Marine bio-actives and health: Functional Foods. ulster.ac.uk

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1 Marine bio-actives and health: Functional Foods ulster.ac.uk Dr. Chris Gill Promac Conference Alesund, Norway, 9 th November 2018

2 N, W

3 Overview Global Seaweed Industry Functional foods Examples of functional food validation Seaweed polysaccharides Seaweed (poly)phenols Summary

Norway lead exploitation of wild stocks of seaweeds.

4 Seaweed production Aquaculture Wet metric tonnes China & Indonesia largest seaweed producers 23+ MT (aggregate) Fisheries Chile, China & Norway lead exploitation of wild stocks of seaweeds. Norway seaweed exploitation largely Laminaria hyperborea, Ascophyllum nodosum) Buschmann et al. EJOP. 2017

5 Global seaweed industry FAO report 2014, Grandview Research Report (2016), FAO report 2012, Hotchkiss et al (2016) 28.5 million wet tonnes of seaweed harvested (cultivated & wild, 2014) In 2015, the global commercial market US$ billion US$ billion by 2024, growing at a CAGR of 8.9%. In million eaten directly as sea vegetables Edible seaweed market in Europe typically growing at 7-10% p.a. Human consumption - edible food products, cosmetics, health & wellness = 81% of market value in highest growth sector

6 Typical use in food Seaweed ingredients added for number of reasons: texture as hydrocolloids (extracts) and whole ingredients; nutrition/supplementation e.g. minerals, vitamins, antioxidants; flavour including sodium replacement; interest/novelty. Seisun et al, IMR, 2016.

7 Food hydrocolloids Primary use of seaweed derived food ingredients = hydrocolloids. In Europe = additives, E Nos not ingredients. The seaweed derived hydrocolloids carrageenan, agar and alginate represent 3/5 top food hydrocolloids market sectors in market value (excluding gelatin & starch) Estimated global production (exc. China) in 2015 (tonnes dw): carrageenan - 68,000; agar 12,500; alginate 16,000. Seisun et al. IMR, 2016, Sanchez et al 2016

Seaweed products value pyramid < 1/kg - dried & milled/flaked only Seaweed meals for feed and horticulture Food grade powders in bulk 1-5/kg - more processing involved Specialist feed & horticulture

8 Seaweed products value pyramid < 1/kg - dried & milled/flaked only Seaweed meals for feed and horticulture Food grade powders in bulk 1-5/kg - more processing involved Specialist feed & horticulture products Higher quality food grade powders /kg depending on purity & use Hydocolloids/bioactive extracts > 2000/kg High purity bioactive extracts (polyphenols/polysaccharides) for nutra/cosmaceutical/research. Desire to increase the value of seaweed & seaweed extracts- functional food?

Sales in billion UD$ We re getting older Increased

material that provides specific functional benefit (health

value Sales of functional foods worldwide from 2015-2024

9 Sales in billion UD$ We re getting older Increased healthcare costs (>8% GDP) A functional food A food material that provides specific functional benefit (health or physiological) additional to its basic nutritional value Sales of functional foods worldwide from Statista 2018 Opportunities for seaweed/seaweed extracts & functional foods?

10 Seaweed & Health

11 Seaweed based functional food studies

12 HYFFI Hydrocolloid derivatives as functional food ingredients European Commission (FP7) Grant agreement No

Seaweed as a source of fibre Increased fibre consumption associated with gut health 8g portion of seaweed ~ 11% Fibre GDA Seaweed is a sustainable source of a range of polysaccharides Alginate, Agar,

13 Seaweed as a source of fibre Increased fibre consumption associated with gut health 8g portion of seaweed ~ 11% Fibre GDA Seaweed is a sustainable source of a range of polysaccharides Alginate, Agar, Carrageenan, Fucoidan, Ulvan Too complex and relatively inaccessible for human microbiota Hydrolysis to form novel LMW derivatives to allow colonic fermentation prebiotic? Applications in food & health, and wellness products M c Artain et al. Nut Rev 2007, Brown et al Nut Rev 2014, Shang et al Carbo poly 2018

Prebiotic potential A non-digestible food ingredient beneficially affects the host, selectively stimulating

owth and/ or activity of one or a limited number of bacteria (e.g.

14 Prebiotic potential A non-digestible food ingredient beneficially affects the host, selectively stimulating the growth and/ or activity of one or a limited number of bacteria (e.g. LAB) in the colon that have the potential to improve host health Modulation of the human gut microbiota increase in Bifidobacterium spp. +/- increase in lactobacilli +/- decrease in harmful bacteria Production of SCFA s Well known food industry examples inulin, derived from chicory root. Potential prebiotics effects seaweed extracts, oligo/polysaccharides- mainly in vitro + animal studies Gibson et al, 2004, Raposo et al, Mar Drug 2016; Rowland et al EJON 2017

15 HYFFI Aim - To develop, from agar & alginate polymers, novel prebiotics. Seaweeds Tested

16 Novel Low molecular weight Polysaccharides LMWPS Overview Alginates and agar are dense hydrocolloid compounds that form gels. Depolymerisation ~ reducing its gelling properties and increasing fermentability & consumer acceptability 19 Candidate LMWPs produced for in vitro screening

17 LMWPs Prebiotic Screening 24h batch culture (37ºC),pH controlled (6.8), 10% faecal slurry, donors (n=3), 1% LMWP.

18 2 best candidates based on in vitro screening. Gelidium seaweed extract CC2253 Agar candidate Significantly increased Bifidobacteria. Extensively fermented, significant increase in Total SCFA Ascophyllum nodosum extract CC2239 Alginate candidate Prebiotic index comparable - inulin Extensively fermented, significant increase in Total SCFA Selection of prebiotics for production scale-up

19 Scale up issues Undegraded MW 130,000 Degraded MW % <50,000. Within EU spec

20 Scale up issues Undegraded MW Degraded MW % <50,000. Within EU spec

21 HYFFI Prebiotic efficacy study Treatment 28 days A Washout 28 days Treatment 28 days B Washout 28 days Treatment 28 days C Tp1 Tp2 Tp3 Tp4 Tp5 Tp6 Double blind randomised placebo controlled 3-way crossover trial 8g LMW Agar, LMW Alginate, maltodextrin (control). Water based Drink, 3.2% fibre, 6% sugar, Na benzoate, Na citrate, citric acid, flavour & colour. N= 60 (30m/30f), BMI >20 <35, years Duration 21 weeks. No antibiotic or supplements use. 6x stool samples, 6x blood samples, 6x 4 d food diary, bowel habits

22 HYFFI Prebiotic efficacy study- Outcomes Increased dietary fibre consumption, stool bulking effects. LMW alginate reduced post prandial glucose, known effect for intact alginate. Allsopp et al EJON in prep

23 Gap between laboratory and industry Industrial scale up Feasibility and practical issues What is your target medium - Product incorporation Consumer acceptability can taste and texture issues be addressed Gelidium seaweed, Ascophyllum never make it to human study?

24 PREMARA (Prebiotics from the sea) Palmaria palmata, Fucus vesiculosus, Laminaria digitate, Ulva spp. Refinement of HYFFI, PreMara in progress FIRM:Premara 13 F 511

25 PREMARA Crude and depolymerised extracts have significantly increased Bifidobacteria

26 Gap between laboratory & industry - Premara Industrial scale up Feasibility and practical issues Consumer acceptability taste and texture issues addressed Premara Human study Study recruited (n=60) Sampling concludes Dec 2018 Analysis starts Jan 2019

27 SWAFAX- Seaweed derived antiinflammatory agents and antioxidants European Commission (FP7) Grant agreement No 26259

28 Seaweed (poly)phenols Green, red algae bromophenols, phenolic acids flavonoids. Brown algae Phlorotannins 6 subclasses: eckols, fuhalols, fucophlorethols, phlorethols, fucols, ishofuhalols Gómez-Guzmán et al Marine Drugs 2018

29 SWAFAX- Seaweed derived antiinflammatory agents and antioxidants Cardiovascular disease (CVD) is the leading cause of death worldwide Evidence suggests a positive effect of (poly)phenol intake on CVD risk (Poly)phenols have antioxidant and anti-inflammatory properties Brown seaweed is a rich source of (poly)phenols No information on the bioavailability of seaweed (poly)phenols, a critical factor influencing in vivo biological activity. WHO 2014, Rangel-Huerta et al 2015, Del Rio et al 2013, Brown et al 2014, Fernando et al 2016, Gómez-Guzmán et al 2018

Seaweed extract Ascophyllum Nodosum (SPE) Preparation of food-grade seaweed polyphenol extract and capsule Solvent extracted 60:40 ethanol water A solvent

30 Seaweed extract Ascophyllum Nodosum (SPE) Preparation of food-grade seaweed polyphenol extract and capsule Solvent extracted 60:40 ethanol water A solvent seaweed ratio of 3:1 Fresh A. nodosum was supplied by The Hebridean Seaweed Company, Isle of Lewis, Scotland in March 2011 Characterised (NP)-HPLC, LC-MS analysis

de-glucosilation methylation glucuronidation SPE capsule - 400mg Capsule, 100mg

31 Gut-bacteria Fermentation Small phenolics Acute bioavailability study 24 healthy volunteers (12 M, 12F), age years. de-glucosilation methylation glucuronidation SPE capsule - 400mg Capsule, 100mg polyphenols ~ Phlorotannins - Hydroxytrifuhalol A, 7-Hydroxyeckol, C-O-C dimer phloroglucinol Cannulation, blood & urine samples collected.

32 SPE Metabolites in Urine LC/MS N - urine post seaweed consumption. Hydroxytrifuhalol A 7-Hydroxyeckol C-O-C dimer of phloroglucinol HPLC-DAD 18 conjugated (glucuronides/sulfates) un-conjugate seaweed metabolites detected Phoroglucinol standard Corona et al BJN 2016

33 Seaweed Metabolites in Plasma HPLC DAD- Seaweed metabolites found in samples collected at 0-8h after capsule ingestion, but majority of the metabolites found in samples collected at 8-24h. Conjugated (glucuronides/sulfates) un-conjugate seaweed metabolites detected. IL-8 Corona et al BJN 2016 Anti- inflammatory activity Ex-vivo LPS + Whole blood culture increase cytokine IL-8 Importance of Colonic Fermentation HMW PPs?

34 ND GID CF Colonic Fermentation HMW PPs Antioxidant activity decreases with digestion, Fermentation. Colonic fermentation increased Anti-genotoxicity (HT29 cells) CF HMW most potent

35 SWAFAX study N= 80, yrs, BMI>25 Registry no. NCT

36 Untargeted analysis of urine and plasma- UHPLC-HR-MS Am J Clin Nutr 2018;108: Biomarkers of seaweed consumption identified in urine only Total seaweed metabolites excreted mmol. 25% low excreters <0.5mmol 20% low excreters <2.0mmol

37 SPE & Risk Biomarker Peripheral Blood Mononuclear Cells (PBMCs) N=78 N=42 N=36 23% Average basal DNA damage /- 1.9 individual treatment effects (pre post supplementation), * P>0.05, (ANOVA, one-tailed test) Seaweed extract significantly reduced basal DNA damage in PBMCs, in population at highest risk (obese). Baldrick et al; AJCN 2018

38 SWAFAX study key outcomes A range of metabolites (un-conjugated & conjugated forms), especially at the later time points. (6 to 24 hours) - due to colonic metabolism of high MW phlorotannins? High & low seaweed phenolic excretors were observed. Decreased lymphocyte DNA damage in obese individuals. Consumption of seaweed phenolics for 8 weeks had no negative effects on: C-Reactive Protein, Plasma total cholesterol, HDLC, LDLC or triglycerides levels. IL-1, IL-2, IL-6, IL-10, IL-12, IFN- and TNF- expression in mononuclear cells

Double blind, randomized controlled, parallel study,

palmata bread stimulated inflammation (CRP), elevated

39 Double blind, randomized controlled, parallel study, n=40, y, 5 g dulse p.d.in 4 weeks. P. palmata extracts tested in vitro first, was anti-inflammatory. P. palmata bread stimulated inflammation (CRP), elevated serum triglycerides, altered thyroid function; changes within clinically normal range not likely to impact health.

40 Randomised Double Blind, Placebo Controlled Crossover Trial: N=38; aged y Fucus vesiculosus extract 500 mg, 2000 mg, Placebo cellulose mg plasma glucose plasma insulin response Seaweed extract no effect on plasma glucose or plasma insulin response

Randomised Double Blind, Placebo Controlled Parallel Trial: N=60; aged 18-65 yrs. 500 mg A. nodosum & F.

41 Randomised Double Blind, Placebo Controlled Parallel Trial: N=60; aged yrs. 500 mg A. nodosum & F. vesiculosus Hot water extract (Sea2 ) Computerized measures of episodic memory, attention/subjective state. Seaweed improved accuracy on digit vigilance (p = 0.035) & choice reaction time (p = 0.043)

Summary Global commercial market for seaweed US$ 10.31 bn Edible food products/cosmetics/health & wellness and pharma for human consumption = the highest market share.

42 Summary Global commercial market for seaweed US$ bn Edible food products/cosmetics/health & wellness and pharma for human consumption = the highest market share. Majority of seaweed and health studies in vitro /animal Functional food requires validation in humans Seaweed fibre (LMWP agar) effects gut function and microbiota Seaweed (poly)phenols are bioavailable & bioactive in humans Opportunities for seaweed/seaweed extracts as functional foods

43 Acknowledgments Professor Sean Strain Professor Ian Rowland Dr Emeir McSorley Dr Pamela Magee Dr Julie Wallace Dr Sonja Nitecki Dr Thomas Smyth Dr Catherine Stanton Mr Chris McLaughlin Prof Paul Ross Dr Gema Periro-Caro Dr Philip Allsopp Dr Kirsty Pourshahidi Dr Emma Brown Dr Mary Slevin Ms Vicky Hegan Mr Ken Collins Dr Anna Soler NutraMara colleagues Dr Mary Rae Dr Sarah Hotchkiss Mr Ross Campbell

44 Any questions?

Introduction to CyberColloids Seaweed Research

Introduction to CyberColloids Seaweed Research April 2016 CyberColloids Ltd, Unit 4A Site 13, Carrigaline Industrial Estate, Carrigaline, County Cork, Ireland. tel: +353-21-4375773 www.cybercolloids.net