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
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.
Type I Primary Cell Wall
Carpita & Gibeaut Model
Present in dicots and non-commelinoid monocots
(type II wall in grasses)
Carpita, N.C., Gibeaut, D.M. 1993. Plant J. 3: 1-30
Pectins
RG I
XGA
Galactose
Glucuronic acid
Arabinose
Galcturonic acid
Rhamnose
Acetyl- methyl-
HGA
Apiose
Fucose
Xylose
RG II
KDO
Aceric acid
DHA
Bifidogenic Properties of Orange Peel
Pectic Oligosaccharides
9.5
* *
*
0h
5h
10 h
24 h
9
Log 10 (cells /g)
8.5
8
7.5
7
6.5
6
Starch
FOS
POS
OA
VO
SB
PSB
Carbohydrate Fraction
•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
Manderson et al. (2005) Appl. Environ. Microbiol. 71:8383-8389.
Citrus Pectic Oligosaccharides
Prebiotic Active Structure
833
MALDI-TOF MS
Monosaccharide and
Linkage Composition
2021
1889
1757
20
1625
1493
1361
40
Orange Peel POS
1229
60
1113 1097
80
981 965
849
% Relative Intensity
100
Sample
Orange Peel
POS
Orange Peel
POS, C
Lemon Peel
POS
0
Ara
Gal
Xyl
Rha Fuc GalA GlcA
48.1
31.2
9.6
2.4
2.1
0.2
6.3
tr
13.6
27.0
4.4
2.6
1.9
0.2
49.6
0.8
32.6
44.9
4.1
7.1
7.9
0.6
1.3
1.5
Glycosyl-linkage
T5- 3,5- 6- 3,6- 2,6- 4,6T-Araf Galp Araf Araf Galp Galp Galp Galp
2418
2285
2153
2021
1889
1229
Sample
Orange Peel
POS
1757
1641 1625
1509 1493
20
1377 1361
1113
40
981
60
Lemon Peel POS
1245
1097
965
833
80
849
% Relative Intensity
100
Glc
0
790
1132
1474
1816
2158
2500
m/z
Pent-(Pent)n-Pent
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
qPCR
6 healthy
human
volunteers
qPCR
Bacteriodetes
Firmicutes
FOS: fructooligosaccharides
SAOS: small arabinooligosaccharides
LAOS: long arabinooligosaccharides
LFAOS: long ferulated
arabinooligosaccharides
SFAOS: small
ferulated arabinooligosaccharides
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
ARA: original mixture
Holck et al. (2011) J. Ag. Food Chem. 59:6511-6519.
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
RG I
XGA
Galactose
Glucuronic acid
Arabinose
Galacturonic acid
Rhamnose
Acetyl- methyl-
HGA
Apiose
Fucose
Xylose
RG II
KDO
Aceric acid
DHA
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.
Preparative HPLC
Pectic Oligosaccharides
qPCR
6 healthy
human
volunteers
FOS: fructooligosaccharides
SAOS: small arabinooligosaccharides
LAOS: long arabinooligosaccharides
LFAOS: long ferulated
arabinooligosaccharides
SFAOS: small
ferulated arabinooligosaccharides
ARA: original mixture
Holck et al. (2011) J. Ag. Food Chem. 59:6511-6519.
Preparative HPLC
Oligogalacturonic Acids
Aminopropylsilica gel, anion-exchange
Hotchkiss et al. (1991) Carbohydr. Res. 215:81-90.
Preparative HPLC
Malto-Oligosaccharides
Aminopropylsilica gel, normal-phase
Hotchkiss et al. (1993) Carbohydr. Res. 242:1-9.
CarboPac PA1, anion-exchange
Hotchkiss et al. (2001) Carbohydr. Res.
334:135-140.
Simulated Moving Bed
Chromatography
Geisser et al (2005) J. Chromatogr. 1092: 17-23.
Synbiotics
Plate counts following refrigerated aerobic
storage
Chaluvadi et al. (2012) Beneficial Microbes 3: 175-187.
•
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
Antiadhesive Oligosaccharides
“Decoy”
oligosaccharides
Bacteria
Toxin
Antiadhesive activity of pectic
oligosaccharides
140
VT1
100
80
70
60
50
*
*
*
30
20
10
0
*
100
*
*
% Cell survivability
Adhesion relative to control (%)
90
40
VT2
120
*
*
*
*
*
*
10
100
*
80
60
40
*
*
20
*
0
No toxin
0.01
0.1
1
Toxin Only
Concentration of POS
mg ml-1
Rhoades et al (2008) Journal of
Food Protection 71: 2272-2277
Antiadhesive activity of pectic
oligosaccharides
Inhibition of adhesion and invasion of CACO-2 cells by
Campylobacter jejuni
1.0
100
%
Undifferentiated
Differentiated
0.1
75
IRC
(%)
50
0.0001
25
0.000001
Adhesion
Invasion
0
0.05
Ganan et al. (2010)
International Journal of Food
Microbiology 137: 181-185
0.5
1
1.5
POS conc. (mg/ml)
2.5
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 antiadhesive activity
Bioactive Pectins
RG I
XGA
Galactose
Glucuronic acid
Arabinose
Galacturonic acid
Rhamnose
Acetyl- methylPrebiotic
HGA
Apiose
Fucose
Xylose
Anti-Adhesion
RG II
KDO
Aceric acid
DHA
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 xylooligosaccharides 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?