Advanced Bio-Conversion and

Separation Technologies in Creation

of New Value-Added Products from

Agro-Industrial Streams (Healthy Bioactive Carbohydrates)

Arland T. Hotchkiss Jr.

U.S. Department of Agriculture, Agricultural Research Service, Dairy and Functional Foods Research Unit, 600 East Mermaid Lane,

Wyndmoor, PA 19038 USA

Global Biorefinery Danish Biotechnology Society Meeting, Vejle, Denmark, 2010

• Multiple co-products from one

feedstock

• US ethanol production from sugar

and citrus crops will be limited;

Florida the most likely location

• Commercial ethanol production:

from sugar cane - Brazil

from sugar beet - Europe

• Biobased products and functional

food ingredients from citrus peel

and sugar beet pulp - US

• A global biorefinery will be

possible where various

components of sugar beet pulp,

sugar cane bagasse and citrus

peel will be utilized in the regions

of the world where markets

support their development

Healthy Carbs

Diabetes Obesity

Prebiotic Products & Foods

Citrus, Sugar Beet and Cranberry Biomass

Biomass:

• That material remaining after

fruit, vegetable, cereal, sugar,

paper, dairy and microbial

processing

• Municipal solid waste (trash,

restaurant food and trap grease)

• Wastewater treatment sludge

Biomass Projections

U.S. Billion-Ton Update: Biomass Supply for a Bioenergy and Bioproducts

Industry. (2011) U.S. Department of Energy, Oak Ridge National Laboratory.

Carpita, N.C., Gibeaut, D.M. 1993. Plant J. 3: 1-30

Type I Primary Cell Wall Carpita & Gibeaut Model

Present in dicots and non-commelinoid monocots (type II wall in grasses)

Pectins

Glucuronic acid Galactose KDO Apiose

Xylose DHA Rhamnose Acetyl- methyl-

Arabinose Fucose Galcturonic acid Aceric acid

RG I XGA HGA RG II

Bifidogenic Properties of Orange Peel Pectic Oligosaccharides

6

6.5

7

7.5

8

8.5

9

9.5

Starch FOS POS OA VO SB PSB

Carbohydrate Fraction

Lo

g 1

0 (

cell

s /

g)

0 h 5 h 10 h 24 h

* **

Manderson et al. (2005) Appl. Environ. Microbiol. 71:8383-8389.

•T test p<0.05 ↑ Bifidobacteria compared to T0

•FISH assay, mixed batch fecal cultures

•Eubacteria rectale also significantly increased

•Butyrate, Propionate, Acetate and Lactate produced

2500 790 1132 1474 1816 2158

m/z

0

20

40

60

80

100

% R

ela

tive I

nte

nsity

833

965

1097

1229

1361

1493

849

981

1113

1245

1625

1377

1757

1509

1889

1641

2021

2153

2285

2418

0

20

40

60

80

100

833

849

965

981

1097

1229

1361

1493

1625

1757

1889

2021

% R

ela

tive I

nte

nsity

1113

Orange Peel POS

Lemon Peel POS

Citrus Pectic Oligosaccharides Prebiotic Active Structure

MALDI-TOF MS

Pent-(Pent)n-Pent

Sample Glc Ara Gal Xyl Rha Fuc GalA GlcA

Orange Peel

POS 48.1 31.2 9.6 2.4 2.1 0.2 6.3 tr

Orange Peel

POS, C 13.6 27.0 4.4 2.6 1.9 0.2 49.6 0.8

Lemon Peel

POS 32.6 44.9 4.1 7.1 7.9 0.6 1.3 1.5

Sample Glycosyl-linkage

Orange Peel

POS T-Araf

T-

Galp

5-

Araf

3,5-

Araf

6-

Galp

3,6-

Galp

2,6-

Galp

4,6-

Galp

Monosaccharide and Linkage Composition

Hotchkiss et al. (2012) Methods of promoting the growth of beneficial bacteria in the gut. U.S. Patent 8,313,789.

Sugar Beet Pectic Oligosaccharides

Holck et al. (2011) J. Ag. Food Chem. 59:6511-6519.

FOS: fructo-oligosaccharides SAOS: small arabino-oligosaccharides LAOS: long arabino-oligosaccharides LFAOS: long ferulated arabino-oligosaccharides SFAOS: small ferulated arabino-oligosaccharides ARA: original mixture

6 healthy

human

volunteers

qPCR

qPCR Bacteriodetes Firmicutes

DP 4 = Degree of polymerization 4 oligogalacturonic

acid with 4,5-unsaturated non-reducing end

DP 5 = Degree of polymerization 5 oligogalacturonic

acid with 4,5-unsaturated non-reducing end

Holck et al. (2011) Process Biochem. 46:1039-1049.

Potato Galactan

a = Bifidobacterium

b = Lactobacillus

c = Bacteriodetes

d = Firmicutes

DNE - Destarched potato pulp, fiber released by addition of No Enzyme

DPP - Destarched potato pulp, fiber released by Pectin lyase and Polygalacturonase

CNE - Crude potato pulp, fiber released by addition of No Enzyme

CPP - Crude potato pulp, fiber released by Pectin lyase and Polygalacturonase

CPP10–100 - Crude potato pulp, fiber released by Pectin lyase and Polygalacturonase, fraction 10–100kDa

CPP>100 - Crude potato pulp, fiber released by Pectin lyase and Polygalacturonase, fraction >100kDa

Thomassen et al. (2011) Appl. Microbiol. Biotechnol. 90:873-884.

Prebiotic Pectins

Glucuronic acid Galactose KDO Apiose

Xylose DHA Rhamnose Acetyl- methyl-

Arabinose Fucose Galacturonic acid Aceric acid

RG I XGA HGA RG II

Onumpai et al. (2011) Appl. Environ. Microbiol. 77:5747-5754.

Preparative HPLC Pectic Oligosaccharides

qPCR Bacteriodetes Firmicutes

DP 4 = Degree of polymerization 4 oligogalacturonic

acid with 4,5-unsaturated non-reducing end

DP 5 = Degree of polymerization 5 oligogalacturonic

acid with 4,5-unsaturated non-reducing end

Holck et al. (2011) Process Biochem. 46:1039-1049.

Holck et al. (2011) J. Ag. Food Chem. 59:6511-6519.

FOS: fructo-oligosaccharides SAOS: small arabino-oligosaccharides LAOS: long arabino-oligosaccharides LFAOS: long ferulated arabino-oligosaccharides SFAOS: small ferulated arabino-oligosaccharides ARA: original mixture

6 healthy

human

volunteers

qPCR

Preparative HPLC Pectic Oligosaccharides

Preparative HPLC Oligogalacturonic Acids

Hotchkiss et al. (1991) Carbohydr. Res. 215:81-90.

Aminopropylsilica gel, anion-exchange

Preparative HPLC Malto-Oligosaccharides

Hotchkiss et al. (1993) Carbohydr. Res. 242:1-9.

Aminopropylsilica gel, normal-phase

Hotchkiss et al. (2001) Carbohydr. Res.

334:135-140.

CarboPac PA1, anion-exchange

Simulated Moving Bed Chromatography

Geisser et al (2005) J. Chromatogr. 1092: 17-23.

Synbiotics

Chaluvadi et al. (2012) Beneficial Microbes 3: 175-187.

Plate counts following refrigerated aerobic

storage

• A peptide from Lactobacillus rhamnosus GG encapsulated in pectin/zein reduced intestinal injury and inflammation in mouse colitis models

• P40 activation of EGFR lead to Akt activation and inhibition of cytokine-induced apoptosis of intestinal epithelial cells

• First report of a probiotic soluble protein solely responsible for this activity

Toxin

“Decoy”

oligosaccharides

Antiadhesive Oligosaccharides

Bacteria

*

0

10

20

30

40

50

60

70

80

90

100

*

* * * *

*

*

Adhesio

n r

ela

tive t

o c

ontr

ol (%

) Antiadhesive activity of pectic

oligosaccharides

Rhoades et al (2008) Journal of Food Protection 71: 2272-2277

0

20

40

60

80

100

120

140

No toxin 0.01 0.1 1 10 100 Toxin Only

Concentration of POS

mg ml-1

% C

ell

surv

ivabili

ty

*

* * * *

* * *

VT1

VT2

0.000001

0.0001

0.1

1.0

Adhesion Invasion

%

0

25

50

75

100

2.5 1.5 1 0.5 0.05

POS conc. (mg/ml)

IRC (%)

Undifferentiated Differentiated

Inhibition of adhesion and invasion of CACO-2 cells by Campylobacter jejuni

Ganan et al. (2010) International Journal of Food Microbiology 137: 181-185

Antiadhesive activity of pectic oligosaccharides

Oligogalacturonic Acid Anti-Adhesive

Oligosaccharides

• Guggenbichler JP, De Bettignies-Dutz A,

Meissner P, Schellmoser S, Jurenitsch J. Acidic

oligosaccharides from natural sources block

adherence of Escherichia coli on uroepithelial

cells. Pharm. Pharmacol. Lett. (1997) 7:35-38.

• Guggenbichler JP, Meissner P, Jurenitsch J, De

Bettignies-Dutz A. Blocking the attachment of

germs to human cells. US Patent (1997)

5,683,991.

• DP 2 and 3 oligogalacturonic acids highest anti-

adhesive activity

Bioactive Pectins

Glucuronic acid Galactose KDO Apiose

Xylose DHA Rhamnose Acetyl- methyl-

Arabinose Fucose Galacturonic acid Aceric acid

RG I XGA HGA RG II

Anti-Adhesion Prebiotic

Resistance to Salmonella Infection

• Fructo-oligosaccharides and inulin inhibited

Salmonella colonization in rats, but tissue

translocation increased (Bovee-Oudenhoven et al.

2003. Gut 52: 1572-1578).

• Fructo-oligosaccharides , inulin and xylo-

oligosaccharides reduced resistance to

Salmonella invasion of epithelial cells (Petersen et

al. 2010. Beneficial Microbes 1: 271-281).

Biomass Healthy Carbohydrates

• Biomass oligosaccharides have potential as prebiotics.

• Some oligosaccharide fractions with in vitro prebiotic

activity also prevent the adhesion of pathogenic bacteria

or may promote probiotic adhesion.

• Modified citrus pectin has anti-cancer, immunostimulatory

and heavy metal binding activity. It is also in a clinical trial

for congestive heart failure.

• New separatory methods to fractionate and scale-up

production of biomass oligosaccharides are needed.

Thanks Questions?