Broccoli Ingredients: Innovation from Agrifood Science to the Spin-

off Experience

Diego A. Moreno-Fernández, Ph.D.

CEBAS-CSIC

1. Broccoli: A Wellness Food, Rich in Health-promoting

Bioactives

2. Broccoli Agri-Food Chain – Research

3. Ingredients from Broccoli and By-products

4. Innovation with broccoli - The spin-off experience

Broccoli Ingredients: Innovation from Agrifood Science to the Spin-off Experience

1. Broccoli: A Wellness Food, Rich in Health-promoting

Bioactives

F&V for healthy diets The consumption of plant-derived

foods (F&V) in the Western countries

is relatively low. It is always below the

recommendations of public health

agencies.

Most countries (government

agencies) and other NGOs are

involved in campaigns to advice the

general public on consuming 5 – 9

portions of F&V a day (375g/d).

Kris-Etherton et al. (2002) Am. J. Med. 113: 71S-88S

Schreiner (2005) Eur. J. Nutr. 44(2): 85-94

Gromis et al. (2007) J. Amer. Diet. Assoc. 8(S1):A25

1. Broccoli: A Wellness Food, Rich in Health-promoting

Bioactives

http://www.aicr.org/foods-that-fight-cancer/broccoli-cruciferous.html

Moreno et al. (2006) J Pharm. Biomed Anal. 41 1508-1522

Cruciferous foods (BROCCOLI):

Wealth of nutrients and bioactive compounds

Moreno et al. (2006) J. Pharm. Biomed. Anal. 41: 1508-1522

Anilakumar et al. (2006) J. Food Sci. Technol. 43: 8-17

Cruciferous foods (BROCCOLI):

Wealth of nutrients and bioactive compounds

GLUCOSINOLATES (in Brassicales): • Characteristics of Brassica spp. and of course, Broccoli -d-thiglucose group, a sulphonated oxime group and a side chain (derived from Met., Phe, Trp)

• The biologically-active compounds are their degradation products: Isothiocyanates (ITCs): Sulforaphane (SFN) and indoles (Indol-3-carbinol, I3C), BITC, PEITC

Phytochemicals for detoxification (Indirect Antioxidants):

S

C

O

N O SO3-

S C6H11O5

Glucoraphanin myrosinase

S

N

O

C S

Sulforaphane

Physiological effects of broccoli consumption

Jeffery & Araya (2009) Phytochem Rev 8: 283-298

Upregulation

(Phase II)

Eating broccoli and cancer: Is there a relationship?

Lung Colorectal Oral cavity Breast Prostate

http://www.aicr.org/foods-that-fight-cancer/broccoli-cruciferous.html

Bioavailability and Metabolism of ITCs (Glucosinolates)

Moreno et al. (2006) J. Pharm. Biomed. Anal. 41: 1508-1522

Tomás-Barberán, Gil-Izquierdo, Moreno D.A. (2009). Designing functional foods- CRC Press

Urine fraction AnalyteZ Control ½ serving 1 serving ANOVA P-value

0-12 h GR n.d. n.d. n.d. n.s.

SFN-GSH n.d. 0.12 ± 0.07a 0.08 ± 0.04a n.s.

SFN-Cys n.d. 1.37 ± 0.38b 2.48 ± 0.42a *

SFN-NAC n.d. 11.03 ± 2.31b 34.32 ±

12.00a

***

SFN n.d. 0.92 ± 0.34b 2.14 ± 0.16a **

12-24 h GR n.d. n.d. n.d. n.s.

SFN-GSH n.d. 0.24 ± 0.09a 0.07 ± 0.05a n.s.

SFN-Cys n.d. 0.09 ± 0.05b 0.25 ± 0.08a **

SFN-NAC n.d. 1.25 ± 0.51b 3.61 ± 0.47a ***

SFN n.d. 0.01 ± 0.00b 0.13 ± 0.01a ***

ZGR: Glucoraphanin, SFN-GSH: Sulforaphane-gluthation, SFN-Cys: Sulforaphane-cysteine, SFN-

NAC: Sulforaphane-N-acetylcysteine.

The amount of

metabolites excreted

(mercapturic acid

pathway) was higher

in the first 12 hours

after broccoli intake

Bioavailability and Metabolism of ITCs (Glucosinolates)

Medina-Escudero et al. (Food Chem), Under review

Lowering Cholesterol(Soluble fibers, ITCs)

Allergies - airways (Flavonoids and ITCs)

Nutrients/Antioxidants

(Vitamin C, flavonols, lutein, -carotene)

Bone health (Calcium, Vitamin K)

Cardiovascular health (ITCs)

Detoxification, Chemoprevention, Autism

(SFN, I3C, other ITCs)

Body weight (dieting)

(Fiber, protein, low caloric food)

Health benefits of Consuming Broccoli

2. Broccoli Agri-Food Chain – Research

Broccoli is important (economically) in the Murcia region (35.000 Has - PROEXPORT Associates)

Murcia is the main producer in Spain and concentrates 65-70% of the production

in the country [broccoli 70% + cauliflower 30%; > 360.000 mT/y]

(> 340.000 mT to EU-27, and ~20.000 mT non-EU).

More than 70% of the production is for fresh broccoli and ~30% is for processed

(frozen) broccoli

Major importers (volume) are the United Kingdom, Germany, Netherlands

2. Broccoli Agri-Food

>> > >

Genetic variability and Nutritive Value

Nutrients and Bioactive Compounds in Brassica oleracea (species) are different among varieties and cultivars

Singh et al. J. Food Comp. Anal. 20, 106-112 (2007)

Domínguez-Perles et al. J. Sci. Food Agric. 91 1638-1647 (2011)

Genetic variability, quality and composition

Cooked inflorescences

Control 40 mM 80 mM

glu

cosi

no

late

s (m

g 1

00

gfw

-1)

0

500

1000

1500

2000

2500

a

b

ab

Steamed florets - 40mM NaCl

Inflorescences

Control 40 mM 80 mM

gluc

osin

olat

es (m

g 10

0mg fw

-1)

0

500

1000

1500

2000

2500

a

b

b

Domestic processing affected the GLS composition of

broccoli grown under NaCl-Stress conditions

GLUCOSINOLATES

PREHARVEST CONDITIONS AFFECT THE POSTHARVEST

QUALITY/COMPOSITION

Control 40 mM

NaCl

80 mM

NaCl

Environment & Processing, quality and composition

Schreiner et al. (2007) J. Food Sci. 72. S585-S589

López-Berenguer et al. (2007) J. Agric. Food Chem. 55: 10001-10007

Processing Effects on Broccoli Phytochemicals

PHENOLIC COMPOUNDS

GLUCOSINOLATES

Cooking:

+ Steaming (blanching)

+ Stir Frying

- Boiling (Trad.)

- Frying

Processing Effects on Broccoli Phytochemicals

2. Broccoli Agri-Food Chain – Research

Food Science & Technology – CEBAS-CSIC

Fernando Vallejo Mellado – Ph.D. Thesis (2002)

Carmen López-Berenguer - Ph.D. Thesis (2006)

Luis López-Pérez – Ph.D. Thesis (2009)

Santiago Pérez-Balibrea – Ph.D. Thesis (2010)

Raúl Domínguez-Perles – Ph.D. Thesis (2011)

Beatriz Muries Bosch – Ph.D. Thesis (2011)

Cesar Mota Cadenas – Ph.D. Thesis (2011)

M. Carmen Rodríguez-Hernández (2010-2013)

Nieves Baenas Navarro – Ph.D. Fellow (2012-)

Dominguez-Perles, R., Ph.D.

Jeffery et al. (2003) J. Food Comp. Anal. 16, 323-30

Moreno et al. (2007) Food 1(2): 297-312

Verkerk et al. (2009) Mol. Nutr. Food Res. 53: S219-S265

Raúl Domínguez-Perles – Ph.D. Thesis (2011)

3. Ingredients from Broccoli and By-products

3. Ingredients from Broccoli and Byproducts

Dominguez-Perles et al. Food Eng. Ingred. 35, 22-25 (2010) Dominguez-Perles et al. J. Food Sci. 75, C383-C392 (2010)

> 350.000 Tm/y

Environmental Stress on Broccoli Phytochemicals

Growth under Adverse conditions (SE Spain) affected Broccoli’s Natural Antioxidants: Vitamin C, Phenolic Compounds & Glucosinolates – In By-products/ harvest remains

Adding value to by-products (harvest remains, side-streams, plant biomass, other co-products)

PHYTOCHEMICALS NUTRIENTS AND MORE Vitamins, Fiber Phenolic acids Flavonoids Glucosinolates Ascorbigen Folates, Tannins, Minerals

4. Innovation with broccoli ingredients

– The spin-off experience

Aquaporins & Ingredients S.L.

CSIC Spin-off

www.aqpingredients.com

4. Innovation with broccoli ingredients – AQP&Ingredients

CONVERGENCE FOOD/PHARMA:

OPPORTUNITIES WITH BRASSICA INGREDIENTS

4. Innovation with broccoli ingredients – AQP&Ingredients

R&D & Innovation: Vegetable crops for Industry – Food/Pharma Industrial Processing (Q.C., Ext. & I.C.): Foods and Ingredients (Nutritive and/or Functional) Gastronomy Developments (Culinary technology) Environment and Life Sciences Applications (Biomolecules) Natural Resources Clinical applications (Wellbeing, Health & Beauty)

Culinary Science & Techniques: From the Lab to La Cuisine • Freeze-drying • Dehydration • Non-aggressive Concentration

New ingredients for taste and textures: • Spices • Fresh products • Textures (powders, emulsions, forms, etc.)

Ingredients for Food/Hedonistic experience

Culinary (gastronomy) and Industrial applications: Phytoconcentrates - Patented procedure (100% Chemical free) - Sustainable practices (use of by-products) - Characterization (HPLC-DAD; HPLC-MS) - No-Solvents Involved in Extraction Fitoconcentrados AQP-CSIC®

• 100% natural powder of Brassica • Purity (bioactives): Glucosinolates (0.2% - 2% d.w.)

Polyphenolics (1% - 10%, d.w.) • Solubility and stability • State Agency – CSIC Spin-off Company • Food and Feed

Ingredients for Food/Industry

CONVERGENCE of FOOD/PHARMA:

OPPORTUNITIES WITH BRASSICA INGREDIENTS

Tendencies in the Development of Ingredients & Therapeutics

100

80

60

40

20

Le

ve

l o

f S

cie

nti

fic K

no

wle

dg

e

Dried Plant

Material (E. & I.C.)

QA/QC

Extracts (E. & I.C.)

QA/QC

Pure

Compounds

Herbs/Teas

Food stores

OTCs

Supplements

Food stores

OTCs

Phytomedicinal

Bot. therapeutic

Stores/OTCs

Drug

Chem/Pharma

Synthetic Drug

Type

of product Traditional use

(Information)

Standardized Extracts

Current Trends

Extraction Purification

Chem.

Synthesis

Original Trend

Ingredients for New Food Product Developments

Broccoli + Green Tea = Benefits (on Colon/Cancer cells)

Caco-2 cells (cancer)

CCD18-co (Control)

Ce

ll v

iab

ilit

y, %

Time (h)

EGCG and SFN combination treatment can inhibit growth of ovarian cancer cells EGCG (20 μmol/L) + SFN (10 μmol/L) treatment can significantly inhibit colony formation, compared with SFN treatment alone. The inhibition effect of EGCG and SFN combination treatment or SFN-alone treatment is significantly stronger in paclitaxel-resistant ovarian cancer cells (SKOV3TR-ip2) than in sensitive cells (SKOV3-ip1). *P<0.05.

S

C

O

N O SO3-

S C6H11O5

Glucoraphanin myrosinase

S

N

O

C S

Sulforaphane

Ingredients for Food/Pharma (Current and future Developments)

Biopeptides and Biomolecules (AQPs) • Cosmetics (skin care; UV protection) • Functional Cosmetics: Cosmeceuticals • Therapeutics and Clinical Applications (Filtration) • Environmental Applications (Water resources) • Green-Chemistry and Agriculture (Biocides)

Research Group on AQPs (at CEBAS-CSIC):

Water – Transport Proteins (in all living organisms)

Abundance in Higher Plants depending on:

• Genotype

• Isoforms

• Organ

• Different expression of the same isoform (upon stress and intensity)

• Different phase of stress

By-products: Source of Biopeptides

Aquaporins (AQPs) – and Stress-response in Plants

Leaves PIP2 (AQPs)

By-products: Source of Biopeptides

Plant biomass for Developing Vesicles (Plasmatic Membrane) rich in AQPs

C Elic1 Elic2

Proteoliposomes – Advantages:

• Liposomes are unstable (Short shelf life)

• Proteins confers stability (to lipid bilayer)

• Non-covalent interactions between

proteins and lipids (strength)

• Amino- and Thiol- groups in proteins bind

elements to substances (Encapsulation)

• Improving the physico-chemical properties

• Improving the permeability of liposomes

• More resistance to osmotic collapse

100% Natural Vesicles (Industrial purposes)

By-products: Source of Biopeptides

AQP&Ingredients SL & CEBAS-CSIC