purification, in vitro digestion and bioactivity of β...
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Food science,au
Purification, in vitro Digestion and
Bioactivity of β-casein Variants
Bjørn Petrat-Melin
PhD student
FOOD AU
AARHUS
UNIVERSITY
Milk Genomics Workshop Oct. 9th 2014 Bjørn Petrat-Melin
Casein Bioactive Peptides
IDJ, 2005
40% aged 25+ hypertensive (WHO)
10 million deaths/year (Lim et al. Lancet, 2012)
AARHUS
UNIVERSITY
Milk Genomics Workshop Oct. 9th 2014 Bjørn Petrat-Melin
ACE inhibitors
Synthetic ACE inhibitors
Benazepril
Captopril
Enalapril
Fosinopril
Lisinopril
Moexipril
Perindopril
Quinapril
Ramipril
Trandolapril
JN, 2004
AARHUS
UNIVERSITY
Milk Genomics Workshop Oct. 9th 2014 Bjørn Petrat-Melin
Identification of milk samples
with pure β-casein variants
Purification of β-casein
In vitro digestion
Characterization of digests Bioactivity of digests/peptides
STUDY OUTLINE
Milk Protein Variants
Milk samples from >800 cows o 4 β-casein variants
Position of amino acid substitutions within the
mature protein of variants of β-casein
Variant(1)
Position A1 A2 B I
67 His Pro Pro
93 Met Leu
122 Ser Arg
(1) A1 is the reference sequence
Caroli et al., JDS, 2009.
AARHUS
UNIVERSITY
Milk Genomics Workshop Oct. 9th 2014 Bjørn Petrat-Melin
Isolating β-casein
1+2: casein pellet
3: Serum
4: Serum with membrane
fragments
β-CN
Frozen 4 °C (48 h)
Ultracentrifugation Isoelectric
precipitation
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UNIVERSITY
Table 2. Relative content of milk proteins in the isolated β-casein variant
preparations. Values were calculated as relative peak areas within each
chromotogram by LC/ESI-MS
β-casein
variant β-casein κ-casein α-casein
Whey
proteins
A1 89.7 ± 1.4 2.3 ± 0.4 4.6 ± 1.7 3.5 ± 2.5
A2 93.2 ± 1.7 0.5 ± 0.3 1.0 ± 0.5 3.2 ± 2.6
B 89.0 ± 0.1 5.6 ± 1.4 3.4 ± 0.3 1.4 ± 1.1
I 90.3 ± 1.1 2.9 ± 0.7 4.8 ± 0.8 1.7 ± 0.2
All values are expressed as mean percentage of total peak area ± SEM (n = 3).
Petrat-Melin et al., JDS, 2014, accepted.
In Vitro digestion of β-CN variants
2. Pancreas
+ 54 mM HCO3-
+ pancreatic enzymes
1. Gastric step
pH = 2.0
T = 37ºC
t = 60 min
Pepsin:CN = 1:200
3. Duodenal step
pH = 6.5, T = 37ºC
t = 5 or 120 minutes
E:CN = 1:200
Questions
• Digestibility?
• Degree of hydrolysis?
• Fragmentation pattern?
• Generation of bioactivity?
Milk Genomics Workshop Oct. 9th 2014 Bjørn Petrat-Melin
Edman sequencing of ≈ 4 kDa A1 peptide:
106H-K-E-M-P-F-P-K-
B variant: 106HKEMPFPKYPVEPFTERQSLTLTDVENLHLP……?
?
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UNIVERSITY
In Vitro digestion of β-CN variants
Peptide profile
Table 1. Peptides from regions with amino acid substitutions identified by LC-MS/MS after in vitro
digestion of bovine β-CN variants A1, A2, B, and I
Digestion
phasea Digested
variant Positionb Sequencec
Genetic
variantsd
Gastric: B 59-80 VYPFPGPIHNSLPQNIPPLTQT A1, B
I 81-93 PVVVPPFLQPEVL I
A1 120-138 TESQSLTLTDVENLHLPLP A1, A2, I
Duodenal 1: I 81-92 PVVVPPFLQPEV A1, A2, B, I
B 108-119 EMPFPKYPVEPF A1, A2, B, I
B 108-122 EMPFPKYPVEPFTER B
B 114-122 YPVEPFTER B
A1, A2 114-125 YPVEPFTESQSL A1, A2, I
A1, A2, I 114-139 YPVEPFTESQSLTLTDVENLHLPLPL A1, A2, I
B 114-139 YPVEPFTERQSLTLTDVENLHLPLPL B
Duodenal 2: I 53-68 AQTQSLVYPFPGPIPN A2, I
I 57-68 SLVYPFPGPIPN A2, I A1, B 59-68 VYPFPGPIHN A1, B A2, I 59-68 VYPFPGPIPN A2, I
A1 59-92 VYPFPGPIHNSLPQNIPPLTQTPVVVPPFLQPEV A1, B
A1, B 67-92 HNSLPQNIPPLTQTPVVVPPFLQPEV A1, B I 69-92 SLPQNIPPLTQTPVVVPPFLQPEV A1, A2, B, I
A1, A2, B 73-92 NIPPLTQTPVVVPPFLQPEV A1, A2, B, I
I 81-92 PVVVPPFLQPEV A1, A2, B, I
B 114-119 YPVEPF A1, A2, B, I
A1 114-124 YPVEPFTESQS A1, A2, I
A1, A2 120-132 TESQSLTLTDVEN A1, A2, I
A1 120-139 TESQSLTLTDVENLHLPLPL A1, A2, I
B 123-132 QSLTLTDVEN A1, A2, B, I
(a) Gastric: 60 minutes pepsin, Duodenal 1: 60 minutes pepsin + 5 minutes pancreatic enzymes, Duodenal 2: 60 minutes
pepsin + 120 minutes pancreatic enzymes.
(b) The position of the peptide within the mature β-casein amino acid sequence.
(c) Peptide amino acid sequence. The residues in parenthesis shows the neighboring amino acids.
(d) Shows which of the four variants contain the sequence of the peptide within their native sequence.
Synthesized peptides
• VYPFPGPIHN f[59-68] Variant A1/B
• VYPFPGPIPN f[59-68] Variant A2/I
• TER f[120-122] Variant B
AARHUS
UNIVERSITY
Milk Genomics Workshop Oct. 9th 2014 Bjørn Petrat-Melin
ACE inhibitory capacity of digested β-CN variants and derived peptides
Table 2. The IC50a
of angiotensin-1 converting enyme inhibitory
peptides derived from β-casein variants
Peptide Variant Positionb
IC50
(µM)c SEM
TER B 120 – 122 090 a 8.8
VYPFPGPIHN A1, B 59 – 68 123 a 14.2
VYPFPGPIPN A2, I 59 – 68 656 b 7.6
(a) Concentration needed to reach half-maximal inhibition.
(b) The position of the peptide within the mature β-casein amino acid sequence.
(c) Different letters within column denote statistically significant difference (P < 0.001).
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UNIVERSITY
ACE inhibition after exposure to differentiated intestinal cells
AARHUS
UNIVERSITY
Milk Genomics Workshop Oct. 9th 2014 Bjørn Petrat-Melin
Caco-2 cells
Conclusions > The amino acid substitutions in β-CN variants A1, A2, B, and I result in changed
cleavage sites for gastrointestinal proteases
> This causes β-CN variants to give rise to peptides with different bioactive potentials
> His rather than Pro at position 67 conveys higher ACE inhibition > The ACE inhibitory peptides VYPFPGPIHN, VYPFPGPIPN, and TER are differently
affected by the intestinal brush-border, modeled by the Caco-2 cell line
> GENETICALLY CONTINGENT VARIATIONS IN BOVINE β-CN HAS IMPLICATIONS FOR THEIR PHYSIOLOGICAL PROCESSING AND EFFECTS
Perspectives > Genetic polymorphisms in casein should at least be considered in the
development of new processes, products, applications, etc.
Conclusions > The amino acid substitutions in β-CN variants A1, A2, B, and I result in changed
cleavage sites for gastrointestinal proteases
> This causes β-CN variants to give rise to peptides with different bioactive potentials
> His rather than Pro at position 67 conveys higher antioxidant capacity and ACE inhibition
> The ACE inhibitory peptides VYPFPGPIHN, VYPFPGPIPN, and TER are differently
affected by the intestinal brush-border, modeled by the Caco-2 cell line
> GENETICALLY CONTINGENT VARIATIONS IN BOVINE β-CN HAS IMPLICATIONS FOR THEIR PHYSIOLOGICAL PROCESSING AND EFFECTS
Perspectives > Genetic polymorphisms in casein should at least be considered in the
development of new processes, products, applications, etc.
Acknowledgements
Pernille Andersen
Thao T. Le Hanne S. Møller
Jan T. Rasmussen Anni Bojsen
Nina A. Poulsen Jette F. Young Lotte B. Larsen