results confirm that β-lg and α-la were very resistant to human gastric and duodenal enzyme digestions, except for the equine milk showing rapid duodenal degradation of β-lg after 30 min leaving only 25% of the protein intact. In contrast, bovine and caprine β-lg were significantly less digested, and more than 60% of the protein was still intact. However, the other whey proteins such as LF and SA were highly degraded by the human gastrointestinal enzymes.

We also observed that high heating of the milk had only minor effect on the degradation of the caseins. This is most probably due to the fact that the caseins have an open and flexible structure, with peptide bonds easily exposed to the gastrointestinal enzymes. Thereby heating seems not to affect the casein structure as opposed to the globular structure of the whey proteins. Heating resulted in 12–20% higher degradation of α-la compared to raw milk. However, in our study β-lg was not much affected by heating. In a previous study by Kim et al. [14] high degradation of both α-la and β-lg was observed in heat-treated whey protein concentrate digested with commercial enzymes.

5. CONCLUSION

Milk from bovine, caprine, equine and human species showed different degradation patterns when digested with human gastrointestinal enzymes at their respective pH values. Caseins in all species were digested very fast after being exposed to both gastric (pH 2.5) and duodenal juices (pH 8.0). Equine and human caseins behaved differently and were easily degraded even in the gastric juice (pH 2.5). In addition equine β-lg was highly degraded after being treated with the gastrointestinal enzymes, in contrast to the bovine and caprine milks where the β-lg was very resistant. Heat treatment of milk did not seem to affect the protein digestion pattern much, except for increased degradation of α-la in all species. These in vitro experiments are continued in our laboratory and need to be confirmed by in vivo results.

Acknowledgments

We would like to thank Mette Johnson/ Equi Libre, Norway, for providing us with equine milk samples, Hilde Almaas for human milk samples and Maxters Catheters, Marseille, France, for tailoring triple lumen tubes enabling simultaneous instillation of a stimulation solution and aspiration of gastric and duodenal juices in humans.

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  27. Park Y.W., Goat milk – chemistry and nutrition, in: Park Y.W., Haenlein G.F.W. (Eds.), Handbook of Milk of Non-Bovine Mammals, Blackwell Publishing, Oxford, UK, 2006, pp. 34–58. [CrossRef] (In the text)
  28. Salami M., Yousefi R., Ehsani M.R., Dalgalarrondo M., Chobert J.-M., Haertlé T., Razavi S.H., Saboury A.A., Niasari-Naslaji A., Moosavi-Movahedi A.A., Kinetic characterization of hydrolysis of camel and bovine milk proteins by pancreatic enzymes, Int. Dairy J. 18 (2008) 1097–1102. [CrossRef] (In the text)
  29. Sanchez-Chiang L., Cisternas E., Ponce O., Partial-purification of pepsins from adult and juvenile salmon fish Oncorhynchus keta. Effect of NaCl on proteolytic activities, Comp. Biochem. Physiol. B 87 (1987) 793–797. [CrossRef] [PubMed] (In the text)
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All Tables

Table I.

Protein content (g·100 g−1) in bovine, caprine, human and equine milks.

Table II.

Remaining protein content (%) in raw milks before digestion (start), after step 1 digested with HGJ at pH 2.5 for 30 min/37 °C and after steps 1 and 2 further digested with HDJ at pH 8.0/37 °C for 30 min. Values are obtained by ImageQuant.

Table III.

Remaining protein content (%) of heated milks (95 °C for 1 min) before digestion (start), after step 1 digested with HGJ at pH 2.5/37 °C for 30 min and after steps 1 and 2 further digested with HDJ at pH 8.0/37 °C for 30 min. Values are obtained by ImageQuant.

All Figures

thumbnail Figure 1.

Protein profiles of milk from bovine, caprine, human and equine species after digestion in two steps. Step 1: HGJ at pH 2.5 for 30 min/37 °C. Step 2: HDJ at pH 8.0/37 °C for 30 min. 1: Low molecular standard, 2: Undigested milk, 3: HGJ for 30 min, and 4–6: HGJ for 30 min followed by HDJ for 5, 15 and 30 min, respectively. Band within circle is uniquely produced in human milk and seems to be a lower molecular weight fragment of the casein band. α-la = α-lactalbumin, β-lg = β-lactoglobulin, CNs = caseins, HDJ = human duodenal juice, HGJ = human gastric juice, IgHc = immunoglobulin heavy chain, LF = lactoferrin, LZ = lysozyme and SA = serum albumin.

In the text
thumbnail Figure 2.

In vitro digestion of caseins in raw milks from bovine (♦), caprine (■), human (▲) and equine (●) species by HGJ at pH 2.5/37 °C for 30 min and HDJ at pH 8.0/37 °C for 5, 15 and 30 min. Values are obtained by ImageQuant. Start: Undigested milk, step 1: HGJ pH 2.5/37 °C for 30 min; step 2: HDJ pH 8.0/37 °C for 5, 15 and 30 min. Error bars are omitted for clarity.

In the text
thumbnail Figure 3.

In vitro digestion of β-lactoglobulin (β-lg) in raw milks from bovine (♦), caprine (■) and equine (●) species by HGJ at pH 2.5/37 °C for 30 min and HDJ at pH 8.0/37 °C for 5, 15 and 30 min. Values are obtained by ImageQuant. Start: Undigested milk, step 1: HGJ pH 2.5/37 °C for 30 min; step 2: HDJ pH 8.0/37 °C for 5, 15 and 30 min. Error bars are omitted for clarity.

In the text
thumbnail Figure 4.

In vitro digestion of α-lactalbumin (α-la) in raw milks from bovine (♦), caprine (■), human (▲) and equine (●) species by HGJ at pH 2.5/37 °C for 30 min and HDJ at pH 8.0/37 °C for 5, 15 and 30 min. Values are obtained by ImageQuant. Start: Undigested milk, step 1: HGJ pH 2.5/37 °C for 30 min; step 2: HDJ pH 8.0/37 °C for 5, 15 and 30 min. Error bars are omitted for clarity.

In the text
thumbnail Figure 5.

Mean size (nm) of casein micelles in raw milk. Bovine: 183.9 ± 0.4 nm, caprine: 220.4 ± 2.8 nm, human: 146.0 ± 0.7 nm, equine: 311.5 ± 1.1 nm. Values are an average of three runs (each containing 10 subruns) obtained by PCS.

In the text

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