Purification, in vitro Digestion and Bioactivity of β...

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

AARHUS

UNIVERSITY


Casein Bioactive Peptides

IDJ, 2005

40% aged 25+ hypertensive (WHO)

10 million deaths/year (Lim et al. Lancet, 2012)

AARHUS

UNIVERSITY


ACE inhibitors

Synthetic ACE inhibitors

Benazepril

Captopril

Enalapril

Fosinopril

Lisinopril

Moexipril

Perindopril

Quinapril

Ramipril

Trandolapril

JN, 2004

AARHUS

UNIVERSITY


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.

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UNIVERSITY


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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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?


Edman sequencing of ≈ 4 kDa A1 peptide:

106H-K-E-M-P-F-P-K-

B variant: 106HKEMPFPKYPVEPFTERQSLTLTDVENLHLP……?

?

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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

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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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ACE inhibition after exposure to differentiated intestinal cells

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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

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