Extraction of proteins from Palmaria palmata

Rósa Jónsdóttir

Background

2

• Increasing demand for high quality protein both for food and feed

• The access to high quality protein is becoming more challenging

• Important to find alternative food sources including protein alternatives

that contain all the essential amino acids fulfilling human requirements

Utilisation of Palmaria palmata

3

• Palmaria palmata - dulse

• Good nutritional value

• Suitable for human food and animal feed application

• Utilized as food in Iceland for centuries

Background

✓High protein content (up to 35%)

– Comparable to high protein vegetables e.g. soybeans

✓Main polysaccharide is xylan (34-35%)

✓ P. palmata has a rigid cell wall consisting mainly of β-(1→4)/β-(1→3)-D-xylans along with some fractions of cellulose and β-(1→4)-xylans

✓Due to the strength of the cell wall, traditional protein extraction methods might give limited results

Objectives

✓Develop fractionation processes for preparation of protein-rich products and extracts to be used as food, nutraceutical or feed ingredients

✓ Explore the use of enzymes (xylanse and protease) as processing tool

✓ Study protein yield, quality and the bioactivity of the different fractions

• P. palmata from Norway (NIBIO)

• Preprocessing: wet-milling

• Enzyme hydrolyzation

• Filtration

• Chemical analysis (water, fat, salt, ash, protein)

• Protein

– SDS-PAGE; Nitrogen;

–Amino acids (SINTEF)

• Bioactivity (TPC, ORAC, DPPH, MC, ACE)

Material and methods

Enzyme ProducerOptimum conditions

Activity

pH Temperature

Umamizyme Amano enzymes Inc. 7.0 50Endo- and exo-peptidase

complex

ProteAX Amano enzymes Inc. 6.0 60Endo- and exo-peptidase

complex

Xylanase Matis ltd 6.5 60 Endo-1,4-β-xylanase

Xylanase is derived from a bacterium belonging to the genus Planifilum, of the familyThermoactinomycetaceae according to 16S rRNA sequence analysis and blasting to the NCBI nrdatabase (Matís, unpublished results).

EnzymesOptimum conditions and activity

Flow chart of the extraction process

Freeze drying Freeze drying

100 µm sieve

Extraction yields of dry matter of both fractions liquid extract and solid phase

0

10

20

30

40

50

60

70

Control Umamizyme Xylanase Xylanase andUmamizyme

Umamizyme

Extr

acti

on

yie

ld (

%d

w)

Yield

Liquid extract

Solid phase

Amino acid composition and protein content of P. palmata

10

Values for untreated seaweed (n=1) are presented as % of total amino acid content.Values for control, xylanase, xylanase + Umamizyme and Umamizyme (n=2) are presented as % of total amino acid content. Values for nitrogen content are presented as % dry weight ± SD (n=4).

Essential amino acids, % of total amino acid content

Amino acid composition and protein content of P. palmata

11

• All EAA were present in the samples

• EAA ratio higher in all solid phase samples compared to liquid extract

• EAA ration higher in liquid extract when treated with protease

― Protease break down proteins into peptides and amino acids passing

the 100 µm sieve after the hydrolysis

=> resulting in higher amino acid content in liquid extract.

Essential amino acids (EAA)

Protein content of P. palmata, untreated and enzymatically treated, using different N conversion factor

54.9

33.4

0

5

10

15

20

25

30

35

40

45

50

55

60

Untreated Liquidextract

Solidphase

Liquidextract

Solidphase

Liquidextract

Solidphase

Liquidextract

Solidphase

Control Xylanase Xylanase +Umamizyme

Umamizyme

Pro

tein

co

nte

nt

(% d

w)

N = 6.25

Calculated conversion factor

Values are presented as

% dry weight ± SD (n=4).

Conversion factor 4.7 3.6 4.1 2.5 3.8 2.9 3.5 3.2 3.4

Bioactivity of different fractions of P. palmata antioxidant activity

13

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

Control ProteAX ProteAX andUmamizyme

Umamizyme Xylanase Xylanase andUmamizyme

Umamizyme

DP

PH

, IC

50

valu

es

Liquid extract

Solid phase

0

10

20

30

40

50

60

70

80

90

100

Control ProteAX ProteAX +Umamizyme

75

Umamizyme75

Xylanase Xylanse +Umamizyme

200

Umamizyme200

Met

al c

hel

atin

g ab

ility

(%

)

Liquid extact

Solid phase

DPPH antioxidant activity, IC50 valuesHigh antioxidant activity in liquid extracts

Metal chelating ability (%) High antioxidant activity in some solid phase and liquid extract

ACE (angiotensin converting enzyme) inhibition

activity of different fractions of P. palmata

14

IC50 (mg/mL)

Liquid extract Solid phase

Control >1.00 >1.00

ProteAX 0.45 ± 0.11 >1.00

ProteAX + Umamizyme 75 0.38 ± 0.02 >1.00

Umamizyme 75 0.69 ± 0.05 >1.00

Xylanase >1.00 >1.00

Xylanase + Umamizyme 200 1.14 ± 0.08 >1.00

Umamizyme 200 >1.00 >1.00

Blood pressure

Conclusion

15

• P. palmata contains high protein content of good quality that can be

extracted with good results using enzymatic treatment with xylanse

• Xylanase disrupted the rigid cell wall, resulting in higher protein yield in

the solid phase

• Protein extract from P. palmata is high in essential amino acids and

would therefore be suitable as food and nutraceutical ingredient

• The results indicate that hydrolysis with protease can be a beneficial

method to extract bioactive hydrolysates from P. palmata

Publications

Acknowledgement

17

Málfríður Bjarnadóttir, MatísBjörn Viðar Aðalbjörnsson, MatísAnna Nilsson, MatísGuðmundur Óli Hreggviðsson, MatísÓlafur H. Friðjónsson, Matís

Rasa Slizyte, SINTEF OceanMichael Y. Roleda, NIBIO

rosa.jonsdottir@matis.is