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Milk protein polymorphism in Amiata donkey
Livestock Science 230 (2019) 103845
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Milk protein polymorphism in Amiata donkey a
b,c
a
T b
d
Rosario Licitra , Stefania Chessa , Federica Salari , Stefano Gattolin , Omar Bulgari , ⁎ Iolanda Altomontea, Mina Martinia,e, a
Department of Veterinary Science, University of Pisa, Pisa, Italy Institute of Agricultural Biology and Biotechnology, National Research Council, Lodi, Italy Department of Veterinary Science, University of Turin, Turin, Italy d Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy e Interdepartmental Research Center Nutrafood “Nutraceuticals and Food for Health”, University of Pisa, Pisa, Italy b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Donkey milk Milk protein Casein Whey protein Milk protein fractions Milk protein polymorphism
Interest in donkey milk for human nutrition has been growing steadily, especially in the therapeutic-diet field, due to its similarity to human milk. Caseins are one of the main allergenic components of milk. Whereas cow milk has a high casein content, in donkey milk the casein concentration is lower and is characterized by a greater digestibility. In fact, recent studies have shown that donkey milk can be consumed by children with cow milk protein allergy. The aim of this study was to evaluate the protein profile of individual samples of Amiata donkey milk and to quantify the different protein fractions in order to better understand the characteristics of donkey milk. Protein fractions of 57 individual whole milk samples were analysed by isoelectric focusing (IEF). The main milk protein fractions were quantified both by IEF gel image analysis and directly on milk using the Agilent 2100 Bioanalyzer. The IEF analysis identified 10 different electrophoretic patterns, which differ from each other in terms of the presence of bands that correspond to homozygous or heterozygous genotypes, as well as the presence/absence of specific protein bands. In addition the quantification of different caseins (αS1, β and κ-CN) and whey proteins (β-LG, α-LA and LZ), highlighted a significant phenotypic variability among individuals which seems to be related to the patterns identified. In particular, different casein:whey protein ratios were identified among jennies and this is known to affect the nutritional, allergenic and technological properties of milk. These findings could be useful in the selection of donkeys with specific milk protein polymorphisms.
1. Introduction Among the various types of milk produced by non-specialized species, donkey milk has recently been rediscovered. This is mainly due to the increasing interest of consumers and scientists in foods with beneficial effects on human health. There is increasing evidence that donkey milk can be successfully used to feed children that suffer from CMPA (Monti et al., 2012; Barni et al., 2018). Donkey milk is similar to human milk, especially in terms of the protein fractions (Altomonte et al., 2019). Milk proteins typically show a high degree of genetic polymorphism, which together with alternative splicing, results in several forms of each protein, characterized by amino acid exchanges and/or deletions of peptides. Post-translational modifications such as phosphorylation or glycosylation further increase this heterogeneity (Ceriotti et al., 2005; Caroli et al., 2009). Caseins represent the main
milk protein fraction in ruminants (about 80% of the total protein) and they are recognized as one of the main allergenic components of milk (Restani et al., 2009). Donkey milk has a lower CN concentration (about 50% of the total protein) (Martini et al., 2014a) and a greater CN digestibility (Tidona et al., 2014) compared to ruminant milk. In donkey milk, the main CN fraction is β-CN, while αS1-CN, αS2-CN and κ-CN are less represented (Cunsolo et al., 2017). The primary structure of donkey αS1-CN, classified as variant A, shows 202 amino acids, a MW of 24,406 Da, a pI of 5.96, a variable degree of phosphorylation, and the presence of isoforms generated by alternative mRNA splicing events. The αS1-CN isoforms are A1 (201 amino acids; MW 24,278 Da; pI 5.96), B (197 amino acids; MW 23,786 Da; pI 5.85) and B1 (196 amino acids; MW 23,658 Da; pI 5.85) (Cunsolo et al., 2017). Two other minor αS1-CN isoforms, with a MW of 25,142 and 25,272 Da, respectively, have also been reported (Cunsolo et al.,
Abbreviations: CN, Casein; WP, Whey protein; CMPA, Cow milk protein allergy; α-LA, Alpha-lactalbumin; β-LG, beta-lactoglobulin; LZ, lysozyme; MW, molecular weight; pI, isoelectric point ⁎ Corresponding author. E-mail address: [email protected] (M. Martini). https://doi.org/10.1016/j.livsci.2019.103845 Received 19 March 2019; Received in revised form 22 October 2019; Accepted 22 October 2019 Available online 25 October 2019 1871-1413/ © 2019 Elsevier B.V. All rights reserved.
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2009a). All donkey αS1-CN isoforms show two levels of phosphorylation with 5 and 6 phosphate groups. Bertino and colleagues (2010) reported that donkey αS1-CN may also exist as glycosylated forms, in contrast to human or bovine αS1-CN, which have never been reported to be glycosylated. Donkey αS2-CN is a very minor component and presents several isoforms. The full-length isoform A (MW 26,028 Da; pI 5.68) contains 221 amino acids and a disulphide bond linking Cys37 and Cys41, whereas the other isoforms described are internally-deleted because of alternative mRNA splicing events: A1 (216 amino acids; MW 25,429 Da; pI 5.50), A1Δ4,5,6 (185 amino acids; MW 21,939 Da; pI 5.65), A2 (214 amino acids; MW 25,203 Da; pI 5.68), and A2 Δ4,5,6 (183 amino acids; MW 21,713 Da; pI 5.85) (Saletti et al., 2012). Like αS1-CN, αS2-CN also shows micro-heterogeneity depending on a variable degree of phosphorylation related to the ten to twelve possible phosphate groups present (Chianese et al., 2010). β-CN is the main fraction in donkey milk and consists of a full-length β-CN named A (226 amino acids; MW 25,529 Da; pI 5.44), and a short form named B (218 amino acids; 24,606 Da; pI 5.24), characterized by the absence of the 27ESITHINK34 peptide due to an alternative splicing of exon 5 (Girardet et al., 2006; Cunsolo et al., 2009b, 2017). Both A and B β-CN fractions show three phosphorylated forms, carrying 5, 6 and 7P, respectively, with the predominant isoform carrying 5P Chianese et al. (2010) described a new variant with a higher mass than the β-CN A and with the same phosphorylation pattern (5, 6, and 7P), but its primary structure was not reported. The κ-CN in donkey milk represents a minor component which has not yet been quantified. It is characterized by a sequence of 162 amino acids, a MW of 18,485 Da, a pI of 9.36 and only one cysteine. Polymorphisms are also present and there is a micro-heterogeneity related to post-translational modifications, as both glycosylated and non-glycosylated forms have also been reported (Chianese et al., 2010). However, it is the only casein not affected by splicing events and therefore without known longer and shorter forms of the same CN. Based on these characteristics, donkey κCN is more similar to human than bovine κ-CN. Compared to ruminant milk, there are higher amounts of whey proteins in donkey milk, and the main whey proteins are β-LG, α-LA and LZ (Vincenzetti et al., 2017). Donkey β-LG exists in two different molecular forms, namely β-LG I (80% of total ß-LG) and β-LG II (20% of total ß-LG), which seem to be the result of gene duplication (GodovacZimmermann et al., 1990). The primary structures of the main isoforms of β-LG I and β-LG II (isoform A) are different: β-LG I consists of 162 amino acids, a MW of 18,524 Da and a pI of 4.53, while β-LG II consists of 163 amino acids, a MW of 18,258 Da and a pI of 4.45 (Herrouin et al., 2000; Cunsolo et al., 2007; Chianese et al., 2013). β-LG I presents a further variant, named B (MW 18,510 Da; pI 4.59), which differs from variant A in relation to three amino acid exchanges E36 → S36, S97 → T97 and V150 → I150 (Herrouin et al., 2000). β-LG II locus shows a higher genetic polymorphism, with six described variants, all characterized by 163 amino acid residues, but differing in terms of amino acid substitution: variant A characterized by D2R18V25D96A100C110M118G162 (MW 18,246 Da; pI 4.45); variant B by D2R18V25D96A100P110T118G162 (MW 18,222 Da; pI 4.45); variant C by D2R18V25E96A100P110T118G162 (MW 18,236 Da; pI 4.47); variant D by D2R18V25D96A100P110M118D162 (MW 18,298 Da; pI 4.39); variant E by N2K18A25D96A100P110M118D162 (MW 18,253 Da; pI 4.45); and variant F characterized by D2R18V25D96T100P110T118D162 (MW 18,310 Da; pI 4.39) and expressed at a very low level (Criscione et al., 2018). Unlike milk of other species (cow, camel and mare), where different variants of α-LA have been described, in donkey milk only one protein form has been identified and characterized, though the existence of two isoforms has been hypothesized (Giuffrida et al., 1992). α-LA consists of 123 amino acid residues with eight cysteines, a MW of 14,214 Da and a pI of 4.88. LZ is an enzyme closely related to α-LA showing a similar amino acid sequence and three-dimensional structure (Tidona et al., 2011). In donkey milk, two LZ variants with 129 amino acid residues have been identified: variant A (MW 14,680 Da; pI 8.01) and variant B (MW
14,631 Da; pI 7.77), differing from variant A in terms of three substitutions (N49 → D49, Y52 → S52 and S61 → N61) (Herrouin et al., 2000). Compared to its bovine counterpart, donkey LZ is more hydrophilic. The genetic polymorphisms of proteins can influence the functional and biological properties of milk and also the involvement of each allergenic protein in clinical symptoms. Although donkey milk has been the subject of intensive research in the last decade, and all these protein isoforms have been described, the different protein fractions and the genetic polymorphisms have still not been fully investigated. Nor is the protein profile completely understood. The aim of this study was thus to clarify which part of the donkey milk protein heterogeneity is easily detectable with the IEF method, and to quantify the different protein fractions in Amiata donkey in order to better characterize this breed. 2. Materials and methods 2.1. Animals and sample collection The research was conducted on 57 healthy jennies of the Amiata donkey, that were daughters of more than 20 stallions. The Amiata donkey is a local breed native to the Mount Amiata area, in Tuscany (central Italy), which consistency was 2457 of which males 114 and 1468 jennies in 2018 (DAD-IS, 2018) Although the increasing trend from 2001 is still endangered and placed in the Local Equine Population List (www.aia.it 2019). The jennies were housed in the same farm reared outdoors in a semiintensive system and were routinely machine-milked once per day. The diet consisted of ad libitum grass hay supplemented with 2 kg/head/day of commercial concentrate. Individual raw milk samples were collected from morning mechanical milking and the foals were separated from their mothers three hours before milking. The milk samples belong from animals that were all between the 6th and 7th month of lactation, with an average milk yield per milking of 0.505 L ± 0.236, a mean age of 10.54 ± 4.46 years and an average parity of 3.49 ± 1.85; the samplings were uniformly distributed throughout the year. After collection, samples were immediately frozen at −20 °C to prevent undesired proteolysis, and then transported to a laboratory for the analysis. 2.2. Milk analysis The individual protein fractions were analysed by IEF on ultrathin gels (250×115×0.3 mm), using a GelBond polyester film as a support. The IEF analysis was performed according to Caroli et al. (2001) with some modifications. In detail, for the screening of donkey milk, the ampholyte mixture was as follows: 1.5% (v/v) Pharmalyte pH 2.5–5.0; 3.3% (v/v) Pharmalyte pH 4.2–4.9; and 2.3% (v/v) Ampholine® pH 5.0–8.0 (Sigma Aldrich, Steinheim, Germany). An acidic precipitation of individual milk samples was also performed in order to separate caseins from whey proteins and run them separately on the same IEF gel condition in order to confirm the identification of the different bands in the whole milk. 2.3. Data analysis The milk protein alleles and haplotype distributions were analysed by the ALLELE and HAPLOTYPE SAS procedures (SAS Institute, 2008). The ALLELE procedure uses the notation and concepts described by Weir (1996). Haplotype frequencies were calculated under the null hypothesis of no linkage disequilibrium and under the alternative hypothesis of associations between genes. The main milk protein fractions were quantified from IEF gels by image acquisition through G:Box (Syngene, Frederick, MD, USA) and using Agilent 2100 Bioanalyzer with the Agilent High Sensitivity Protein 250 kit (Agilent Technologies, Santa Clara, CA, USA). For each milk protein fraction, means and 2
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Fig. 1. Isoelectrofocusing of 12 individual Amiata donkey milk samples. The eight IEF patterns determined by βLG I and β-LG II genotypes are reported and indicated with Roman numerals. The black dot corresponds to β-LG II*D, the black diamond to β-LG II*B (or A or E), the black star to β-LG II*C, the white dot to β-LG I*A, and the white diamond to βLG I*B. Genotypes are written in black for β-LG II, and in white for β-LG I. IIa = pattern II lacking β-CN; IIb = =pattern lacking αS1-CN.
to β-LG I-β-LG II AB-BD, AA-BB, and AB-BC, respectively, and are the least represented patterns due to the low frequency of β-LG I*A (0.11, Table 1); patterns V and VII (BB-BC and BB-BB, respectively) were found both with a frequency of 0.11. The most represented patterns were, in descending order, VI, II and VIII (BB-CC, BB-CD, BB-DD). Pattern II (BC-BD) presented two variations, which led to a total of ten different protein patterns: IIa lacking in β-CN and IIb lacking in αS1-CN (Fig. 1). The β-LG I*A variant was found in combination with β-LG II BD, BB and BC genotypes, and in particular in one sample β-LG I*A was in haplotype with β-LG II*B (pattern III AA-BB). Since the two β-LG forms seem to have originated from gene duplication (GodovacZimmermann et al., 1990), it is not surprising that β-LG I and β-LG II could be in haplotype. Haplotype analysis showed that of the six possible haplotypes found in the hypothesis of no associations (Table 2), only four β-LG I- β-LG II haplotypes are the most probable due to association: A-B (0.11, all the β-LG I*A seems to be associated with β-LG II*B), B-B, B-C, and B-D. Analysis of a larger population or of genealogic data, together with the possibility of testing parents for β-LG I and β-LG II variants, could confirm the association that we found. Although various approaches have been used for CN quantification in human and cow milk, such as urea-polyacrylamide gel electrophoresis, capillary zone electrophoresis, and liquid chromatograph coupled with mass spectrometry (Kroening et al., 1998; Gustavsson et al., 2014; Altendorfer et al., 2015; Liao et al., 2017), there are still very few quantitative data on donkey milk in the literature. Some results on donkey milk proteins quantification using Agilent 2100 Bioanalyzer
standard deviations were calculated for the eight patterns. 3. Results and discussion The IEF analysis allowed to identify β-LG I, β-LG II, α-LA, LZ, αs1CN, β-CN fractions in line with the isoelectric point reported in the literature for the various milk proteins, and in particular referring to paper published by Criscione et al. (2009). These authors already used the IEF methodology to identify the main casein fractions and confirmed the results using RP-HPLC and MALDI-TOF techniques. κ-CN was deduced thanks to the pI and its behaviour in the IEF gels of whole milk and of the fractions of the whole milk made up of separated caseins and whey proteins respectively, while αS2-CN was not detectable. In agreement with previous studies on the Amiata donkey (Caroli et al., 2015), a genetic polymorphism for β-LG I was found. Two alleles for β-LG I (named A and B) and three alleles for β-LG II were highlighted (Fig. 1). Considering the IEF migration pattern and according to the pI of each variant reported by Criscione et al. (2018), we called the β-LG II IEF variants: B, C and D. The BIEF variant, with an intermediate pI, could correspond to A, B or E variants, which all have a pI of 4.45. The CIEF variant, with the least acid migration, must correspond with variant C, since it is the only variant with a pI of 4.47. Finally the DIEF variant, the most acidic one, could correspond to either D or F variants, which are both characterized by a pI of 4.39. A further molecular characterization should be carried out to confirm the association between the detected patterns and the corresponding genetic variants. The most frequent IEF variants were β-LG I*B and β-LG II*C, with a frequency of 0.89 and 0.42, respectively (Table 1), whereas β-LG II*B and β-LG II*D showed similar frequencies (0.27 and 0.31, respectively). The combination of β-LG I and β-LG II genotypes was responsible for eight of the ten observed IEF patterns: patterns I, III and IV correspond
Table 2 Frequencies of IEF patterns identified due to the β-LG I - β-LG II genotype combination and haplotype frequencies calculated both under the hypothesis of loci independence (H0) and taking association into account (H1). Pattern III was represented by only one sample. Pattern
Table 1 β- LG allele frequencies in the Amiata breed. Gene
Allele
Frequency
β-LG I
A B B C D
0.11 0.89 0.27 0.42 0.31
β-LG II
I II III IV V VI VII VIII
3
Genotypes β-LG I β-LG II
Freq.
AB BB AA AB BB BB BB BB
0.09 0.20 0.02 0.07 0.11 0.25 0.11 0.15
BD CD BB BC BC CC BB DD
Haplotype β-LG I β-LG II
H0
H1
A A A B B B
0.03 0.04 0.03 0.24 0.38 0.27
0.11 – – 0.16 0.43 0.31
B C D B C D
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(73.01% of total WP), and as shown by IEF (Fig. 1) β-LG I was much more represented than β-LG II, in line with the literature (GodovacZimmermann et al., 1990). The total content of β-LG in Amiata donkey was higher than the mean value obtained by the RP-HPLC by Vincenzetti et al. (2008) on Ragusana (3.7 g/L). However, a wide range of quantitative variation of β-LG (1.3–5.5 g/L) has been reported in has been reported in the literature in equids (Malacarne et al., 2002; Miranda et al., 2004; Brumini et al., 2016), and could be due to different genotypes of the individual animals as well as to differences among breeds and the different quantification methods applied. β-LG, which is absent in human milk, is present in cow milk with about 5 g/L (Hallén et al., 2008), and is considered one of main milk allergens, together with CN (Restani et al. 2009), although its role in donkey milk allergy has never been demonstrated. Moreover, donkey β-LG has been found to be highly degraded (70%) in vitro by human gastric and duodenal juice compared to its cow counterpart (Marletta et al., 2016). The α-LA mean concentration in Amiata donkey was 1.22 g/L ± 0.405 (14.69% of total WP), which is more similar to the value of 1.32 g/L reported in the Martina Franca breed (D'Alessandro et al., 2011), than to the 1.80 g/L reported in the Ragusana donkey (Vincenzetti et al., 2008), which were both measured by reversed-phase high-performance liquid chromatography. α-LA is higher is higher in human and mare milk (between 2.6 and 4.2 g/L; 2.36–3.3 g/L) and lower in cow milk (about 1 g/L) (Hallén et al., 2008; Malacarne et al., 2002; Miranda et al., 2004; Lönnerdal et al., 2017). Finally, the average LZ concentration was 1.07 g/L ± 1.226 (12.90% of total WP) which is similar to the findings of Vincenzetti et al. (2008) in the Ragusana breed and mare milk by Miranda et al. (2004) and in the range of human milk (0.3–1.1 g/L; Lönnerdal et al., 2017). In bovine milk, only LZ traces have been reported (Martini et al., 2018). This high variability could be due to genetic polymorphism, as mutations in non-coding regions of the LZ gene have been reported in other asinine breeds. These polymorphisms may have an effect on the expression of this gene (Cosenza et al., 2018). In the IEF pattern II, we found variations in the CN:WP ratio due to the absence of β and αS1-CN. In both cow and goat milk, protein polymorphism has been found to be associated with a different expression levels and relative content of the various proteins (Hallén et al., 2008). We thus estimated by Agilent quantification the mean content of each fraction based on the eight IEF patterns identified, excluding patterns IIa and IIb, which were represented by just one sample each. We then tested whether there was a similar trend in donkey milk. Our results (Table 3) showed that the pattern variability seems associated with the protein fraction content. Patterns I and IV are those with the lowest CN:WP ratio. These two patterns have β-LG I*A in common. Unfortunately pattern III, which is homozygous for the β-LG I*A, was represented by only one sample, and had the highest CN:WP ratio with Agilent quantification and the lowest CN:WP ratio with IEF gel quantification. All the other patterns had similar ratios both by Agilent and in IEF gel quantification. Thus, for this sample, the Agilent assay could have overestimated the CN content (especially αS1-CN). Finding other individuals carrying β-LG I*A at the homozygous state
with the Protein 80 kit were recently published (Gubić et al., 2016). An application note of this kit demonstrate its ability to discriminate the main protein fraction in different species, showing that sheep, cows and goats milk main protein, although presenting many differences in amino acidic sequences, have similar behaviour on the chip. We used previous data obtained with goat, cattle and donkey milk (data not published) on Protein 80 kit to assign the corresponding peaks in the High Sensitivity Protein 250 kit. We decide to use this kit because of the very low content of some milk proteins in donkey milk. Four samples were replicated twice in different chips and the mean correlation of the two quantifications was 0.97 ± 0.02. We also compared the quantification obtained by image analysis of the IEF to confirm the results obtained by the Agilent 2100 Bioanalyzer. Furthermore, since the quantification on the IEF gels is much more time consuming and the software is not always able to identify the bands if they are too weak, to close to each other or not perfectly straight, we had to exclude about the 30% of the individuals samples because of too much lacking data; therefore we shown the detailed quantification results obtained with the Agilent 2100 Bioanalyzer and used the IEF quantification data only to confirm the obtained results. One of the most important parameters regarding donkey milk proteins is the CN:WP ratio. In fact this ratio is one of the parameters that makes donkey milk more similar to human milk than that of other dairy animals (Martini et al., 2018). In the present study the mean CN:WP ratio was 1.08 ± 0.364 and 1.12 ± 0.207, with Agilent and IEF gel, respectively (Table 3), in line with our previous work (Martini et al., 2014b). Agilent quantification revealed that β-CN was the most represented protein, with an average content of 6.11 g/L ± 1.030 (62.34% of total CN), which is an intermediate value between the minimum values found in human (1.25–4.72 g/L) (Liao et al., 2017; Kroening et al., 1998) and the maximum in cow milk (11.85–12.87 g/L) and mare milk (11 g/L) (Hallén et al., 2008; Miranda et al., 2004). Our results confirm Cunsolo et al. (2017) who found that β-CN was the main CN fraction in donkey milk, followed by αS1-CN. The mean concentration of αS1-CN was 2.54 g/L ± 0.673 (25.92% of total CN) similarly to mare milk (Miranda et al., 2004), but higher than in human milk (0.33–0.50 g/L) (Altendorfer et al., 2015; Liao et al., 2017), and lower than in cow milk (8.52–9.16 g/L) (Hallén et al., 2008). Although αS2eCN has been detected as a minor component in donkey milk (Bertino et al., 2010; Marletta et al., 2016) and in mare (Miranda et al., 2004), in our study it was not detectable. The absence of αS2-CN has also been reported in human milk, where it is caused by a premature stop codon (Rijnkels et al., 2003) resulting in no αS2-CN expression, whereas in cow milk the average content is 1.48 g/L (Hallén et al., 2008; Gustavsson et al., 2014). To our knowledge, the κ-CN content has never been quantified in donkey milk. In our study κ-CN was the minor CN fraction, with a mean content of 1.18 g/L ± 0.685 (12.04% of total CN), higher than the human average content of 0.75 g/L and 0.25 g/L respectively (Liao et al., 2017; Miranda et al., 2004) and lower than the 3.07–3.82 g/L of cow milk (Hallén et al., 2008). β-LG was the predominant whey protein with an average content of 6.06 g/L ± 1.610
Table 3 CN:WP ratio and milk protein fraction content for each pattern (g/L) as obtained from Agilent 2100 Bioanalyzer quantification. Pattern III was represented by only one sample. Data are expressed as mean ± standard deviation. Pattern
I II III IV V VI VII VIII total
CN:WP IEF
Agilent
0.97 1.06 0.88 0.85 1.21 1.18 1.29 1.25 1.12
0.96 1.16 1.33 0.94 1.23 1.02 1.14 1.09 1.08
± 0.04 ± 0.10 ± ± ± ± ± ±
0.07 0.15 0.15 0.05 0.12 0.21
± 0.23 ± 0.29 ± ± ± ± ± ±
0.26 0.36 0.43 0.27 0.27 0.36
αs1-CN
β-CN
2.26 2.25 3.15 2.39 2.20 2.58 2.73 2.65 2.54
6.12 5.87 5.07 6.32 6.40 6.23 5.94 6.21 6.11
± 0.98 ± 0.90 ± 0.55 ± 1.00 ± 0.49 ± 0.17 ± 0.21 ± 0.67
κ-CN
± 0.70 ± 1.92 ± ± ± ± ± ±
0.77 0.94 0.51 0.20 0.24 1.03
4
1.33 1.45 1.49 1.00 1.16 1.07 1.06 0.93 1.18
α-LA
ß-LG
± 0.41 ± 1.34 ± ± ± ± ± ±
0.28 0.15 0.24 0.15 0.32 0.69
3.88 6.48 2.81 4.31 6.07 6.67 6.86 6.64 6.06
± 0.50 ± 1.96 ± ± ± ± ± ±
0.32 1.28 1.26 0.35 0.75 1.61
1.17 1.05 1.93 1.10 1.32 1.12 1.01 1.55 1.22
LZ
± 0.42 ± 0.33 ± ± ± ± ± ±
0.34 0.54 0.40 0.26 0.33 0.41
3.02 0.51 3.47 3.72 0.84 0.43 0.22 0.44 1.07
± 0.59 ± 0.43 ± ± ± ± ± ±
0.24 0.12 0.21 0.02 0.23 1.23
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Lönnerdal, B., Erdmann, P., Thakkar, S.K., Sauser, J., Destaillats, F., 2017. Longitudinal evolution of true protein, amino acids and bioactive proteins in breastmilk: a developmental perspective. J. Nutr. Biochem.41, 1–11. Malacarne, M., Martuzzi, F., Summer, A., Mariani, P., 2002. Protein and fat composition of mare’s milk: some nutritional remarks with reference to human and cow’s milk. Int. Dairy J. 12, 869–877. Martini, M., Altomonte, I., Salari, F., Caroli, A.M., 2014a. Short communication: monitoring nutritional quality of Amiata donkey milk: effects of lactation and productive season. J. Dairy Sci. 97, 6819–6822. Martini, M., Altomonte, I., Salari, F., 2014b. Amiata donkeys: fat globule characteristics, milk gross composition and fatty acids. Ital. J. Anim. Sci 13, 123–126. Martini, M., Altomonte, I., Licitra, R., Salari, F., 2018. Nutritional and nutraceutical quality of donkey milk. J. Equine Vet. Sci. 65, 33–37. Miranda, G., Mahé, M., Leroux, C., Martin, P., 2004. Proteomic tools to characterize the protein fraction of Equidae milk. Proteomics 4, 2496–2509. Monti, G., Viola, S., Baro, C., Cresi, F., Tovo, P.A., Moro, G., Ferrero, M.P., Conti, A., Bertino, E., 2012. Tolerability of donkeys'milk in 92 highly-problematic cows’ milk allergic children. J. Biol. Regul. Homeost. Agents 26, 75–82. Marletta, D., Tidona, F., Bordonaro, S., 2016. Donkey milk proteins: digestibility and nutritional significance. In: Gigli, I. (Ed.), Milk Proteins - From Structure to Biological Properties and Health Aspects. InTech., Rijeka, pp. 199–209. Restani, P., Ballabio, C., Lorenzo, Di, C., Tripodi, S., Fiocchi, 2009. Molecular aspects of milk allergens and their role in clinical events. Anal. Bioanal. Chem. 395, 47–56. Rijnkels, M., Elnitski, L., Miller, W., Rosen, J.M., 2003. Multispecies comparative analysis of a mammalian-specific genomic domain encoding secretory proteins. Genomics 82, 417–432. Saletti, R., Muccilli, V., Cunsolo, V., Fontanini, D., Capocchi, A., Foti, S., 2012. MS- based characterization of α(s2)-casein isoforms in donkey's milk. J. Mass Spectrom. 47, 1150–1159. SAS Institute, 2008. SAS/STAT User's Guide. SAS Institute Inc., Cary Release 9.2. Tidona, F., Sekse, C., Criscione, A., Jacobsen, M., Bordonaro, S., Marletta, D., Vegarud, G.E., 2011. Antimicrobial effect of donkeys' milk digested in vitro with human
could help in understanding whether an association really exists. These three patterns have another common feature: they have the highest LZ values and the lowest β-LG quantity. This suggests that β-LG I*A could be associated with a reduction in β-LG content which seems to be balanced by an increase in LZ. By contrast, the homozygous BB pattern VII for β-LG I and β-LG II had a higher β-LG content and less LZ. 4. Conclusions The present work demonstrates the interesting genetic variability of Amiata donkey milk proteins. The IEF analysis highlighted the genetic polymorphism of β-LG I and of β-LG II, together with the presence of putative null alleles both for β and αS1-CN. Although further molecular characterization is necessary to confirm the association between the detected patterns and the corresponding genetic variants, and to discover the mutation responsible for the lack of β and αS1-CN, the IEF technique was shown to be a cheap and fast method able to detect most of the phenotypic and the majority of the genetic variability of donkey milk proteins. We also managed to quantify the κ-CN, whose content has never been measured before in donkey milk, and we showed that the protein fraction content could be related to genetic polymorphism. The methods applied highlighted a variability that seems to influence the CN:WP ratio and the content of specific caseins, LZ and β-LG. This information could be exploited to increase the knowledge on the nutritional quality of donkey milk and could thus be used when selecting donkeys to produce milk with proven nutraceutical effects. Declaration of Competing Interest None of the authors have any conflicts of interest to declare. Acknowledgments This work was supported by PRA 2017 (AteneoResearch Project, University of Pisa). References Altendorfer, I., König, S., Braukmann, A., Saenger, T., Bleck, E., Vordenbäumen, S., Kubiak, A., Schneider, M., Jose, J., 2015. Quantification of αS1-casein in breast milk using a targeted mass spectrometry-based approach. J. Pharm. Biomed. Anal. 103, 52–58. Altomonte, I., Salari, F., Licitra, R., Martini, M., 2019. Donkey and human milk: insights into their compositional similarities. Int. Dairy J. 89, 111–118. Barni, S., Sarti, L., Mori, F., Muscas, G., Belli, F., Pucci, N., Novembre, E., 2018. Tolerability and palatability of donkey's milk in children with cow's milk allergy. Pediatr. Allergy Immunol. 29, 329–331. Bertino, E., Gastaldi, D., Monti, G., Baro, C., Fortunato, D., Perono Garoffo, L., Coscia, A., Fabris, C, Mussap, M, Conti, A, 2010. Detailed proteomic analysis on DM: insight into its hypoallergenicity. Front. Biosci. (Elite Ed.) 2, 526–536. Brumini, D., Criscione, A., Bordonaro, S., Vegarud, G.E., Marletta, D., 2016. Whey proteins and their antimicrobial properties in donkey milk: a brief review. Dairy Sci. Technol. 96, 1–14. Caroli, A.M., Jann, O., Budelli, E., Bolla, P., Jäger, S, Erhardt, G, 2001. Genetic polymorphism of goat kappa-casein (CSN3) in different breeds and characterization at DNA level. Anim. Genet. 32, 226–230. Caroli, A.M., Chessa, S., Erhardt, G.J., 2009. Invited review: milk protein polymorphisms in cattle: effect on animal breeding and human nutrition. J. Dairy Sci. 92, 5335–5352. Caroli, A.M., Bulgari, O., Gigliotti, C., Altomonte, I., Salari, F., Martini, M., 2015. Profilo lattoproteico e attività proteasica totale del latte di asina. Scienza e Tecnica LattieroCasearia 66, 11–16. Ceriotti, G., Chiatti, F., Bolla, P., Martini, M., Caroli, A., 2005. Genetic variability of the ovine αs1-casein. Ital. J. Anim. Sci. 4, 64–66. Chianese, L., Calabrese, M.G., Ferranti, P., Mauriello, R., Garro, G., De Simone, C., Ramunno, L., 2010. Proteomic characterization of donkey milk “caseome”. J. Chromatogr. A 1217, 4834–4840. Chianese, L., De Simone, C., Ferranti, P., Mauriello, R., Costanzo, A., Quarto, M., Ramunno, L., 2013. Occurrence of qualitative and quantitative polymorphism at donkey beta-Lactoglobulin II locus. Food Res. Int. 54, 1273–1279. Cosenza, G., Ciampolini, R., Iannaccone, M., Gallo, D., Auzino, B., Pauciullo, A., 2018. Sequence variation and detection of a functional promoter polymorphism in the lysozyme c-type gene from Ragusano and Grigio Siciliano donkeys. Anim. Genet. 49, 270–271. Criscione, A, Cunsolo, V, Tumino, S., Di, Francesco, A, Bordonaro, S, Muccilli, V, Saletti,
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Vincenzetti, S., Pucciarelli, S., Polzonetti, V., Polidori, P., 2017. Role of proteins and of some bioactive peptides on the nutritional quality of donkey milk and their impact on human health. Beverages 3, 1–20. Weir, B.S., 1996. Genetic Data Analysis II: Methods For Discrete Population Genetic Data, second ed. Sinauer Associates Inc., Sunderland.
gastrointestinal enzymes. Int. Dairy J. 21, 158–165. Tidona, F., Criscione, A., Devold, T.G., Bordonaro, S., Marletta, D., Vegarud, G.E., 2014. Protein composition and micelle size of donkey milk with different protein patterns: effects on digestibility. Int. Dairy J. 35, 57–62. Vincenzetti, S., Polidori, P., Mariani, P., Cammertoni, N., Fantuz, F., Vita, A., 2008. Donkey's milk protein fractions characterization. Food Chem 106, 640–649.
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Milk protein polymorphism in Amiata donkey a
b,c
a
T b
d
Rosario Licitra , Stefania Chessa , Federica Salari , Stefano Gattolin , Omar Bulgari , ⁎ Iolanda Altomontea, Mina Martinia,e, a
Department of Veterinary Science, University of Pisa, Pisa, Italy Institute of Agricultural Biology and Biotechnology, National Research Council, Lodi, Italy Department of Veterinary Science, University of Turin, Turin, Italy d Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy e Interdepartmental Research Center Nutrafood “Nutraceuticals and Food for Health”, University of Pisa, Pisa, Italy b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Donkey milk Milk protein Casein Whey protein Milk protein fractions Milk protein polymorphism
Interest in donkey milk for human nutrition has been growing steadily, especially in the therapeutic-diet field, due to its similarity to human milk. Caseins are one of the main allergenic components of milk. Whereas cow milk has a high casein content, in donkey milk the casein concentration is lower and is characterized by a greater digestibility. In fact, recent studies have shown that donkey milk can be consumed by children with cow milk protein allergy. The aim of this study was to evaluate the protein profile of individual samples of Amiata donkey milk and to quantify the different protein fractions in order to better understand the characteristics of donkey milk. Protein fractions of 57 individual whole milk samples were analysed by isoelectric focusing (IEF). The main milk protein fractions were quantified both by IEF gel image analysis and directly on milk using the Agilent 2100 Bioanalyzer. The IEF analysis identified 10 different electrophoretic patterns, which differ from each other in terms of the presence of bands that correspond to homozygous or heterozygous genotypes, as well as the presence/absence of specific protein bands. In addition the quantification of different caseins (αS1, β and κ-CN) and whey proteins (β-LG, α-LA and LZ), highlighted a significant phenotypic variability among individuals which seems to be related to the patterns identified. In particular, different casein:whey protein ratios were identified among jennies and this is known to affect the nutritional, allergenic and technological properties of milk. These findings could be useful in the selection of donkeys with specific milk protein polymorphisms.
1. Introduction Among the various types of milk produced by non-specialized species, donkey milk has recently been rediscovered. This is mainly due to the increasing interest of consumers and scientists in foods with beneficial effects on human health. There is increasing evidence that donkey milk can be successfully used to feed children that suffer from CMPA (Monti et al., 2012; Barni et al., 2018). Donkey milk is similar to human milk, especially in terms of the protein fractions (Altomonte et al., 2019). Milk proteins typically show a high degree of genetic polymorphism, which together with alternative splicing, results in several forms of each protein, characterized by amino acid exchanges and/or deletions of peptides. Post-translational modifications such as phosphorylation or glycosylation further increase this heterogeneity (Ceriotti et al., 2005; Caroli et al., 2009). Caseins represent the main
milk protein fraction in ruminants (about 80% of the total protein) and they are recognized as one of the main allergenic components of milk (Restani et al., 2009). Donkey milk has a lower CN concentration (about 50% of the total protein) (Martini et al., 2014a) and a greater CN digestibility (Tidona et al., 2014) compared to ruminant milk. In donkey milk, the main CN fraction is β-CN, while αS1-CN, αS2-CN and κ-CN are less represented (Cunsolo et al., 2017). The primary structure of donkey αS1-CN, classified as variant A, shows 202 amino acids, a MW of 24,406 Da, a pI of 5.96, a variable degree of phosphorylation, and the presence of isoforms generated by alternative mRNA splicing events. The αS1-CN isoforms are A1 (201 amino acids; MW 24,278 Da; pI 5.96), B (197 amino acids; MW 23,786 Da; pI 5.85) and B1 (196 amino acids; MW 23,658 Da; pI 5.85) (Cunsolo et al., 2017). Two other minor αS1-CN isoforms, with a MW of 25,142 and 25,272 Da, respectively, have also been reported (Cunsolo et al.,
Abbreviations: CN, Casein; WP, Whey protein; CMPA, Cow milk protein allergy; α-LA, Alpha-lactalbumin; β-LG, beta-lactoglobulin; LZ, lysozyme; MW, molecular weight; pI, isoelectric point ⁎ Corresponding author. E-mail address: [email protected] (M. Martini). https://doi.org/10.1016/j.livsci.2019.103845 Received 19 March 2019; Received in revised form 22 October 2019; Accepted 22 October 2019 Available online 25 October 2019 1871-1413/ © 2019 Elsevier B.V. All rights reserved.
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2009a). All donkey αS1-CN isoforms show two levels of phosphorylation with 5 and 6 phosphate groups. Bertino and colleagues (2010) reported that donkey αS1-CN may also exist as glycosylated forms, in contrast to human or bovine αS1-CN, which have never been reported to be glycosylated. Donkey αS2-CN is a very minor component and presents several isoforms. The full-length isoform A (MW 26,028 Da; pI 5.68) contains 221 amino acids and a disulphide bond linking Cys37 and Cys41, whereas the other isoforms described are internally-deleted because of alternative mRNA splicing events: A1 (216 amino acids; MW 25,429 Da; pI 5.50), A1Δ4,5,6 (185 amino acids; MW 21,939 Da; pI 5.65), A2 (214 amino acids; MW 25,203 Da; pI 5.68), and A2 Δ4,5,6 (183 amino acids; MW 21,713 Da; pI 5.85) (Saletti et al., 2012). Like αS1-CN, αS2-CN also shows micro-heterogeneity depending on a variable degree of phosphorylation related to the ten to twelve possible phosphate groups present (Chianese et al., 2010). β-CN is the main fraction in donkey milk and consists of a full-length β-CN named A (226 amino acids; MW 25,529 Da; pI 5.44), and a short form named B (218 amino acids; 24,606 Da; pI 5.24), characterized by the absence of the 27ESITHINK34 peptide due to an alternative splicing of exon 5 (Girardet et al., 2006; Cunsolo et al., 2009b, 2017). Both A and B β-CN fractions show three phosphorylated forms, carrying 5, 6 and 7P, respectively, with the predominant isoform carrying 5P Chianese et al. (2010) described a new variant with a higher mass than the β-CN A and with the same phosphorylation pattern (5, 6, and 7P), but its primary structure was not reported. The κ-CN in donkey milk represents a minor component which has not yet been quantified. It is characterized by a sequence of 162 amino acids, a MW of 18,485 Da, a pI of 9.36 and only one cysteine. Polymorphisms are also present and there is a micro-heterogeneity related to post-translational modifications, as both glycosylated and non-glycosylated forms have also been reported (Chianese et al., 2010). However, it is the only casein not affected by splicing events and therefore without known longer and shorter forms of the same CN. Based on these characteristics, donkey κCN is more similar to human than bovine κ-CN. Compared to ruminant milk, there are higher amounts of whey proteins in donkey milk, and the main whey proteins are β-LG, α-LA and LZ (Vincenzetti et al., 2017). Donkey β-LG exists in two different molecular forms, namely β-LG I (80% of total ß-LG) and β-LG II (20% of total ß-LG), which seem to be the result of gene duplication (GodovacZimmermann et al., 1990). The primary structures of the main isoforms of β-LG I and β-LG II (isoform A) are different: β-LG I consists of 162 amino acids, a MW of 18,524 Da and a pI of 4.53, while β-LG II consists of 163 amino acids, a MW of 18,258 Da and a pI of 4.45 (Herrouin et al., 2000; Cunsolo et al., 2007; Chianese et al., 2013). β-LG I presents a further variant, named B (MW 18,510 Da; pI 4.59), which differs from variant A in relation to three amino acid exchanges E36 → S36, S97 → T97 and V150 → I150 (Herrouin et al., 2000). β-LG II locus shows a higher genetic polymorphism, with six described variants, all characterized by 163 amino acid residues, but differing in terms of amino acid substitution: variant A characterized by D2R18V25D96A100C110M118G162 (MW 18,246 Da; pI 4.45); variant B by D2R18V25D96A100P110T118G162 (MW 18,222 Da; pI 4.45); variant C by D2R18V25E96A100P110T118G162 (MW 18,236 Da; pI 4.47); variant D by D2R18V25D96A100P110M118D162 (MW 18,298 Da; pI 4.39); variant E by N2K18A25D96A100P110M118D162 (MW 18,253 Da; pI 4.45); and variant F characterized by D2R18V25D96T100P110T118D162 (MW 18,310 Da; pI 4.39) and expressed at a very low level (Criscione et al., 2018). Unlike milk of other species (cow, camel and mare), where different variants of α-LA have been described, in donkey milk only one protein form has been identified and characterized, though the existence of two isoforms has been hypothesized (Giuffrida et al., 1992). α-LA consists of 123 amino acid residues with eight cysteines, a MW of 14,214 Da and a pI of 4.88. LZ is an enzyme closely related to α-LA showing a similar amino acid sequence and three-dimensional structure (Tidona et al., 2011). In donkey milk, two LZ variants with 129 amino acid residues have been identified: variant A (MW 14,680 Da; pI 8.01) and variant B (MW
14,631 Da; pI 7.77), differing from variant A in terms of three substitutions (N49 → D49, Y52 → S52 and S61 → N61) (Herrouin et al., 2000). Compared to its bovine counterpart, donkey LZ is more hydrophilic. The genetic polymorphisms of proteins can influence the functional and biological properties of milk and also the involvement of each allergenic protein in clinical symptoms. Although donkey milk has been the subject of intensive research in the last decade, and all these protein isoforms have been described, the different protein fractions and the genetic polymorphisms have still not been fully investigated. Nor is the protein profile completely understood. The aim of this study was thus to clarify which part of the donkey milk protein heterogeneity is easily detectable with the IEF method, and to quantify the different protein fractions in Amiata donkey in order to better characterize this breed. 2. Materials and methods 2.1. Animals and sample collection The research was conducted on 57 healthy jennies of the Amiata donkey, that were daughters of more than 20 stallions. The Amiata donkey is a local breed native to the Mount Amiata area, in Tuscany (central Italy), which consistency was 2457 of which males 114 and 1468 jennies in 2018 (DAD-IS, 2018) Although the increasing trend from 2001 is still endangered and placed in the Local Equine Population List (www.aia.it 2019). The jennies were housed in the same farm reared outdoors in a semiintensive system and were routinely machine-milked once per day. The diet consisted of ad libitum grass hay supplemented with 2 kg/head/day of commercial concentrate. Individual raw milk samples were collected from morning mechanical milking and the foals were separated from their mothers three hours before milking. The milk samples belong from animals that were all between the 6th and 7th month of lactation, with an average milk yield per milking of 0.505 L ± 0.236, a mean age of 10.54 ± 4.46 years and an average parity of 3.49 ± 1.85; the samplings were uniformly distributed throughout the year. After collection, samples were immediately frozen at −20 °C to prevent undesired proteolysis, and then transported to a laboratory for the analysis. 2.2. Milk analysis The individual protein fractions were analysed by IEF on ultrathin gels (250×115×0.3 mm), using a GelBond polyester film as a support. The IEF analysis was performed according to Caroli et al. (2001) with some modifications. In detail, for the screening of donkey milk, the ampholyte mixture was as follows: 1.5% (v/v) Pharmalyte pH 2.5–5.0; 3.3% (v/v) Pharmalyte pH 4.2–4.9; and 2.3% (v/v) Ampholine® pH 5.0–8.0 (Sigma Aldrich, Steinheim, Germany). An acidic precipitation of individual milk samples was also performed in order to separate caseins from whey proteins and run them separately on the same IEF gel condition in order to confirm the identification of the different bands in the whole milk. 2.3. Data analysis The milk protein alleles and haplotype distributions were analysed by the ALLELE and HAPLOTYPE SAS procedures (SAS Institute, 2008). The ALLELE procedure uses the notation and concepts described by Weir (1996). Haplotype frequencies were calculated under the null hypothesis of no linkage disequilibrium and under the alternative hypothesis of associations between genes. The main milk protein fractions were quantified from IEF gels by image acquisition through G:Box (Syngene, Frederick, MD, USA) and using Agilent 2100 Bioanalyzer with the Agilent High Sensitivity Protein 250 kit (Agilent Technologies, Santa Clara, CA, USA). For each milk protein fraction, means and 2
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Fig. 1. Isoelectrofocusing of 12 individual Amiata donkey milk samples. The eight IEF patterns determined by βLG I and β-LG II genotypes are reported and indicated with Roman numerals. The black dot corresponds to β-LG II*D, the black diamond to β-LG II*B (or A or E), the black star to β-LG II*C, the white dot to β-LG I*A, and the white diamond to βLG I*B. Genotypes are written in black for β-LG II, and in white for β-LG I. IIa = pattern II lacking β-CN; IIb = =pattern lacking αS1-CN.
to β-LG I-β-LG II AB-BD, AA-BB, and AB-BC, respectively, and are the least represented patterns due to the low frequency of β-LG I*A (0.11, Table 1); patterns V and VII (BB-BC and BB-BB, respectively) were found both with a frequency of 0.11. The most represented patterns were, in descending order, VI, II and VIII (BB-CC, BB-CD, BB-DD). Pattern II (BC-BD) presented two variations, which led to a total of ten different protein patterns: IIa lacking in β-CN and IIb lacking in αS1-CN (Fig. 1). The β-LG I*A variant was found in combination with β-LG II BD, BB and BC genotypes, and in particular in one sample β-LG I*A was in haplotype with β-LG II*B (pattern III AA-BB). Since the two β-LG forms seem to have originated from gene duplication (GodovacZimmermann et al., 1990), it is not surprising that β-LG I and β-LG II could be in haplotype. Haplotype analysis showed that of the six possible haplotypes found in the hypothesis of no associations (Table 2), only four β-LG I- β-LG II haplotypes are the most probable due to association: A-B (0.11, all the β-LG I*A seems to be associated with β-LG II*B), B-B, B-C, and B-D. Analysis of a larger population or of genealogic data, together with the possibility of testing parents for β-LG I and β-LG II variants, could confirm the association that we found. Although various approaches have been used for CN quantification in human and cow milk, such as urea-polyacrylamide gel electrophoresis, capillary zone electrophoresis, and liquid chromatograph coupled with mass spectrometry (Kroening et al., 1998; Gustavsson et al., 2014; Altendorfer et al., 2015; Liao et al., 2017), there are still very few quantitative data on donkey milk in the literature. Some results on donkey milk proteins quantification using Agilent 2100 Bioanalyzer
standard deviations were calculated for the eight patterns. 3. Results and discussion The IEF analysis allowed to identify β-LG I, β-LG II, α-LA, LZ, αs1CN, β-CN fractions in line with the isoelectric point reported in the literature for the various milk proteins, and in particular referring to paper published by Criscione et al. (2009). These authors already used the IEF methodology to identify the main casein fractions and confirmed the results using RP-HPLC and MALDI-TOF techniques. κ-CN was deduced thanks to the pI and its behaviour in the IEF gels of whole milk and of the fractions of the whole milk made up of separated caseins and whey proteins respectively, while αS2-CN was not detectable. In agreement with previous studies on the Amiata donkey (Caroli et al., 2015), a genetic polymorphism for β-LG I was found. Two alleles for β-LG I (named A and B) and three alleles for β-LG II were highlighted (Fig. 1). Considering the IEF migration pattern and according to the pI of each variant reported by Criscione et al. (2018), we called the β-LG II IEF variants: B, C and D. The BIEF variant, with an intermediate pI, could correspond to A, B or E variants, which all have a pI of 4.45. The CIEF variant, with the least acid migration, must correspond with variant C, since it is the only variant with a pI of 4.47. Finally the DIEF variant, the most acidic one, could correspond to either D or F variants, which are both characterized by a pI of 4.39. A further molecular characterization should be carried out to confirm the association between the detected patterns and the corresponding genetic variants. The most frequent IEF variants were β-LG I*B and β-LG II*C, with a frequency of 0.89 and 0.42, respectively (Table 1), whereas β-LG II*B and β-LG II*D showed similar frequencies (0.27 and 0.31, respectively). The combination of β-LG I and β-LG II genotypes was responsible for eight of the ten observed IEF patterns: patterns I, III and IV correspond
Table 2 Frequencies of IEF patterns identified due to the β-LG I - β-LG II genotype combination and haplotype frequencies calculated both under the hypothesis of loci independence (H0) and taking association into account (H1). Pattern III was represented by only one sample. Pattern
Table 1 β- LG allele frequencies in the Amiata breed. Gene
Allele
Frequency
β-LG I
A B B C D
0.11 0.89 0.27 0.42 0.31
β-LG II
I II III IV V VI VII VIII
3
Genotypes β-LG I β-LG II
Freq.
AB BB AA AB BB BB BB BB
0.09 0.20 0.02 0.07 0.11 0.25 0.11 0.15
BD CD BB BC BC CC BB DD
Haplotype β-LG I β-LG II
H0
H1
A A A B B B
0.03 0.04 0.03 0.24 0.38 0.27
0.11 – – 0.16 0.43 0.31
B C D B C D
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(73.01% of total WP), and as shown by IEF (Fig. 1) β-LG I was much more represented than β-LG II, in line with the literature (GodovacZimmermann et al., 1990). The total content of β-LG in Amiata donkey was higher than the mean value obtained by the RP-HPLC by Vincenzetti et al. (2008) on Ragusana (3.7 g/L). However, a wide range of quantitative variation of β-LG (1.3–5.5 g/L) has been reported in has been reported in the literature in equids (Malacarne et al., 2002; Miranda et al., 2004; Brumini et al., 2016), and could be due to different genotypes of the individual animals as well as to differences among breeds and the different quantification methods applied. β-LG, which is absent in human milk, is present in cow milk with about 5 g/L (Hallén et al., 2008), and is considered one of main milk allergens, together with CN (Restani et al. 2009), although its role in donkey milk allergy has never been demonstrated. Moreover, donkey β-LG has been found to be highly degraded (70%) in vitro by human gastric and duodenal juice compared to its cow counterpart (Marletta et al., 2016). The α-LA mean concentration in Amiata donkey was 1.22 g/L ± 0.405 (14.69% of total WP), which is more similar to the value of 1.32 g/L reported in the Martina Franca breed (D'Alessandro et al., 2011), than to the 1.80 g/L reported in the Ragusana donkey (Vincenzetti et al., 2008), which were both measured by reversed-phase high-performance liquid chromatography. α-LA is higher is higher in human and mare milk (between 2.6 and 4.2 g/L; 2.36–3.3 g/L) and lower in cow milk (about 1 g/L) (Hallén et al., 2008; Malacarne et al., 2002; Miranda et al., 2004; Lönnerdal et al., 2017). Finally, the average LZ concentration was 1.07 g/L ± 1.226 (12.90% of total WP) which is similar to the findings of Vincenzetti et al. (2008) in the Ragusana breed and mare milk by Miranda et al. (2004) and in the range of human milk (0.3–1.1 g/L; Lönnerdal et al., 2017). In bovine milk, only LZ traces have been reported (Martini et al., 2018). This high variability could be due to genetic polymorphism, as mutations in non-coding regions of the LZ gene have been reported in other asinine breeds. These polymorphisms may have an effect on the expression of this gene (Cosenza et al., 2018). In the IEF pattern II, we found variations in the CN:WP ratio due to the absence of β and αS1-CN. In both cow and goat milk, protein polymorphism has been found to be associated with a different expression levels and relative content of the various proteins (Hallén et al., 2008). We thus estimated by Agilent quantification the mean content of each fraction based on the eight IEF patterns identified, excluding patterns IIa and IIb, which were represented by just one sample each. We then tested whether there was a similar trend in donkey milk. Our results (Table 3) showed that the pattern variability seems associated with the protein fraction content. Patterns I and IV are those with the lowest CN:WP ratio. These two patterns have β-LG I*A in common. Unfortunately pattern III, which is homozygous for the β-LG I*A, was represented by only one sample, and had the highest CN:WP ratio with Agilent quantification and the lowest CN:WP ratio with IEF gel quantification. All the other patterns had similar ratios both by Agilent and in IEF gel quantification. Thus, for this sample, the Agilent assay could have overestimated the CN content (especially αS1-CN). Finding other individuals carrying β-LG I*A at the homozygous state
with the Protein 80 kit were recently published (Gubić et al., 2016). An application note of this kit demonstrate its ability to discriminate the main protein fraction in different species, showing that sheep, cows and goats milk main protein, although presenting many differences in amino acidic sequences, have similar behaviour on the chip. We used previous data obtained with goat, cattle and donkey milk (data not published) on Protein 80 kit to assign the corresponding peaks in the High Sensitivity Protein 250 kit. We decide to use this kit because of the very low content of some milk proteins in donkey milk. Four samples were replicated twice in different chips and the mean correlation of the two quantifications was 0.97 ± 0.02. We also compared the quantification obtained by image analysis of the IEF to confirm the results obtained by the Agilent 2100 Bioanalyzer. Furthermore, since the quantification on the IEF gels is much more time consuming and the software is not always able to identify the bands if they are too weak, to close to each other or not perfectly straight, we had to exclude about the 30% of the individuals samples because of too much lacking data; therefore we shown the detailed quantification results obtained with the Agilent 2100 Bioanalyzer and used the IEF quantification data only to confirm the obtained results. One of the most important parameters regarding donkey milk proteins is the CN:WP ratio. In fact this ratio is one of the parameters that makes donkey milk more similar to human milk than that of other dairy animals (Martini et al., 2018). In the present study the mean CN:WP ratio was 1.08 ± 0.364 and 1.12 ± 0.207, with Agilent and IEF gel, respectively (Table 3), in line with our previous work (Martini et al., 2014b). Agilent quantification revealed that β-CN was the most represented protein, with an average content of 6.11 g/L ± 1.030 (62.34% of total CN), which is an intermediate value between the minimum values found in human (1.25–4.72 g/L) (Liao et al., 2017; Kroening et al., 1998) and the maximum in cow milk (11.85–12.87 g/L) and mare milk (11 g/L) (Hallén et al., 2008; Miranda et al., 2004). Our results confirm Cunsolo et al. (2017) who found that β-CN was the main CN fraction in donkey milk, followed by αS1-CN. The mean concentration of αS1-CN was 2.54 g/L ± 0.673 (25.92% of total CN) similarly to mare milk (Miranda et al., 2004), but higher than in human milk (0.33–0.50 g/L) (Altendorfer et al., 2015; Liao et al., 2017), and lower than in cow milk (8.52–9.16 g/L) (Hallén et al., 2008). Although αS2eCN has been detected as a minor component in donkey milk (Bertino et al., 2010; Marletta et al., 2016) and in mare (Miranda et al., 2004), in our study it was not detectable. The absence of αS2-CN has also been reported in human milk, where it is caused by a premature stop codon (Rijnkels et al., 2003) resulting in no αS2-CN expression, whereas in cow milk the average content is 1.48 g/L (Hallén et al., 2008; Gustavsson et al., 2014). To our knowledge, the κ-CN content has never been quantified in donkey milk. In our study κ-CN was the minor CN fraction, with a mean content of 1.18 g/L ± 0.685 (12.04% of total CN), higher than the human average content of 0.75 g/L and 0.25 g/L respectively (Liao et al., 2017; Miranda et al., 2004) and lower than the 3.07–3.82 g/L of cow milk (Hallén et al., 2008). β-LG was the predominant whey protein with an average content of 6.06 g/L ± 1.610
Table 3 CN:WP ratio and milk protein fraction content for each pattern (g/L) as obtained from Agilent 2100 Bioanalyzer quantification. Pattern III was represented by only one sample. Data are expressed as mean ± standard deviation. Pattern
I II III IV V VI VII VIII total
CN:WP IEF
Agilent
0.97 1.06 0.88 0.85 1.21 1.18 1.29 1.25 1.12
0.96 1.16 1.33 0.94 1.23 1.02 1.14 1.09 1.08
± 0.04 ± 0.10 ± ± ± ± ± ±
0.07 0.15 0.15 0.05 0.12 0.21
± 0.23 ± 0.29 ± ± ± ± ± ±
0.26 0.36 0.43 0.27 0.27 0.36
αs1-CN
β-CN
2.26 2.25 3.15 2.39 2.20 2.58 2.73 2.65 2.54
6.12 5.87 5.07 6.32 6.40 6.23 5.94 6.21 6.11
± 0.98 ± 0.90 ± 0.55 ± 1.00 ± 0.49 ± 0.17 ± 0.21 ± 0.67
κ-CN
± 0.70 ± 1.92 ± ± ± ± ± ±
0.77 0.94 0.51 0.20 0.24 1.03
4
1.33 1.45 1.49 1.00 1.16 1.07 1.06 0.93 1.18
α-LA
ß-LG
± 0.41 ± 1.34 ± ± ± ± ± ±
0.28 0.15 0.24 0.15 0.32 0.69
3.88 6.48 2.81 4.31 6.07 6.67 6.86 6.64 6.06
± 0.50 ± 1.96 ± ± ± ± ± ±
0.32 1.28 1.26 0.35 0.75 1.61
1.17 1.05 1.93 1.10 1.32 1.12 1.01 1.55 1.22
LZ
± 0.42 ± 0.33 ± ± ± ± ± ±
0.34 0.54 0.40 0.26 0.33 0.41
3.02 0.51 3.47 3.72 0.84 0.43 0.22 0.44 1.07
± 0.59 ± 0.43 ± ± ± ± ± ±
0.24 0.12 0.21 0.02 0.23 1.23
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could help in understanding whether an association really exists. These three patterns have another common feature: they have the highest LZ values and the lowest β-LG quantity. This suggests that β-LG I*A could be associated with a reduction in β-LG content which seems to be balanced by an increase in LZ. By contrast, the homozygous BB pattern VII for β-LG I and β-LG II had a higher β-LG content and less LZ. 4. Conclusions The present work demonstrates the interesting genetic variability of Amiata donkey milk proteins. The IEF analysis highlighted the genetic polymorphism of β-LG I and of β-LG II, together with the presence of putative null alleles both for β and αS1-CN. Although further molecular characterization is necessary to confirm the association between the detected patterns and the corresponding genetic variants, and to discover the mutation responsible for the lack of β and αS1-CN, the IEF technique was shown to be a cheap and fast method able to detect most of the phenotypic and the majority of the genetic variability of donkey milk proteins. We also managed to quantify the κ-CN, whose content has never been measured before in donkey milk, and we showed that the protein fraction content could be related to genetic polymorphism. The methods applied highlighted a variability that seems to influence the CN:WP ratio and the content of specific caseins, LZ and β-LG. This information could be exploited to increase the knowledge on the nutritional quality of donkey milk and could thus be used when selecting donkeys to produce milk with proven nutraceutical effects. Declaration of Competing Interest None of the authors have any conflicts of interest to declare. Acknowledgments This work was supported by PRA 2017 (AteneoResearch Project, University of Pisa). References Altendorfer, I., König, S., Braukmann, A., Saenger, T., Bleck, E., Vordenbäumen, S., Kubiak, A., Schneider, M., Jose, J., 2015. Quantification of αS1-casein in breast milk using a targeted mass spectrometry-based approach. J. Pharm. Biomed. Anal. 103, 52–58. Altomonte, I., Salari, F., Licitra, R., Martini, M., 2019. Donkey and human milk: insights into their compositional similarities. Int. Dairy J. 89, 111–118. Barni, S., Sarti, L., Mori, F., Muscas, G., Belli, F., Pucci, N., Novembre, E., 2018. Tolerability and palatability of donkey's milk in children with cow's milk allergy. Pediatr. Allergy Immunol. 29, 329–331. Bertino, E., Gastaldi, D., Monti, G., Baro, C., Fortunato, D., Perono Garoffo, L., Coscia, A., Fabris, C, Mussap, M, Conti, A, 2010. Detailed proteomic analysis on DM: insight into its hypoallergenicity. Front. Biosci. (Elite Ed.) 2, 526–536. Brumini, D., Criscione, A., Bordonaro, S., Vegarud, G.E., Marletta, D., 2016. Whey proteins and their antimicrobial properties in donkey milk: a brief review. Dairy Sci. Technol. 96, 1–14. Caroli, A.M., Jann, O., Budelli, E., Bolla, P., Jäger, S, Erhardt, G, 2001. Genetic polymorphism of goat kappa-casein (CSN3) in different breeds and characterization at DNA level. Anim. Genet. 32, 226–230. Caroli, A.M., Chessa, S., Erhardt, G.J., 2009. Invited review: milk protein polymorphisms in cattle: effect on animal breeding and human nutrition. J. Dairy Sci. 92, 5335–5352. Caroli, A.M., Bulgari, O., Gigliotti, C., Altomonte, I., Salari, F., Martini, M., 2015. Profilo lattoproteico e attività proteasica totale del latte di asina. Scienza e Tecnica LattieroCasearia 66, 11–16. Ceriotti, G., Chiatti, F., Bolla, P., Martini, M., Caroli, A., 2005. Genetic variability of the ovine αs1-casein. Ital. J. Anim. Sci. 4, 64–66. Chianese, L., Calabrese, M.G., Ferranti, P., Mauriello, R., Garro, G., De Simone, C., Ramunno, L., 2010. Proteomic characterization of donkey milk “caseome”. J. Chromatogr. A 1217, 4834–4840. Chianese, L., De Simone, C., Ferranti, P., Mauriello, R., Costanzo, A., Quarto, M., Ramunno, L., 2013. Occurrence of qualitative and quantitative polymorphism at donkey beta-Lactoglobulin II locus. Food Res. Int. 54, 1273–1279. Cosenza, G., Ciampolini, R., Iannaccone, M., Gallo, D., Auzino, B., Pauciullo, A., 2018. Sequence variation and detection of a functional promoter polymorphism in the lysozyme c-type gene from Ragusano and Grigio Siciliano donkeys. Anim. Genet. 49, 270–271. Criscione, A, Cunsolo, V, Tumino, S., Di, Francesco, A, Bordonaro, S, Muccilli, V, Saletti,
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