Advanced Bio-Conversion and Separation Technologies in...
Transcript of Advanced Bio-Conversion and Separation Technologies in...
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
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
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6.5
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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
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% R
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tive I
nte
nsity
833
965
1097
1229
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1493
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1245
1625
1377
1757
1509
1889
1641
2021
2153
2285
2418
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1889
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% R
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tive I
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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
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
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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
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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.