functional, natural, sustainable industrial application of ...eprints.whiterose.ac.uk/115423/1/8th...

Industrial application of

anthocyanins extracted

from food waste

@keracol

Richard Blackburna, Meryem Benohoudb, Chris Raynerc aSustainable Materials Research Group, University of Leeds, UK bKeracol Limited, University of Leeds, UK cSchool of Chemistry, University of Leeds, UK

Keracol Functional, natural, sustainable

Spin-out company from The University of Leeds, UK

We develop cosmetics and personal care products that

- achieve high performance

- are safe

- are sustainable

Keracol Limited

Exploit the fantastic array of chemistry that nature provides

Develop a new generation of products through our understanding of

- the fundamental chemistry of natural products

- methods to extract and purify them in the cleanest way

- utilising their functionality to achieve performance

Keracol Functional, natural, sustainable cosmetics

Extraction of anthocyanins

Idealised extraction from plant material


LIQUID

SOLID

SOLVENT • Target compound (active) exhaustively removed from source

• Active is as free as possible from interfering or undesirable

compounds extracted from the same source

1) Mass transfer process: solvent is transferred into the solid phase

2) Molecular diffusion: solvent penetrates the solid matrix

3) Solvation of soluble material and return to the surface of the solid

4) Transfer of solvated active to bulk solution via natural/coerced convection

Complications

• interactions of target active with other compounds within

the chemical matrix

• enzymatic processes that may degrade target active

before it is able to be extracted

Extraction & Purification

Clean extraction

• Polar metabolites such as anthocyanins can be extracted using

water, ethanol or blends of the two solvents


Non-toxic solvents that allow efficient extractions in optimised conditions

Acceptable solvents for food or personal care and cosmetic applications

No-regulatory limitations

Non-selective solvents

Free sugars, proteins and low-polarity metabolites are extracted too

Solid-Phase Extraction (SPE): strategy for extract purification

• Anthocyanins interact with solid phase via H-bonding and hydrophobic interactions

• Resin allows for removal of interferents via preferential sorption of active Free sugars removed with acidified water Anthocyanins subsequently eluted with acidified ethanol

Simple, safe and low cost

Allows high recovery of active

Reduces consumption of solvents

Source needs to be loaded in water

Scale-up limitation?

Extraction-Purification

Industrial-scale process


Co

lum

nC

om

pr. a

ir

Eth

ano

l

(pum

pe

d)

Waste

3-way3-way

Skins

Ho

t w

ate

r

Eva

po

rato

r

Spray

dryer

Pro

du

ct

Integrated Extraction-Purification

Lab-scale process


Berry skins +

Acidified water

Pump

Heater stirrer

Adsorption column

WHICH RESIN?

TEMPERATURE?

TEMPERATURE?

FLOW?

SOLID:SOLVENT RATIO?

OPTIMAL pH?

PARTICLE SIZE?

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0

-100

0

100

200

300

400 #39 [modified by chmhplc] PMR 083A Hyper 2 UV_VIS_5mAU

min

1 - Peak 2 - 10.175

WVL:520 nm

Flow: 1.000 ml/min

%B: 0.0 %

5.0

100.0

BLACKBERRY (Rubus fruticosus)

1. cyanidin-3-O-glucoside (>95%)

STRAWBERRY (Fragaria × ananassa)

1. Pelargonidin 3-O-glucoside (86.35%) 2. Pelargonidin 3-O-rutinoside (13.65%)

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0

-40

-20

0

20

40

60

80

100 #58 [modified by chmhplc]PMR 086 4000mg Hyper 2 UV_VIS_5mAU

min

1 - Peak 2 - 10.217

2 - 12.717

WVL:520 nm

Flow: 1.000 ml/min

%B: 0.0 %

5.0

100.0

1. cyanidin-3-O-glucoside (73.83%) 2. cyanidin-3-O-rutinoside (26.17%)

BLACK MULBERRY (Morus nigra)

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0

-30.0

-12.5

0.0

12.5

25.0

37.5

60.0 #26 [modified by chmhplc] PMR 047/1 Hyper 2 UV_VIS_5mAU

min

1 - 8.334

2 - 12.267

3 - 15.134

4 - 17.9425 - 21.942

6 - 25.042

WVL:520 nm

Flow: 1.000 ml/min

%B: 0.0 %

5.0

100.0 100.0

BLUEBERRY (Vaccinium corymbosum)

1. malvidin-3-O-galactoside (18.1%) 2. delphinidin-3-O-galactoside (13.86%) 3. delphinidin-3-O-arabinoside (12.38%) 4. petunidin-3-O-galactoside (1.74%) 5. petunidin-3-O-arabinoside (5.52%) 6. malvidin-3-O-arabinoside (48.38%)

Black mulberry (Morus nigra)

King James I mulberry tree


White Mulberry

(Morus alba)

‘Caught red

handed’

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0

-40

-20

0

20

40

70 #26 [modified by chmhplc] PMR 112A/0 Hyper 2 UV_VIS_5mAU

min

1 - 10.025

2 - 14.675

WVL:520 nm

Flow: 1.000 ml/min

%B: 0.0 %

5.0

100.0

1. cyanidin-3-O-galactoside (68%) 2. cyanidin-3-O-arabinoside (30%)

ARONIA (Aronia melanocarpa)

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0

-40

0

25

50

75

100

140 #21 [modified by chmhplc] PMR 046/1 Hyper 2 UV_VIS_5mAU

min

1 - 9.642

2 - 11.442

3 - 14.358

4 - 17.300

WVL:520 nm

Flow: 1.000 ml/min

%B: 0.0 %

5.0

100.0

BLACKCURRANT (Ribes nigrum)

1. delphinidin-3-O-glucoside (15.71%) 2. delphinidin-3-O-rutinoside (43.25%) 3. cyanidin-3-O-glucoside (7.03%) 4. cyanidin-3-O-rutinoside (34.00%)

1. Petunidin-3-coumaroylrutinoside-5-glucoside

PURPLE POTATO (Solanum tuberosum)

GRAPE (Vitis vinifera)

1. Cyanidin-3-O-glucoside (5.84%) 2. Delphinidin-3-O-glucoside (7.27%) 3. Malvidin-3-O-glucoside (65.30%) 4. Peonidin-3-O-glucoside (5.95%) 5. Petunidin-3-O-glucoside (15.64%)

Case Study 1: Natural hair dyes

Natural dyes

• Extract from blackcurrants (Ribes nigrum) grown in UK

– sustainably sourced

– waste from blackcurrant juice process (Ribena)

• Extracted and purified using green technology

– Aqueous process, clean, energy efficient

• High levels of both anthocyanins and flavonoids

• Patented (WO2010131049) semi-permanent hair

colorants and coloration process

– Range of shades, fast to 12+ washes



Dyeing from acidic medium (pH 3-4)

• lmax in aqueous solution at pH 3.0: cyanidin 517 nm; delphinidin 526 nm

– purple/violet colour consistent with flavylium cation


• lmax when adsorbed onto hair from aqueous medium: 570-580 nm

– Blue colour consistent with quinonoidal base

– in situ neutralisation by basic sites on hair surface leading to formation of anhydrobase

– Stable over 12+ washes, minimal colour loss, no colour change


Sorption studies

• sorption significantly in excess of theoretical monolayer capacity

• Formation of hemimicellar (side-by-side) and admicellar (stacking)

aggregates


0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

0 1 2 3 4 5 6 7 8

lnCe

qe u

mol g

-1

Monolayer

Hemimicellar

Admicellar

Blackcurrant anthocyanins Keracol Functional, natural, sustainable cosmetics

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0

-40

0

25

50

75

100

140 #21 [modified by chmhplc] PMR 046/1 Hyper 2 UV_VIS_5mAU

min

1 - 9.642

2 - 11.442

3 - 14.358

4 - 17.300

WVL:520 nm

Flow: 1.000 ml/min

%B: 0.0 %

5.0

100.0

O

O-Glu

OH

HO

OH

OH

OHO

O-Rut

OH

HO

OH

OH

OH

Delphinidin-3-Rutinoside Delphinidin-3-Glucoside

O

O-Glu

OH

HO

OH

OH

O

O-Rut

OH

HO

OH

OH

Cyanidin-3-Glucoside Cyanidin-3-Rutinoside

O OHOH

OH

HO

Glucoside Unit

O OHOH

O

HO

O OHOH

CH3

HO

Rutinoside Unit


Successive dyeings from solution (amount remaining)

• All anthocyanins adsorb onto bleached hair


0

20

40

60

80

100

120

140

160

180

Ab

so

lute

am

ou

nt

um

ol

dm

-3

Del-3-Glu Del-3-Rut Cy-3-Glu Cy-3-Rut

PMR 046 Successive dyeing in same dye bath (BC Anth)

Pre Dye

Post 1st Dye

Post 2nd Dye

Post 3rd Dye

Post 4th Dye


Successive dyeings from solution (amount adsorbed)

• Apparent preferential adsorption in favour of monosaccharides

(glucosides) – ca. 2-fold over disaccharides (rutinosides)


0.00

0.02

0.04

0.06

0.08

0.10

0.12

Ab

so

lute

am

ou

nt

rem

ov

ed

fro

m d

ye

bath

(u

mo

l)

Del-3-Glu Del-3-Rut Cy-3-Glu Cy-3-Rut

PMR 046 Successive dyeing in same dye bath (BC Anth)

Post 1st Dye

Post 2nd Dye

Post 3rd Dye

Post 4th Dye


Blackcurrant glycoside sorption

• Isotherm study: cyanidin-3-O-glucoside higher adsorption energy in

comparison with cyanidin-3-O-rutinoside

• Superior H-bonding through primary hydroxyl? Steric effects?


-5

-4

-3

-2

-1

0

1

0 1 2 3 4 5 6

lnq

e

lnCe

Cy-3-O-glc

Cy-3-O-rut

Δμ0 = -5.1 kJ mol-1

Δμ0 = -3.5 kJ mol-1


Grape glucoside sorption

• Isotherm study: Most glucosides show consistent sorption properties

• Unexplained sorption differences for petunidin-3-O-glucoside

• However, generally anthocyanin parent structure does not have significant

effect on sorption – glycosylation more important


0

5

10

15

20

25

30

-2 0 2 4 6 8 10

qe

lnCe

Cy-3-O-glc

Del-3-O-glc

Mal-3-O-glc

Peo-3-O-glc

Pet-3-O-glc

-5

-4

-3

-2

-1

0

1

2

3

4

5

-2 0 2 4 6 8 10lnq

e

lnCe

Cy-3-O-glc

Del-3-O-glc

Mal-3-O-glc

Peo-3-O-glc

Pet-3-O-glc

Δμ0 = -7.8 kJ mol-1

Δμ0 = -7.4 kJ mol-1

Δμ0 = -7.2 kJ mol-1

Δμ0 = -7.5 kJ mol-1

Δμ0 = -9.8 kJ mol-1

Case Study 2: Food colorants Keracol Functional, natural, sustainable cosmetics

The Challenge • Blue food colorants

dominated by

Brilliant Blue FCF (E133)

• Can induce allergic reactions

• Regulation a big issue

• Blue from nature most difficult to

achieve

The Market Opportunity • B. Blue FCF ca. 1,300 tpa

• Market value >$260m

• Industry → natural colorants

• Spirulina only current natural blue,

but has application and stability

problems

• Stable, natural blue highly

desirable

The technology • Anthocyanins extracted from sustainable source plant materials

• Lake pigment formed using novel “biomimicry” process

• inspired by plant pigment formation in flowers

• Pigments in a range of colours suitable food application

• Both water soluble and water insoluble pigments are possible

Plant sources identified

Pigments formed (water-soluble and

water-insoluble)

Fe3+, Al3+

Mg2+, Ca2+


Formation of metal complexes with blackcurrant

anthocyanins

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

400 450 500 550 600 650 700 750 800

A

l (nm)

[Ca]

[Ti]

[Mo]

[Mn]

[Co]

[Cu]

[Ag]

[Al]

[Fe]

[Mg]

Blackcurrant

[Fe]

[Al]

[Ag]

[Mg] [Mo]


Stability of complex to pH changes

• Stability of Al-complex in aqueous solution with changing pH

• Blue colour is stable down to pH 2.8; only below pH 2.8 does

complex break down and revert to the red form (non-complexed)

<--1 M HCl Original 1M NaOH-->

2.0 2.1 2.3 2.5 2.8 3.8 4.1 5.7 7.1 8.4 9.5 10.5 12.2

Red Red Red/

Purple

Blue/

Purple Blue Blue Blue Blue Blue Blue Blue Blue Blue


Stability of complex to citric acid • Very low concentrations of citric acid cause a colour change to red,

significantly higher than pH 2.8

• Effect with malic and lactic acid was not as extreme, but still a colour change was observed at relatively low concentrations

• Acetic acid could be used in much higher concentrations before any colour change was observed

• Effect not directly related to pH - citric acid has a propensity to complex Al3+, abstract the metal from the colorant complex, and cause the complex to break down

Acid Ratio (mmol acid per g colorant)

Citric 0.00 0.11 0.53 1.60 3.71

Blue Blue/ Purple Red/ Purple Red Red

Malic 0.00 0.27 0.78 1.34 7.39

Blue Blue/ Purple Red/ Purple Red Red

Lactic 0.00 0.70 1.67 2.78 10.89

Blue Blue/ Purple Red/ Purple Red/ Purple Red

Acetic 0.00 3.67 8.50 11.17 13.33

Blue Blue Blue/ Purple Blue/ Purple Blue/ Purple


Example formulations in confectionary products

No acid

Citric acid

Acetic acid

Formulation of inks for egg coding applications

using dyes extracted from waste food products

• Inks developed using colorants extracted from natural waste

materials (WO2015128646)

• Increase in the information placed on an egg

• Reduced environmental and toxicological impact

– Some current concerns over erythrosine

• Enhance security, safety, and traceability

Case Study 3: Marking eggshell Keracol Functional, natural, sustainable cosmetics

red

blue


Cy-3-O-glc

Cy-3-O-rut

Del-3-O-glc

Del-3-O-rut

black

Fe3+

Cy-3-O-gal

Cy-3-O-arab

Al3+

New inks technically superior to erythrosine

• Good adhesion, excellent water fastness, no penetration of

colorant into egg interior

– Binding with Ca2+ in CaCO3 eggshell forms stable complex

(known that Ca2+ involved in anthocyanin complex formation in blue

flower petals; Shiono et al., Nature. 2005, 436, 791)

– Also protein in eggshell matrix may contribute to interactions with

anthocyanins

• Provide high print definition to achieve text and barcoding

• Increase in the information placed on an egg

• Technology being trialled by egg producer in UK for rollout in

2016


The Team

Dr Meryem Benohoud

Lead Development Scientist

Dr Alenka Tidder

Coloration Specialist

Dr Robert Hefford

Cosmetics Consultant

Keracol University of Leeds

Dr Richard Blackburn

UoL & Co-Founder Director

Prof Christopher Rayner

UoL & Co-Founder Director

Lauren Ford

Natural colorants research

Lissie Dufton

Skincare research

Eefke van Eden

Haircare research


www.keracol.co.uk

Keracol Functional, natural, sustainable

@keracol

[email protected]

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