Digestibility of Selected Resistant Starches in Humans · 2.1.1: Coining and Defining of the Term ‘Resistant Starch’ 4 2.1.2: Formation of Resistant Starch in High-Amylose Maize
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Digestibility of Selected Resistant Starches in Humans
by
Asmaa Alraefaei
A thesis submitted in conformity with the requirements for the degree of Master of Science
Digestibility of Selected Resistant Starches in Humans
Asmaa Alraefaei
Master of Science
Department of Nutritional Sciences University of Toronto
2015
Abstract
Resistant starches (RSs) are food ingredient sources of fiber. Their fiber content is measured in
vitro, which may not accurately reflect in vivo digestibility. Carbohydrate content of commercial
starches (Hi-Maize® 260 [HM], Hylon® VII [HY] and Amioca [AM]) was determined in vivo.
Fiber values for HM, HY and AM were: 32.8 g, 11.5 g and 0 g in vitro, 12.5 g, 40.8 g and 6.2 g
from blood glucose study (n=10) and 18.7 g, 21.6 g and 1.7 g from ileostomy study (n=3).
13.4% and 43.5% of RS in HM and HY was recovered in effluent. Total bacterial count (TBC)
was 109 CFU/ g of effluent and positive correlation (r=0.4955) existed between TBC and
carbohydrates by difference for HM and HY. The in vitro AOAC 991.43 method underestimates
fiber content in HY and AM and overestimates fiber content in HM and does not accurately
reflect fiber content of RS products.
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Acknowledgements
It feels like yesterday when I received an e-mail from the Department of Nutritional
Sciences at the University of Toronto to inform me that I was admitted to the M.Sc. program.
It was one of the most powerful moments in my life and ultimately, one of the saddest.
If I was going to accept the offer of admission, I knew I had to leave my 2 year old son
and 8 month daughter for a while in Kuwait until things have resolved.
I still remember the night I left vividly, I picked up my daughter from her crib, took her
upstairs to my mother’s room, kissed her and lay her down to sleep next to my mother.
With a shattered heart, I walked away to pursue an education.
To my brilliant supervisor, Dr. Thomas Wolever, I want to thank you sincerely for
offering me a position in your lab and I hope I have lived up to your expectations.
To my advisory committee, Dr. Young-In Kim, Dr. Deborah O’Connor and Dr. Elena
Comelli, thank you for your patience and invaluable guidance.
To Dr. David Jenkins, it is a profound honor to have you as my external examiner.
To the sponsor of my project, Ingredion, thank you for allowing this project to take place.
To Glycemic Index Laboratories, thank you for being wonderful people to work with.
To Dr. Wolever’s lab members, Ahmad, Judlyn, Shannan, Sari, Tracy, Evan, Halah and
Kervan, thank you all for your wonderful feedback and training.
A very special thank you to Amel Taibi for training me religiously during my time in
Dr. Comelli’s lab.
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To my husband, Ammar, for your endless support and children, Abdulmalek and Dalya.
I love you all more and more each day and I hope my actions have spoken volumes.
To my sisters, my best friends, Sara, Noha, Buthayna and Hadeel.
To Buthayna especially, for always knowing how to put me back together
when I was a million little pieces.
To my mother, Mona, there is nothing I can every say and nothing I can ever do to express my
utmost love and respect for you, you are simply my universe.
To my father, Majid, for teaching me persistence and determination.
To my grandmothers, Moyassar and Sara Margie, may you rest in eternal peace.
To R.E., you will forever hold a very special place in my heart, M.N.M.K.
I dedicate this degree to every Muslim female seeking the right to an education.
This degree is whole-heartedly for you.
Above all, I thank God for all the blessings in my life and pray that may He continue blessing me
with health, wealth, knowledge, peace and happiness in my life.
May this degree be the start to boundless devotion and contribution to academia.
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Table of Contents
Contents Page No.
Abstract ii
Acknowledgements iii
List of Tables ix
List of Figures x
List of Abbreviations xi
List of Appendices xii
Chapter 1: Introduction 1
Chapter 2: Literature Review 4
2.1: Resistant Starch 4
2.1.1: Coining and Defining of the Term ‘Resistant Starch’ 4
2.1.2: Formation of Resistant Starch in High-Amylose Maize Starch 5
2.1.3: Classification of Resistant Starch 6
2.1.4: Health Benefits of Resistant Starch 7
2.2: Measurement of Resistant Starch 10
2.2.1: In Vitro Methods 10
2.2.1.1: Background 10
2.2.1.2: Initial Methods Developed for the Determination of Resistant Starch 11
2.2.1.3: Development of AOAC Official Method 2002.02 15
2.2.1.4: AOAC 991.43 Method vs. AOAC 2002.02 Method 17
2.2.1.5: Incorporation of Resistant Starch into Dietary Fiber Measurements and the Development of AOAC 2009.01 18
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2.2.2: In Vivo Methods 21
2.2.2.1: Breath Hydrogen Test 22
2.2.2.2: Ileostomy Model 23
2.2.2.2.1: Potential Problems with the Ileostomy Model 26
2.3: Resistant Starch as a Food Ingredient 26
2.4: Resistant Starch and Dietary Fiber Labeling 28
Chapter 3: Rationale, Objectives and Hypotheses 31
3.1: Rationale 31
3.2: Objectives 34
3.3: Hypotheses 34
Chapter 4: Study 1 – Digestibility of Selected Resistant Starches Estimated from the Glycemic Responses they Elicit 35
4.1: Background 35
4.2: Methods 36
4.2.1: Subjects 36
4.2.2: Protocol 37
4.2.3: Test Meals 38
4.2.4: Palatability 40
4.2.5: Blood Samples 41
4.2.6: Available Carbohydrates Calculations 41
4.2.7: Statistical Analysis 43
4.3: Results 44
4.3.1: Baseline Characteristics 44
4.3.2: Blood Glucose Analysis 44
4.3.3: Glycemic Response Elicited by Reference Food 45
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4.3.4: Palatability 45
4.3.5: Blood Glucose Responses 47
4.3.6: Incremental Areas Under the Blood Glucose Response Curves 48
4.3.7: Relative Glycemic Responses 49
4.3.8: Available Carbohydrates Calculated from Blood Glucose Responses 50
4.3.9: Estimates of Available Carbohydrates in the Test Starches 51
4.4: Discussion 52
Chapter 5: Study 2 – Digestibility of Selected Resistant Starches in Ileostomates 57
5.1: Background 57
5.2: Methods 57
5.2.1: Subjects 57
5.2.2: Protocol 58
5.2.3: Test Meals 60
5.2.4: Freeze-Drying Ileal Effluent 60
5.2.5: Ileal Effluent Analysis 61
5.2.5.1: Proximate Analysis 61
5.2.5.2: AOAC Official Method 2002.02 62
5.2.5.3: Total Bacterial Count Determined by Real-Time Quantitative Polymerase Chain Reaction 62
5.2.6: Statistical Analysis 63
5.3: Results 63
5.3.1: Baseline Characteristics 64
5.3.2: Control Diets 65
5.3.2.1: Summary of the Control Diets 65
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5.3.3: Amount of Ileal Effluent Collected 65
5.3.3.1: Amount of Dry Matter Produced 66
5.3.3.2: Mean Percent Dry Matter 67
5.3.4: Proximate Analysis of Ileal Effluent 67
5.3.5: Comparison of Resistant Starch Fed vs. Recovered 69
5.3.6: Comparison of Carbohydrates by Difference Fed and Recovered 70 5.3.7: Amount of Fiber and Available Carbohydrates by Different Methods 71
5.3.8: Total Bacterial Count Determined by Real-Time Quantitative Polymerase Chain Reaction 72
5.3.8.1: Comparison of Log Transformed Values of Total Bacterial Count 73
5.3.8.2: Correlations Between Total Bacterial Count and Carbohydrate Measure 74
5.3.8.2.1: Correlation Between Total Bacterial Count and Resistant Starch 74
5.3.8.2.2: Correlation Between Total Bacterial Count and Carbohydrates by Difference 75
5.4: Discussion 77
Chapter 6: Conclusion and Implications 82
Chapter 7: Limitations and Future Directions 85
References 87
Appendices 101
ix
List of Tables
Tables Page No.
Table 1 – Comparison of Main Initial In Vitro Methods Used to Determine Resistant Starch Content 14
Table 2 – Comparison of Resistant Starch Values Obtained Using Several In Vitro
Methods and In Vivo Results 16 Table 3 – Total Dietary Fiber and Resistant Starch Content of a Range of Samples 18 Table 4 – Comparison of Resistant Starch Values Obtained Using Several In Vitro
Methods (Including AOAC 2009.02 Method) and In Vivo Results 20 Table 5 – Composition of the Test Meals 40 Table 6 – Baseline Characteristics of Study 1 44 Table 7 – Palatability, Incremental Areas Under the Blood Glucose Response Curves
and Relative Glycemic Responses of the Test Meals 46
Table 8 – Available Carbohydrate Calculated from Blood Glucose Responses 50 Table 9 – Estimates of Available Carbohydrate in Test Starches 52 Table 10 – Baseline Characteristics of Study 2 64 Table 11 – Summary of the Control Diets 65 Table 12 – Amount of Ileal Effluent Collected in 10 Hours 66 Table 13 – Amount of Dry Matter Produced in 10 Hours 66 Table 14 – Mean Percent Dry Matter Produced in 10 Hours 67 Table 15 – Proximate Analysis of Ileal Effluent 68 Table 16 – Comparison of Resistant Starch Fed vs. Recovered 69 Table 17 – Comparison of Carbohydrates by Difference Fed and Recovered 70 Table 18 – Amount of Fiber and Available Carbohydrates by Different Methods 71 Table 19 – Total Bacterial Count of Ileal Effluent 73 Table 20 – Comparison of Log Transformed Values of Total Bacterial Count 74
x
List of Figures
Figures Page No.
Figure 1 – Dietary fiber components measured by AOAC Method 991.43 21 Figure 2 – Dietary fiber components measured by AOAC Method 2009.01 21 Figure 3 – Illustration of the Protocol for the Blood Glucose Response Study 38 Figure 4 – Palatability of the Test Meals 46 Figure 5 – Blood Glucose Responses Elicited by the Test Meals 47 Figure 6 – Incremental Areas Under the Blood Glucose Response Curves 48 Figure 7 – Relative Glycemic Responses 49 Figure 8 – Available Carbohydrate Calculated from Blood Glucose Responses 51 Figure 9 – Illustration of the Protocol for the Ileostomy Study 60 Figure 10 – Amount of Fiber and Available Carbohydrates by Different Methods 72 Figure 11 – Correlation Between Total Bacterial Count and Resistant Starch 75 Figure 12 – Correlation Between Total Bacterial Count and Carbohydrates by 76
Difference
xi
List of Abbreviations
Abbreviation Full Term
AM Amioca
ANOVA Analysis of Variance
AOAC Association of Official Analytical Chemists
avCHO Available Carbohydrates
BGR Blood Glucose Response
BMI Body Mass Index
CFU Colony Forming Units
CHOD Carbohydrates by Difference
CV Coefficient of Variation
GI Glycemic Index
HM Hi-maize® 260
HY Hylon® VII
iAUC Incremental Area Under the Blood Glucose Response Curve
No Amyloglucosidase Amyloglucosidase (pH 4.75 at 60°C for 30 minutes)
Termamyl + Amyloglucosidase
Amyloglucosidase (14 units/ml at
70°C for 30 minutes + 100°C for 10 minutes)
Glucose Determination
Enzymatic, GOD-PODd
Enzymatic, GOD-PAPe
Enzymatic, GOD-PAPe
Enzymatic, GOD-PAPe
Enzymatic, GOD-PODd
Enzymatic, GOD-PAPe
Validation In vivo data obtained with
antibiotic-treated rats
In vivo ileostomy data
In vivo ileostomy data
In vivo ileostomy data
(from literature)
In vivo ileostomy data
(from literature)
In vivo ileostomy and intubation data
Table adapted from Champ et al. (2003) c RT – Room Temperature a Ø – Diameter d GOD-POD – glucose oxidase peroxidase b 2 M KOH – 2 Molar potassium hydroxide e GOD-PAP – glucose oxidase-peroxidase-4-aminoantipyrine
15
2.2.1.3: Development of AOAC Official Method 2002.02
While significant steps were made in the development of in vitro methods for the measurement
of RS during the 1990s, none of these methods were successfully subjected to interlaboratory
evaluation (McCleary, 2013). This prompted the development of a new method that gave values
in line with those obtained from ileostomy patients and that could survive the rigors of
interlaboratory evaluation.
A survey of scientists initiated in 1993 by Lee & Prosky (1995) showed that 80% favored the
inclusion of RS in the definition of dietary fiber, which led to the development of AOAC Official
Method 2002.02 for the measurement of RS (McCleary & Monaghan, 2002). The incubation
conditions, level of pancreatic α-amylase and amyloglucosidase used, protease pretreatment and
procedure for recovery of RS, among others, were adjusted to optimize an assay for the direct
measurement of RS. This method was subjected to evaluation under the auspices of AOAC
International and American Association of Cereal Chemists International to determine its
interlaboratory performance. Thirty-seven laboratories tested eight pairs of blind duplicate
starch or plant material samples with RS values between 0.6-64% fresh weight basis (McCleary
et al., 2002). Two statistical measurements of precision, the relative standard deviation
repeatability (RSDr) and relative standard deviation reproducibility (RSDR) were used in order to
validate the method. The RSDr expresses the measurement results under repeatability conditions
where independent results are obtained with the same method on identical test items using the
same laboratory, operator and equipment within short intervals of time (Horie et al., 2008). The
ideal RSDr value is 4% and the reported RSDr values for this method ranged from 1.97-4.2%
(McCleary & Monaghan, 2002). The RSDR expresses the measurement results under
reproducibility conditions where results are obtained with the same method on identical test
items using different laboratories, operators and equipment (Horie et al., 2008). The ideal RSDR
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value is 6% and the reported RSDR values for this method ranged from 4.58-10.9% (McCleary et
al., 2002). The method was not suitable for samples with less than 1% RS because for such
samples, the RSDr and RSDR values were very high and considered unacceptable; therefore, this
method was applicable to samples containing 2-64% RS content. On the basis of the previous
evaluation, the method was accepted as AOAC Official Method 2002.02 and American
Association of Cereal Chemists Recommended Method 32-40.01 (McCleary et al., 2002).
Table 2 – Comparison of Resistant Starch Values Obtained Using Several In Vitro Methods and In Vivo Results: Resistant Starch (in vitro results, %)
ActiStar 63j – 57j 58.0 57j 59j Table adapted from McCleary & Monaghan (2002) a Englyst et al., 1992a b Faisant et al., 1995a c Champ et al., 1999a d McCleary & Monaghan, 2002 e Goñi et al., 1996 (assuming a starch content of 40% for bean flakes and 70% for corn flakes) f In vivo results from ileostomy patients g Champ et al., 2003 (personal communication between Champ et al., 2003 and Langkilde, A.M., Andersson, H. and Bouns, F.) h Schweizer et al., 1988 i Muir & O’Dea (1993) and Englyst et al., 1992a j Results provided by Bernd Kettlitz, Cerestar (Vilvoorde, Belgium). Englyst et al., 1992a value produced by Englyst Carbohydrate Services, Champ value at INRA (Nantes, France) and Goñi value at Cerestar (Vilvoorde, Belgium) In Table 2, McCleary & Monaghan (2002) reported the values for 4 different in vitro methods
for the determination of RS, as a percentage of total starch, and compared them to RS
17
percentages obtained in vivo from ileostomy patients. For amylomaize starch (native), the RS
percentage of the sample obtained in vivo was 50.3% as opposed to 72.2% from the Faisant et al.
(1995b) method, 71.4% from the Englyst et al. (1992a) method, 52.8% from the Champ (1992)
method and 51.7% from the McCleary & Monaghan (2002) AOAC 2002.02 method. Thus, the
Faisant et al. (1995b) and Englyst et al. (1992a) methods greatly overestimated RS percentage in
amylomaize starch (native) while the Champ (1992) and McCleary & Monaghan (2002) AOAC
2002.02 methods marginally overestimated RS content. In addition, the inconsistency is not only
apparent between the in vitro methods but also within the same in vitro method when
determining RS content from different sources of starch. For example: when comparing the
Englyst et al., (1992a) RS percentages with in vivo RS percentages, this method underestimated
RS by 12.3% for potato starch (native), overestimated RS by 21.1% for amylomaize starch
(native) and closely resembled the in vivo percentage for amylomaize starch (retrograded),
30.5% in vitro and 30.1% in vivo. Overall, AOAC 2002.02 method developed by McCleary &
Monaghan (2002) seems to be the in vitro method whose values closely resemble RS recoveries
in vivo.
2.2.1.4: AOAC 991.43 Method vs. AOAC 2002.02 Method
Lee et al. (1992) developed the AOAC Official Method 991.43 for the determination of total,
soluble, and insoluble dietary fiber in foods, an enzymatic-gravimetric based method. This
method underestimated the total dietary fiber (TDF) content of the majority of food materials
because it incompletely analyzed RS content (McCleary & Rossiter, 2004). This was
demonstrated when McCleary & Rossiter (2004) compared the TDF percentage determined by
AOAC 991.43 and a modified version of AOAC 991.43 where dimethyl sulfoxide was added to
solubilize RS. RS was determined by AOAC 2002.02 method (Table 3) and expressed as a
percentage of total starch. For Hylon VII, RS percentage determined by AOAC 2002.02 was
18
53.7% but was not reflected in the TDF percentages because AOAC 991.43 method determined
25.9% TDF and the modified AOAC 991.43 method determined 1.0% TDF. This comparison
was also true for ActiStar, native potato starch and CrystaLean. As for Hi-maize 1043, Hi-maize
bread, rye crispbread and kidney beans, the results were inconsistent. For Hi-maize 1043 and
Hi-maize bread, AOAC 991.43 percentages were higher than the AOAC 2002.02 percentages;
however, the modified AOAC 991.43 percentages were lower than the AOAC 2002.02
percentages. For rye crispbread and kidney beans, both AOAC 991.43 and modified AOAC
991.43 had TDF percentages higher than AOAC 2002.02 method. Hence, the results of AOAC
991.43 and modified AOAC 991.43 methods in Table 3 suggest that these methods are
unsuitable for the determination of RS content in RS containing food materials.
Table 3 – Total Dietary Fiber and Resistant Starch Content of a Range of Samples:
Table adapted from McCleary & Rossiter (2004) a Modification to AOAC 991.43 was made in which the sample was first cooked with dimethyl sulfoxide to solubilize resistant starch 2.2.1.5: Incorporation of Resistant Starch into Dietary Fiber
Measurements and the Development of AOAC 2009.01
Hipsley (1953) coined the term ‘dietary fiber’ to describe the non-digestible constituents of
plants that make up the plant cell wall such as cellulose, hemicellulose and lignin. Twenty-three
years later, Trowell et al. (1976), broadened the definition into a physiological one based on
edibility and resistance to digestion in the human small intestine. This new definition included
indigestible polysaccharides such as gums, modified celluloses, mucilages and pectin and non-
19
digestible oligosaccharides (NDO). In 1990, the British Nutrition Foundation Task Force
proposed that complex carbohydrates be defined as the sum of starches and non-starch
polysacchardies (British Nutrition Foundation, 1990). In this proposal, starches are considered
avCHO and all other fractions are considered unavailable carbohydrates. This proposal was
dismissed because this classification underestimated the importance of the physiological function
of RS; simply, “the contribution of RS to the body’s physiological functions is much too
significant to be buried under the general term available carbohydrates” (Schweizer et al., 1990).
The first official method for the measurement of dietary fiber was the AOAC Official Method
985.29 – ‘Total dietary fibre in foods; enzymatic-gravimetric method’ developed by Prosky et al.
(1985). This method was later extended by Lee et al. (1992) to allow measurements of TDF,
soluble dietary fiber and insoluble dietary fiber in foods and was known as AOAC Official
Method 991.43 (Figure 1). The recurrent problem with these methods is the partial measurement
of RS content in some food materials; subsequently, underestimating RS content in foods (Tables
3 & 4).
The fact that a single method to measure all dietary fiber components (including RS) is needed,
has been known for some time. While it is possible to measure many individual fiber
components with specific and non-specific methods, TDF cannot simply be calculated by adding
the values for these specific components to the determined value of high molecular weight
dietary fiber as measured by AOAC 991.43. Since AOAC 991.43 also measures some RS in
food materials, summation leads to “double counting” of this material and an eventual
overestimation of RS content (McCleary, 2013). To resolve this issue, an integrated method for
the measurement of total dietary fiber (ITDF), AOAC 2009.01 (Figure 2), was published by
McCleary (2007) and it allowed the accurate measurement of total high molecular weight dietary
fiber, which includes insoluble dietary fiber, including RS, and higher molecular weight soluble
20
dietary fiber. For most food and ingredient samples analyzed, the RS values obtained with
AOAC 2009.01 were higher than those obtained from AOAC 991.43. This method was
successfully subjected to interlaboratory evaluation and accepted as AOAC Method 2009.01
(McCleary et al., 2009) and has been recommended for use in dietary fiber labeling as it takes
into account all dietary fiber fractions including RS (Figure 2).
Table 4 – Comparison of Resistant Starch Values Obtained Using Several In Vitro Methods (Including AOAC 2009.02 Method) and In Vivo Results: Resistant Starch (in vitro results, %)
Source of Starch
Englyst AOAC 991.43
AOAC 2002.02
AOAC 2009.01
Resistant Starch (in vivo
results, %)c Raw potato
starch 66.5a 0.9b 64.9b 56.8b 67.9d
High-amylose corn
starch
71.4a 25.6b 50.0b 49.3b 43.7d
Corn flakes 3.9a 3.3e 2.2b 2.4b 3.1-5.0e Raw green
banana 54.2b 7.5b 51b 38b 55.3d
Table adapted from Shukri et al. (2013) a Englyst et al., 1992a b McCleary (2007) c In vivo results from ileostomy patients d Langkilde & Andersson (1995) e Muir & O’Dea (1992) In Table 4, Shukri et al. (2013) reported the values for 4 different in vitro methods for the
determination of RS, as a percentage of total starch, and compared them to RS percentages
obtained in vivo from ileostomy patients. When comparing RS percentage determined in vitro
by AOAC 2009.01 method with in vivo RS percentages, this method underestimated RS content
for raw potato starch and raw green banana and overestimated RS content in high-amylose corn
starch. Out of the four in vitro methods, AOAC 991.43 was the method that greatly
underestimated RS content when compared to in vivo results. Overall, Table 4 shows that no one
in vitro method accurately reflects in vivo digestibility of RS and results remain inconsistent
within the same method when comparing different sources of RS. However, AOAC 2009.01 as
21
a general method for the determination of TDF and AOAC 2002.02 as a specific method for the
determination of RS content seem to be the methods that closely reflect RS content in vivo when
compared to other in vitro methods.
2.2.2: In Vivo Methods
The two main in vivo methods for determining starch digestibility include the breath hydrogen
test and the ileostomy model. The breath hydrogen test exhibits a number of limitations and the
ileostomy model is generally accepted as the “golden standard” for the measurement of the
undigested fraction of carbohydrates.
Figure 2 – Dietary fiber components measured by AOAC Official Method 2009.01: This method takes into account the complete fraction of RS as a component of dietary fiber. * FOS – Fructo-oligosaccharides Source: Medallion Labs. (2014). Dietary Fiber. Retrieved December 3, 2014 from http://www.medallionlabs.com/TestOfferings/Fiber.aspx Used with permission from Medallion Labs.
Figure 1 – Dietary fiber components measured by AOAC Official Method 991.43: This method underestimates RS content because it only measures a fraction of it. * FOS – Fructo-oligosaccharides Source: Megazyme. (2014). Measurement of Dietary Fiber. Retrieved December 3, 2014 from http://www.megazyme.com/resources/dietary-fiber/measurement-of-dietary-fiber Used with permission from Megazyme.
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2.2.2.1: Breath Hydrogen Test
Undigested carbohydrates in the small intestine are fermented by the gut microbiota in the colon
and the products of carbohydrate fermentation are methane, hydrogen gas, carbon dioxide and
SCFAs (Hijova & Chmelarova, 2007). Since hydrogen gas is one of the four products of
carbohydrate fermentation, a large number of studies have used the breath hydrogen test to
assess the undigested fraction of carbohydrates. Breath hydrogen tests are simple, convenient,
inexpensive, non-invasive and do not disturb the normal physiological functions of the intestine
(Rumessen, 1992).
In the 1990s, several studies were conducted on RS and the breath hydrogen test. Hylla et al.
(1998) and Muir et al. (1995b) reported that the AUC of breath hydrogen was significantly
higher during the high-RS diet compared to the low-RS diet and van Munster et al. (1994)
reported that the mean rise in breath hydrogen relative to placebo was 35% (p=0.03) for all
subjects and 60% for the eight non-methane producing subjects (p=0.02) in the study. Wolever
et al. (1986) reported that direct measurement of the available carbohydrate in ileal effluent after
the consumption of three test meals by three ileostomates gave values similar to those
determined by the breath hydrogen method (white bread 10% from effluent and 11% from breath
hydrogen, wholemeal bread 8% from both effluent and breath hydrogen and red lentils 22% from
effluent and 18% from breath hydrogen). However, a study by Jenkins et al. (1998) reported that
commercial RS2 and RS3 supplements from high-amylose corn starch did not alter breath
hydrogen and methane when compared to the control treatment (low-fiber supplement). Since
this project involves commercial RS products, the results from the Jenkins et al. (1998) study
directed the methodology of this project to use the ileostomy model, instead of breath hydrogen,
as an in vivo model of determining starch digestibility.
23
In addition to the inconsistency in the breath hydrogen results mentioned above, other limitations
are associated with this in vivo method including:
1) 20% of subjects are non-producers of hydrogen gas (Gilat et al., 1978; Saltzberg et al.,
1988) and 5% are low-producers (Bond & Levitt, 1977; Vogelsang et al., 1988) and this
largely depends on the type of microbiota dominating the gut (i.e.: methanogens) (Gilat et
al., 1978);
2) Acidic colon microclimate (Perman et al., 1981; Vogelsang et al., 1988) antibiotic use
(Gilat et al., 1978; Murphy & Calloway, 1972), enemas and colonoscopy preparation
(Gilat et al., 1978) have all shown to reduce hydrogen gas production;
3) The nature of the indigestible carbohydrate such as decreased fiber particle size (Ehle et
Starch composition values based on in vitro analysis provided by the sponsor a Fiber reported “as is” and analyzed by AOAC Official Method 991.43 b Proximate analysis results from Gelda Scientific for March 2013 (provided by Glycemic Index Labs)
4.2.4: Palatability
After consuming the test meal, subjects rated the palatability of the test meal using a visual
analogue scale consisting of a 100 mm line anchored at the left end by “very unpalatable” and
the right end by “very palatable”. Subjects made a vertical mark along the line to indicate their
perceived palatability. The distance from the left end of the line to the mark made by the subject
is the palatability rating. The longer the line, the higher the perceived palatability.
41
4.2.5: Blood Samples
Blood samples (2-3 drops for each time point) were collected into 5ml tubes containing a small
amount of sodium fluoride/potassium oxalate as an anticoagulant and preservative. The samples
were mixed by rotating the tube vigorously and refrigerated during the testing session. After
completion of the test session, the samples were stored at -20°C prior to glucose analysis. Blood
glucose analysis, using a YSI Model 2300 Glucose Analyzer (Yellow Spring Instruments, OH,
U.S.A) took place within 3 days of sample collection.
4.2.6: Available Carbohydrate Calculations
The trapezoid rule was used to calculate the iAUC, ignoring the area beneath baseline (Wolever,
2006). For the purpose of the iAUC calculation, fasting glucose was taken to be the mean of the
glucose concentrations at time -5 and 0 minutes.
In order to calculate the avCHOs from glycemic responses, the GI of each product needs to be
known. The dose of AM fed was assumed to contain 50 g avCHO (value provided by the
sponsor); thus, the GI of AM was determined in this experiment and was assumed to reflect the
GI of HM and HY. The amount of digestible carbohydrates absorbed from the test meals was
determined based on the rationale outlined below.
Wolever et al. (2006) showed that the iAUC elicited by various doses of carbohydrates from
white bread could be expressed as:
[1] iAUC = Z x (1 – e-0.0233 g) + 7.8
where ‘g’ is the amount of avCHO in the test meal. The value ‘Z’ depends upon the GI of the
food fed and the carbohydrate tolerance of the subject, ‘S’. Hence, equation [1] can be re-written
as follows:
[2] iAUC = GI x S x (1 – e-0.0233 g) + 7.8
42
If the test carbohydrate is glucose (GI=100), the value for ‘Z’ can be derived for each subject by
substituting ‘R’ (mean iAUC after 50 g dextrose) for iAUC and 50 for ‘g’ in equation [1] and
rearranging the equation as follows:
R = Z x (1 – e-1.165 g) + 7.8
Therefore, expressing the equation in terms of ‘Z’ yields:
[3] Z = (R – 7.8) 0.688
Since ‘Z = GI x S’; thus, ‘S = Z/GI’. Since the GI of glucose = 100, from equation [3], we can
calculate the carbohydrate tolerance ‘S’ as:
[4] S = (R – 7.8) 68.8
Thus, the amount of avCHO contained in the test carbohydrate was calculated by rearranging
equation [2] to solve for ‘g’ as follows:
[5] avCHO = {ln(1-[(F-7.8)/(GI x S)])}/-0.0233
where ‘F’ is the iAUC elicited by the test carbohydrate and ‘S’ is derived from equation [4]. The
accuracy of this equation was tested by calculating the avCHO for 10 g dextrose (GI=100) and
55 g white bread (GI=71) from their iAUC and GI values in which a value of 10 was expected
for 10 g dextrose and 24.8 for 55 g white bread.
When the experiment was completed, we found that equation [5] did not work for 7 of the 90
glycemic responses because the value (F-7.8)/(GIxS) was greater than 1 so that the value of 1-
[(F-7.8)/(GIxS)] was less than 0 and it is not mathematically possible to take the natural log of a
negative number. The problem reflects the large day-to-day variation of glycemic responses and
was caused by high iAUC values relative to GI and/or the carbohydrate tolerance ‘S’. Two
solutions to this problem were implemented: 1) the mean iAUC was used in equation [5] instead
of each subject’s iAUC and 2) another equation [5a] derived from iAUCs after the consumption
43
of various doses of different sources of carbohydrates was developed by Wolever (2006); thus,
equation [5a] was derived as follows:
[1a] iAUC = Z x (1-e-0.0222 g)
[2a] iAUC = GI x S x (1-e-0.0222 g)
R = Z x (1-e-1.11)
[3a] Z = (R) 0.670
[4a] S = (R) 67.0
[5a] avCHO = {ln(1-[(F)/(GI x S)])}/-0.0222
Use of equation [5a] resulted in somewhat different estimates of avCHO, but (F)/(GIxS)
remained greater than 1 for the 7 problematic glycemic responses described above. Because
mean iAUC was used in equation [5], mean iAUC was also used in equation [5a] and the mean
of these 2 equations was taken as the final mean BGR avCHO content of the test meals. Use of
the mean iAUC generated 2 avCHO values, 1 for each equation; hence, a measure of variability
for the avCHO content was not possible and affected how this data was statistically analyzed in
section 4.3.8 below.
4.2.7: Statistical Analysis
The results were assessed using repeated measures analysis of variance (RM-ANOVA) and if a
statistically significant difference was found, this was followed by pairwise comparisons using
Tukey’s test. Outliers were excluded for values >2xSD above the mean.
44
4.3: Results
4.3.1: Baseline Characteristics
A sample of 10 healthy, normoglycemic subjects (4 males and 6 females) aged 39±13 years with
a body mass index (BMI) of 26.5±4.2 kg/m2 participated in this study. One subject dropped out
at the beginning of the study due to new employment responsibilities. Three subjects were
taking medication or supplements on a daily basis. Subject ID 452 was taking one-a-day
multivitamin and 1,500 IU vitamin D3/ day. Subject ID 559 was taking 400 mg Seroquel, an
atypical antipsychotic drug, once a day. Subject ID 564 was taking 50 mg Tradozone as an
antidepressant, 75 mg Arthrotec for her arthritis and 40 mg Tecta to reduce gastric acid once a
day. The individual details are shown in Table 6 below.
Table 6 – Baseline Characteristics of Study 1:
Subject ID
Gender Ethnicity Age (yrs)
Height (m)
Weight (kg)
BMI (kg/m2)
336 M Latin American 48 1.77 70.0 22.3 341 M South Asian 47 1.88 110.5 31.3 354 F Caucasian 43 1.66 71.2 25.8 452 M Caucasian 63 1.65 75.8 27.8 538 M Caucasian 21 1.83 81.8 24.4 550 F Arab/West Asian 34 1.51 51.5 22.6 559 F Caucasian 37 1.64 92.7 34.5 560 F Arab/West Asian 32 1.62 66.3 25.3 563 F Caucasian 22 1.73 65.9 22.0 564 F Caucasian 47 1.58 73.2 29.3
Mean±SD 39±13 1.69±0.11 75.9±16.2 26.5±4.2
4.3.2: Blood Glucose Analysis
Of the 720 blood samples, only 1 (0.1%) BGR value was missing (subject ID 563 at 45 minute
blood collection on the first 50 g dextrose test) and was replaced by the mean of the 30 and 60
minute BGR values. Due to a low volume of blood, duplicate analysis could not be done on 10
of the 90 0 minute samples. For the remaining 80 0 minute BGRs, the mean±SD was 4.57±0.33
45
mmol/L yielding a coefficient of variation (CV%) of 7.27%, well over 3% as a result of
analytical variation. The mean±SD for the 90 -5 and 0 minute BGRs was 4.62±0.32 mmol/L
yielding a CV% of 6.86%, reflecting both analytical variation and minute-to-minute variation in
BGRs.
4.3.3: Glycemic Response Elicited by Reference Food
The Dex50 test meal was consumed 3 times by each subject. The mean±SEM of the iAUC
values were 221.2±29.0, 212.4±31.4 and 199.1±28.7 mmolxmin/L for the 1st, 2nd and 3rd tests of
Dex50 respectively and no significant differences were observed among them (F=0.19, p=0.77).
The mean±SEM of the within-subject CV% of the iAUC values after the Dex50 test meals was
25.6±3.1% and this is satisfactory since the mean within-subject CV for the repeated reference
food tests should be less than 30%.
4.3.4: Palatability
Palatability, iAUCs and relative glycemic responses (RGRs) for every treatment are shown in
Table 7 below. A one-way RM-ANOVA was performed on the palatability results (F=8.3,
p=0.002) with significant differences observed among the 6 test meals. WB was significantly
different from AM (p≤0.01) and HM (p≤0.001) and Dex50 was significantly different from HY
and HM (p≤0.05). The starch products scored fairly low on the palatability scale with HM
scoring the lowest, 20.2±5.7 mm and HY scoring the highest, 30.7±9.6 mm among the products
(Figure 4).
46
Table 7 – Palatability, Incremental Areas Under the Blood Glucose Response Curves and
Relative Glycemic Responses of the Test Meals:
Values are expressed as means±SEM for each treatment
A) Bars represent mean±SEM of the palatability values for each treatment for n=10 subjects B) Abbreviations: WB – White Bread, Dex50 – Dextrose 50, Dex10 – Dextrose 10, HY – Hylon® VII, AM – Amioca, HM – Hi-maize® 260 C) Bars with different letters differ significantly
1 1 1 1 1 1
0
10
20
30
40
50
60
70
80
90
100
Treatment
Pala
tabi
lity
(mm
)
WB Dex50 Dex10 HY AM HM
abad
ae
be
cde
ce
47
4.3.5: Blood Glucose Responses
A two-way RM-ANOVA was performed on the BGRs with a significant time (F=56.6,
p<0.0001), treatment (F=7.8, p<0.0001), and time x treatment interaction (F=11.1, p<0.0001).
Significant differences in the BGRs were observed among the 6 test meals at 15, 30, 45 and 60
minutes. AM and HM elicited nearly identical BGRs at 30 minutes, 5.62±0.1 mmol/L and
5.63±0.25 mmol/L respectively, and did not differ significantly at any time point (p>0.05).
Among the 3 investigational starch products, AM elicited the highest BGR, followed closely by
HM and HY elicited the lowest BGR.
Figure 5 – Blood Glucose Responses Elicited by the Test Meals:
A) Blood glucose response curves represent the mean±SEM for each treatment at each time point (0, 15, 30, 45, 60, 90 and 120 minutes) for n=10 subjects B) Abbreviations: Dex50 – Dextrose 50, WB – White Bread, Dex10 – Dextrose 10, HM – Hi-maize® 260, AM – Amioca, HY – Hylon® VII
0 30 60 90 1204
5
6
7
8HM
HYAM
WBDex10
Dex50
Time (minutes)
Blo
od
Glu
cose
Res
po
nse
(mm
ol/L
)
48
4.3.6: Incremental Areas Under the Blood Glucose Response Curves
iAUC values for the treatments are shown in Table 7 above. A one-way RM-ANOVA was
performed on the iAUC values (F=19.2, p<0.0001) and Dex50 had a significantly higher iAUC,
210.9±25.6 mmolxmin/L, than all the other treatments (Figure 6). AM’s iAUC was significantly
higher than HY (p≤0.01) and the iAUC of HY was 34% that of AM and 37% that of HM. The
GI of AM was calculated to be 38 (low) and assumed to reflect the GI of HY and HM.
Figure 6 – Incremental Areas Under the Blood Glucose Response Curves:
A) Bars represent the mean±SEM of the iAUC values for each treatment for n=10 subjects B) Abbreviations: Dex50 – Dextrose 50, WB – White Bread, AM – Amioca, HM – Hi-maize® 260, Dex10 – Dextrose 10, HY – Hylon® VII C) Bars with different letters differ significantly
0
25
50
75
100
125
150
175
200
225
250
Treatment
Blo
od
Glu
cose
iAU
C (m
mo
lxm
in/L
)
Dex50 WB AM HM Dex10 HY
a
bc
b bc
b
c
49
4.3.7: Relative Glycemic Responses
RGR values are shown in Table 7 above. A one-way RM-ANOVA was performed on the RGRs
(F=28.2, p<0.0001) with Dex50 (100%) significantly different from all the other treatments. The
RGR of AM was significantly different from HY (p≤0.01) and remained significant after the
outlier was excluded from HY (one outlier only for subject 341). The highest RGR of the
starches was from AM (37.7%) followed by HM (32.4%) and HY (9.7% and excluding the
outlier: 6.3%).
Figure 7 – Relative Glycemic Responses:
A) Bars represent the relative glycemic response percentages for each treatment for n=10 subjects B) Abbreviations: Dex50 – Dextrose 50, WB – White Bread, AM – Amioca, HM – Hi-maize® 260, Dex10 – Dextrose 10, HY – Hylon® VII C) Bars with different letters differ significantly
1 1 1 1 1 1
0
20
40
60
80
100
Treatment
Rel
ativ
e R
esp
on
se (%
)
Dex50 WB AM HM Dex10 HY
a
b
b bcb
c
50
4.3.8: Available Carbohydrate Calculated from Blood Glucose Responses
avCHO values are shown in Table 8 and Figure 8 below and values are expressed as means only
(explained in section 4.2.6 above). Hence, a two-way ANOVA and multiple comparisons were
not relevant to perform on these data. The mean BGR values from equations [5] and [5a] in
Table 8, column 5 are the final BGR avCHO values and will be referred to as ‘mean BGR’
values in this and the following sections. The mean BGR avCHO values for the starches are as
follows: HM was 37.5 g (2.2 times higher than the in vitro value), AM was 43.8 g (0.9 times or
10% less than the in vitro value) and HY was 9.2 g (0.2 times or 80% less than the in vitro
value).
Table 8 – Available Carbohydrate Calculated from Blood Glucose Responses:
Available Carbohydrates (g) Test Meal In vitroa BGR
Equation [5]b BGR
Equation [5a]b Mean BGR Equations [5] & [5a]
HM 17.2 35.1 40.0 37.5 AM 50.0 41.2 46.4 43.8 HY 38.5 7.5 10.9 9.2
Dex50 50.0 50.1 49.9 49.9 WB 25.0 27.1 28.9 27.9
Dex10 10.0 8.6 9.8 9.2 a In vitro values provided by the sponsor b BGR values expressed as means for n=10 subjects Abbreviations: HM - Hi-maize® 260, AM – Amioca, HY - Hylon® VII, Dex50 – Dextrose 50, WB – White Bread, Dex10 – Dextrose 10
51
Figure 8 – Available Carbohydrate Calculated from Blood Glucose Responses:
4.3.9: Estimates of Available Carbohydrate in Test Starches
Estimates of avCHOs in the test starches are shown in Table 9 below. The amount of avCHOs
per 100 g of the test starches are as follows (in vitro vs. mean BGR): HM was 31.1 g vs 67.9 g,
AM was 88.7 g vs 77.7 g and HY was 67.7 g vs. 16.2 g. The largest difference observed in the
in vitro vs. mean BGR values was for HY.
A) Bars represent the mean avCHO content of each treatment by each method for n=10 subjects B) Abbreviations: Dex50 – Dextrose 50, WB – White Bread, Dex10 – Dextrose10, AM – Amioca, HY - Hylon® VII, HM - Hi-maize® 260
1 1 1 1 1 1
0
5
10
15
20
25
30
35
40
45
50
55
60
Treatment
Ava
ilab
le C
arb
oh
ydra
te (g
/test
mea
l)
Dex50 WB Dex10 AM HY HM
In-vitroMean BGR [5]Mean BGR [5a]Mean BGR [5&5a]
52
Table 9 – Estimates of Available Carbohydrate in Test Starches:
Available Carbohydrates (g/portion)
Available Carbohydrates (g/100 g)
Test Meal Weight (g) In vitroa Mean BGRb In vitroa Mean BGRb HM 55.3 17.2 37.5 31.1 67.9 AM 56.4 50.0 43.8 88.7 77.7 HY 56.9 38.5 9.2 67.7 16.2
a In vitro values provided by the sponsor b Mean BGR values represent mean of equations [5] and [5a] and are expressed as means for n=10 subjects Abbreviations: HM - Hi-maize® 260, AM – Amioca, HY - Hylon® VII 4.4: Discussion
The research question for this study was: Do in vitro methods accurately reflect true in vivo
digestibility of RS? To answer this question, our results showed that the iAUCs and RGRs of the
3 test starches were significantly less than that of Dex50 and suggest that the in vitro AOAC
991.43 method greatly underestimated the fiber content in HY, marginally underestimated the
fiber content in AM and greatly overestimated the fiber content in HM. No previous studies
have been done on estimating avCHO content of RS products from glycemic responses; hence no
direct comparison of our data with data from the literature was possible. Nonetheless, our results
for HM were confirmed in Table 4 when AOAC 991.43 method overestimated RS content of
high-amylose corn starch in comparison to in vivo data from ileostomy patients.
The first parameter of interest in this study was the perceived taste of the raw starch products
measured by the palatability scale. The test starches scored fairly low on the palatability scale
with HM scoring the lowest (20%), HY scoring the highest (31%) and AM scoring in between
(29%). The taste of the raw starch mixed in water was predominantly described as “wallpaper
paste.” It is important to note that these starch products are not manufactured in order to be
consumed raw; however, are food ingredients meant to be incorporated into cakes, cookies,
biscuits etc. to increase fiber content without effect on taste and texture. Hence, these
53
palatability results are not representative of the effect that these starch products would have on
taste and texture had they been added to cake for example, instead of being consumed raw.
The iAUC of Dex50, 210.9±25.6 mmolxmin/L, and RGR, 100%, were significantly higher than
all the other treatments. WB had half the amount of avCHO of Dex50 (50 g vs. 25 g) and this
clearly shows from the iAUC values in which the iAUC of WB, 105.8 mmolxmin/L, was half
that of Dex50. Dex10 had a similar BGR to WB at 30 minutes but reduced below fasting levels
after 45 minutes up until 120 minutes. When calculating the iAUC, the area below fasting levels
is disregarded, possibly explaining why the iAUC of Dex10 (61.4±11.0 mmolxmin/L) is similar
to HM (70.4±20.4 mmolxmin/L). Surprisingly, the BGRs of HM (iAUC=70.4±20.4
mmolxmin/L) and AM (iAUC=77.0±14.2 mmolxmin/L) were very similar and nearly identical at
30 minutes although in vitro analysis has indicated that HM contains 60% RS as dietary fiber and
Amioca has 0% dietary fiber. HY elicited the lowest BGR (iAUC=25.8±13.0 mmolxmin/L)
although in vitro analysis indicated an avCHO content of 38.5 g per 50 g tCHO content,
theoretically placing its BGR closer to AM.
Of interest was the comparison of the RGR and mean BGR avCHO content of HM, Dex10 and
HY. The RGR of HM and Dex10 were very similar, 32% vs. 31% respectively, although the
estimated mean BGR avCHO content was 37.5 g per 50 g tCHO portion of HM and 10 g for
Dex10. On the other hand, the estimated mean BGR avCHO content of HY was 9.2 g per 50 g
tCHO portion, identical to the value estimated for Dex10, 9.2 g. Taking a look back at the RGR
of Dex10 and HY, they were 31% vs. 10%. This confirms the very concept of GI in which the
same amount of ingested avCHO from different carbohydrate sources, i.e.: dextrose and starch,
elicit completely varying BGR, iAUC and ultimately RGR values.
54
The similarity in the BGR, iAUC and RGR values of HM and AM was alarming, due to the fact
that, based on in vitro analysis, HM contained 17.2 g avCHO per 50 g tCHO portion while AM
contained 50 g avCHO per 50 g tCHO portion. This suggests that there are more avCHO in HM
and less avCHO in AM than accounted for in vitro and this was confirmed by the mean BGR
avCHO calculations in which HM contained 37.5 g of avCHO and AM contained 43.8 g of
avCHO per 50 g tCHO, a mere 6.3 g difference between them. This also suggests that a large
fraction of the starch in HM is slowly digestible starch, as opposed to RS, that is slowly but
completely digested and absorbed into the blood stream. Also, AM might not only contain
readily digestible starch but also slowly digestible starch which might explain why HM and AM
elicited similar glycemic responses. As for HY, the in vitro method indicated that it contained
38.5 g avCHO per 50 g tCHO portion; however, mean BGR avCHO calculations showed that
HY only contained 9.2 g avCHO, explaining why it elicited the lowest BGR, iAUC and RGR
values.
The avCHOs calculated for Dex10 (9.2 g) was a better representation of the accuracy of the
method than WB because it was within 8% of its in vitro value as opposed to WB (27.9 g) that
was 12% higher than its in vitro value. A possible reason for this is that subjects vary in their
ability to process avCHOs from different carbohydrate sources, i.e.: dextrose and flour. If the
individual iAUC values, as opposed to the mean values, for Dex10 and WB were used to
determine the avCHO content of these test meals, more accurate values could have been
observed, possibly within 2-3% of their in vitro values. For example: using individual iAUCs
for Dex10 for equation [5] yielded an average of 10 g avCHOs and equation [5a] yielded an
average of 11 g avCHOs and the mean of the equations was 10.5 g avCHO content. Hence, a
better representation of the avCHO content of Dex10 could have been achieved had individual
iAUCs, rather than mean iAUC, been used. This was not possible to do with our data because of
55
the 7 problematic glycemic responses described in section 4.2.6; thus, mean iAUCs were used to
estimate the avCHO content of all the test meals.
When it comes to determining the digestibility of RS, two main in vivo methods are used, the
breath hydrogen method (Jenkins et al., 1998) and the ileostomy model (Englyst et al, 1992a).
The breath hydrogen method assesses digestibility based on how much hydrogen gas is produced
from the RS that escapes digestion in the small intestine. The ileostomy model assesses
digestibility of RS by direct collection of ileal effluent and further analysis to determine amount
of RS recovered. Both of these methods do not take into account the affect that RS has on
glycemic responses when it replaces a portion of avCHOs. This novel in vivo method has
several advantages including the use of healthy, normoglycemic subjects, sample collection takes
place in 2 hours as opposed to 10 hours for the ileostomy model and it avoids the issue of the
type of gut microbiota inhabiting the colon of subjects, i.e.: methanogens (Gilat et al., 1978),
from the breath hydrogen method.
The results of this novel in vivo approach further emphasize the importance of using the relevant
in vitro methods for the dietary fiber fraction of interest. This ties directly with the implications
of this project in which accurate dietary fiber labeling of novel RS products is essential to ensure
accurate fiber labeling of products which incorporate RS.
There was one main limitation of the BGR method used in this study, i.e.: accurate knowledge of
the product GI was necessary to give accurate estimates of the avCHO content of the starch
products. For example: the GI of Amioca was calculated to be 38 and the mean BGR avCHO
content was 43.8 g/ 50 g tCHO content of the product. Let’s assume that the GI of Amioca was
calculated to be 36 or 40 (±2). If this was the case, the mean BGR avCHO content for AM for a
56
GI of 36 would have been 48 g and 37 g if the GI was 42. Therefore, a -2 in GI resulted in a +5
g in the avCHO content of AM and a +2 in GI resulted in a -5 g in the avCHO content of AM.
It remains unclear whether the novel in vivo approach to estimating avCHO content of RS
products used in this study produces accurate results of the true avCHO content of commercial
RS containing products. Having said that, this novel approach needs to be validated by
comparing its results with the results from the ileostomy model since it is considered the golden
standard of studying starch digestibility.
57
Chapter 5 Study 2
Digestibility of Selected Resistant Starches in Ileostomates
5.0: Digestibility of Selected Resistant Starches in Ileostomates
5.1: Background
Study 2 was the ileostomy study conducted from October 2013 – April 2014. This study was
conducted in order to validate the results from the novel in vivo BGR method described in
Chapter 4. The best way to conclude whether the method described in Chapter 4 truly reflects
the bioavailability of RSs is by comparing its results to the results from the golden standard of
studying starch digestibility, i.e.: the ileostomy model. This study will help us answer the
following question: Does the novel BGR method accurately reflect the bioavailability of novel
RS products? This study was registered in ClinicalTrials.gov (NCT01939600).
5.2: Methods
5.2.1: Subjects
We aimed to have a total of 10 subjects to complete this study and the subjects were recruited
from the Division of Gastroenterology at St. Michael’s Hospital. The gastroenterologists
referred patients with a conventional ileostomy to the study coordinator who briefly assessed
them for eligibility via telephone. Patients interested in the study and who met the inclusion
criteria were invited to the Risk Factor Modification Center of St. Michael’s Hospital to
complete the consent form, subject screening form as well as the meal plan. All the study visits
took place at GI Labs and monetary compensation was provided for the subjects for their time
and effort.
58
The inclusion criteria included males or non-pregnant females with a conventional ileostomy and
who are clinically stable with no clinical evidence of malabsorption. The exclusion criteria was
as follows: 1) short bowel syndrome (defined as having less than 200 cm of intact small
intestines left), 2) prone to high ileostomy output with change in diet, 3) ileostomy created less
than 6 months from the first study visit unless output is stable, 4) subjects using medications
which influence gastrointestinal motility or absorption, 5) any condition which might make
participation dangerous to the subjects themselves or to others or which may affect the results in
any way, 6) subjects who cannot or will not comply with the experimental procedures and 7)
severe food allergies of any kind. All subjects provided written informed consent before
participation and the protocol was approved by St. Michael’s Hospital Research Ethics board as
well as the Human Subjects Review Committee at the University of Toronto.
5.2.2: Protocol
The study was open-label with a randomized, cross-over design with each subject studied on 4
occasions (control and 3 investigational starch products) over a minimum of 4 weeks (one test
per week) and a maximum of 7 weeks. On the evening before each test day, subjects consumed
a starch- and fiber-free dinner. The meal was planned by the study coordinator and the subject
and selected from a menu (Appendix 1) suitable for subjects who consume animal products as
well as vegetarians. The dinner meal was picked up by the subject from Glycemic Index
Laboratories (3rd floor, 20 Victoria Street, Toronto) or was delivered to them at home or work 1
or 2 days before each test day. Subjects were provided with a Test Meal Record Sheet (TMRS)
to fill out indicating the composition of all the study meals consumed, the time they were
consumed, any changes to medication and any unusual activities.
Each test day started at approximately 8:00 am after a 10-14 hour overnight fast where the
subject first changed their ileostomy pouch, started a timer and consumed a breakfast test meal
59
consisting of a polysaccharide-free breakfast meal alone (control) or a breakfast test meal with
one of the 3 investigational starch products in randomized order (Table 5). A starch- and fiber-
free lunch meal (similar to the dinner meal) was consumed 4 hours after the breakfast meal and
the subjects were allowed a mid-morning snack before lunch consisting of either cheese, yogurt
or pudding with fruit juice or the meal-replacement. When the ileostomy pouch was a third full,
effluent was collected and placed immediately in the -80ºC deep freezer and the subjects
continued to collect ileostomy effluent for 10 hours.
Subjects had the choice to complete their visits at GI Labs or another location such as their home
or workplace. If the latter, a study staff delivered the test breakfast and lunch meals and a box of
dry ice to the subject’s location, supervised the breakfast meal consumption and showed the
subject how to handle and store the box of dry ice safely. Ileostomy effluent collected at home
or work was delivered to GI Labs within 24 hours of collection either by the subject or by taxi or
was picked up from the subject's home or work by the study staff.
The dinner, breakfast and lunch meals chosen remained constant over the 4 test occasions and
were recorded in the TMRS. Subjects were allowed drinks of water, coffee or tea freely during
the day. The protocol is illustrated in Figure 9 below.
60
Figure 9 – Illustration of the Protocol for the Ileostomy Study:
5.2.3: Test Meals
The 3 investigational starch products as well as the amount fed in this study were equivalent to
the ones used in study 1 (Table 5). Subjects chose breakfast from a menu (Appendix 1) of items
including: milk-based cheese or cheese-alternative, milk- or soy-based yogurt, pudding, fruit
salad, pastries, fruit juice, meal-replacement (Ensure Nutrition Plus), milk and coffee or tea. The
test starches were mixed into milk, yogurt, pudding, fruit juice, water or were taken in the meal-
replacement. The selected breakfast meal was the same for all 4 test occasions.
5.2.4: Freeze-Drying Effluent
Ileal effluent samples were removed from the deep-freezer and placed in a shaking water bath at
room temperature for 15-20 minutes to thaw. After thawing, the samples were weighed and the
weight was recorded. Each sample consisted of 2-3 collection bags which were emptied into a
blender and homogenized for 1 minute at lowest speed. The freeze-drier flask was weighed and
350 g of homogenized effluent was poured into the flask. The sample was rotated in the flask to
Fasting 10-‐14 hours
Night Before Test Day 1
Night Before Test Day 2
1
Night Before Test Day 3
1
Night Before Test Day 4
Dinner
Test Meal Record Sheet (TMRS)
Control Breakfast or Breakfast with Added Resistant Starch
Snack
Lunch
TMRS
Ileal Effluent Collection
10 hours
Control Breakfast or Breakfast with Added Resistant Starch
Snack
Lunch
TMRS
Ileal Effluent Collection
10 hours
Control Breakfast or Breakfast with Added Resistant Starch
Snack
Lunch
TMRS
Ileal Effluent Collection
10 hours
Control Breakfast or Breakfast with Added Resistant Starch
Snack
Lunch
TMRS
Ileal Effluent Collection
10 hours
Dinner
Test Meal Record Sheet
Dinner
Test Meal Record Sheet
Dinner
Test Meal Record Sheet
Visits
Fasting 10-‐14 hours
Fasting 10-‐14 hours
Fasting 10-‐14 hours
61
reduce thickness and increase surface area then placed at a 45º angle in the -80ºC freezer
overnight. The next day, the freeze-dryer was turned on and the frozen samples were loaded
onto the freeze-dryer. Samples from the control diet took approximately 4 days to dry and
samples from the starch test days took approximately 7 days to dry. The samples were dry when
the external side of the flask was warm and dry and when big cracks appeared through the
effluent. After the samples had dried they were removed from the freeze-dryer and weighed.
The dry material was transferred into a plastic bag and lightly pounded into powder which was
later transferred into hinged-lid plastic containers. The freeze-dried samples were sent to Dr.
Suzanne Hendrich’s lab, Professor of Food Science and Human Nutrition at Iowa State
University for proximate and RS analysis.
5.2.5: Ileal Effluent Analysis
5.2.5.1: Proximate Analysis
The moisture content of the freeze-dried effluent samples was determined using the Oven-Drying
Method (Horwitz, 2007) where the percent moisture was calculated as follows:
% Moisture = (weight (g) of sample before drying – weight (g) of sample after drying)/
weight (g) of sample before drying x 100%.
The fat content was determined using the Goldfisch Method (Horwitz, 2007) in which:
% Oil = % oil (wet basis)/ [1-(% moisture/100)]
The protein content was determined by the DUMAS Nitrogen Combustion Method (Simonne et
al., 1997) in which:
% Protein = weight of nitrogen (N) (g) × 6.25 × 100%/ weight (g) of sample
The ash content (Horwitz, 2007) was determined by placing 5g of the sample in a crucible in a
550ºC furnace overnight and the:
% Ash = weight of ash (g)/ weight of sample (g) x 100%
62
The carbohydrate by difference (Horwitz, 2007) was calculated as:
100 - (weight in grams [protein + fat + moisture + ash]) in 100g of the effluent
Analysis was performed on duplicate samples and the mean was taken as the final value for
further calculations.
5.2.5.2: AOAC Official Method 2002.02
The RS content of the ileal effluent samples was determined by AOAC Official Method 2002.02
(McCleary et al., 2002) and this method is applicable to samples containing more than 2%
weight/weight (w/w) of RS. The samples were first prepared with the assumption that they
contained <10% RS. If the absorbance read over 1 absorbance unit, it was indicative that the
sample contained >10% RS and was diluted to 100 ml with a second reading of the absorbance.
For samples >10% RS:
A) RS (g/100 g of the sample) = ∆E x F/W x 90
where ‘E’ is the absorbance read against the reagent blank, ‘F’ is the conversion from absorbance
to micrograms of D-glucose and ‘W’ is the dry weight of the sample analyzed.
For samples <10% RS:
B) RS (g/100 g of the sample) = ∆E x F/W x 9.27
The difference in the multiplication factor of 90 in equation A and 9.27 in equation B is due to
the volume correction of the solution. For equation A, 0.1 ml was taken from 100 ml of the
solution and for equation B, 0.1 ml was taken from 10.3 ml of the solution.
5.2.5.3: Total Bacterial Count Determined by Real-Time Quantitative Polymerase Chain Reaction
Bacterial DNA was extracted from 500 µg of undiluted ileal effluent using E.Z.N.A.™ Stool
DNA Isolation Kit (Omega Bio-Tek, Georgia, U.S.A) according to the manufacturer’s
instructions, but modified to include a lysozyme digestion step (20 mg/ml at 37ºC for 30
63
minutes). The lysozyme digestion step was included to facilitate maximal bacterial cell wall
lysis in order to improve efficiency of DNA extraction (Singh et al., 2013). DNA concentration
and purity were measured using Thermo Scientific NanoDrop 2000 spectrophotometer (Thermo
Fisher Scientific, Massachusetts, U.S.A) at 230, 260 and 280 nm. Purity ratios above 1.7 were
considered of good quality and used for the real-time quantitative polymerase chain reaction (R-
T qPCR).
R-T qPCR was employed using 10 ng of total DNA, total bacteria TaqMan assay (Furet et al.,
2009; Suzuki et al., 2000) and TaqMan Gene Expression Master Mix (Applied Biosystems,
U.S.A.) in 10 µl PCR reaction. All reactions were run in triplicates in a 384-well optical plate in
Applied Biosystems 7900 HT Real-Time PCR machine with default thermocycling conditions as
per the manufacturer’s instructions.
5.2.6: Statistical Analysis
The results were assessed using repeated measures analysis of variance (RM-ANOVA) and if a
statistically significant difference was found, this was followed by pairwise comparisons using
Tukey’s test. Pearson correlations were performed on the total bacterial count, carbohydrates by
difference and RS.
5.3: Results
We aimed to recruit 10 subjects for this study but only 3 ileostomates were enrolled. The
recruitment period extended for 5 months where 7 ileostomates were approached and 3 agreed to
participate. It was difficult to find patients with conventional ileostomies because this surgical
procedure is becoming less commonly performed and is being replaced by newer procedures
aimed at enhancing the quality of life of these patients. Of the 4 ileostomates that declined
participation, 2 had difficulties with transportation, one was on a strict dietary regimen and the
64
last ileostomate did not have stable output due to her ileostomy being created less than 2 months
from the time of contact. Therefore, only 3 ileostomates completed the study.
5.3.1: Baseline Characteristics
A sample of 3 Caucasian, female patients with conventional ileostomies completed the study.
The mean±SD age of the ileostomates was 44.3±26.8 years with a normal BMI of 23.5±6.2
kg/m2. Subject 1 (AR001) had her ileostomy for 30 years due to Crohn’s disease with 76 cm of
her small bowel removed and occasionally takes domperidone to relieve nausea. Subject 2
(AR002) has had her ileostomy for 16 years due to Crohn’s disease with 15 cm of her small
bowel removed and takes multivitamins, vitamin D and fish oil supplements. Subject 3 (AR003)
has had her ileostomy for 4 months due to dysmotility of the large intestine as well as
gastroparesis without the resection of any of her small bowel and takes a combination of
medication to relieve nausea and pain. The individual details are shown in Table 10 below.
Table 10 – Baseline Characteristics of Study 2:
Subject ID
Gender Ethnicity Age (yrs)
Height (m)
Weight (kg)
BMI (kg/m2)
Amount of Small Bowel
Resected (cm)
AR001 F Caucasian 73 1.5 50.0 21.9 76 AR002 F Caucasian 40 1.6 46.7 18.2 15 AR003 F Caucasian 20 1.7 85.4 30.3 0
SD for (R-F) 0.35 3.10 3.67 0.23 95% CI for (R-F) -0.36,1.36 -36.10, -20.69 -15.64, 2.61 -0.42, 0.70
ª ‘F’ are the fed fiber values based on in vitro analysis (AOAC Official Method 991.43) provided by the sponsor (g/50 g total carbohydrates) b ‘R’ are the recovered RS values in ileal effluent and analyzed based on AOAC Official Method 2002.02 c ‘R’ values are adjusted (R = RS recovered with starch – RS recovered on control day) Values with different letters differ significantly Abbreviations: HM – Hi-maize® 260, HY – Hylon® VII, AM – Amioca
70
5.3.6: Comparison of Carbohydrates by Difference Fed and Recovered
Amounts of CHOD fed (in vitro) vs. recovered (in vivo) are shown in Table 17 below. A one-
way RM-ANOVA was performed on the results (F=34.14, p=0.01) and there were significant
differences in the means of the control treatment and HM (p≤0.05), HM and HY (p≤0.05) and
HM and AM (p≤0.01). The largest difference between CHOD fed vs. recovered values for the 3
starches was for HM (-14.07 g) followed by HY (10.05 g) and AM (1.72 g). Approximately
57% of the CHOD fed in HM was recovered in ileal effluent and the percentage increased to
187% in HY.
Table 17 – Comparison of Carbohydrates by Difference Fed and Recovered: Treatment Control (g) HM (g) HY (g) AM (g)
Mean Difference (R-F)* 10.18d -14.07e 10.05d 1.72d
SD for (R-F) 1.58 4.74 3.30 6.37 95% CI for (R-F) 6.27, 14.10 -25.85, -2.31 1.86, 18.25 -14.09, 17.54
ª ‘F’ are the fed fiber values based on in vitro analysis (AOAC Official Method 991.43) provided by the sponsor (g/50 g total carbohydrates) b ‘R’ are the recovered CHOD values in ileal effluent and analyzed based on proximate analysis c ‘R’ values are adjusted (R = CHOD recovered with starch – CHOD recovered on control day) Values with different letters differ significantly Abbreviations: HM – Hi-maize® 260, HY – Hylon® VII, AM – Amioca
71
5.3.7: Amount of Fiber and Available Carbohydrates by Different Methods
The amount of fiber and avCHOs by different methods (in vitro, in vivo BGR study and in vivo
ileostomy study) are shown in Table 18 and Figure 10 below. The values for each method were
expressed as means only since a measure of variability was not provided for the in vitro values
and not possible from the in vivo BGR study as discussed in 4.2.6 above. Hence, a two-way
ANOVA and multiple comparisons were not relevant to perform on these type of data. The fiber
values for HY and AM from the ileostomy study are less than those from the BGR study and the
fiber value for HM from the ileostomy study was more than the fiber value from the BGR study.
Table 18 – Amount of Fiber and Available Carbohydrates by Different Methods:
Fiber Available Carbohydrates Method HM (g) HY (g) AM (g) Method HM (g) HY (g) AM (g) In vitroa 32.8 11.5 0 In vitroa 17.2 38.5 50 In vivo BGR
Studyb
12.5 40.8 6.2 In vivo BGR
Studyc
37.5 9.2 43.8
In vivo Ileostomy Studyd,e
18.7 21.6 1.7 In vivo Ileostomy
Studyf
31.3 28.4 48.3
ª Values provided by the sponsor (g/50 g total carbohydrates) and fiber analyzed based on AOAC Official Method 991.43 b Fiber (g) = 50 g total carbohydrates – avCHO (g) c Mean BGR values of equations 5 and 5a d Fiber values are the CHOD values from ileal effluent calculated from proximate analysis e Fiber values are adjusted (Fiber (g) = Calculated CHOD value (g) with starch – calculated CHOD value (g) on control day) f avCHO (g) = 50 g total carbohydrates – fiber (g) (CHOD value from ileal effluent calculated from proximate analysis)
Abbreviations: HM – Hi-maize® 260, HY – Hylon® VII, AM – Amioca
72
Figure 10 – Amount of Fiber and Available Carbohydrates by Different Methods:
5.3.8: Total Bacterial Count Determined by Real-Time Quantitative
Polymerase Chain Reaction
The total bacterial count (TBC) of the ileal effluent after each treatment is shown in Table 19
below. The TBC after each treatment indicated a mean value of 109 colony forming units
(CFU)/g of ileal effluent, substantially higher than the amount of total bacteria in the small
intestines of healthy subjects (<104 (CFU)/ml of intestinal fluid). A two way RM-ANOVA was
performed on the values and a significant main effect of subjects (F=49.5, p=0.02), treatment
(F=37, p=0.007) and subject x treatment interaction (F=37.4, p=0.0002) were observed.
AHMinvitro
HMBGR
HMileo
HYinvitro
HYBGRHYile
o
AMinvitro
AMBGR
AMileo
FHMinvitro
HMBGR
HMileo
HYinvitro
HYBGRHYile
o
AMinvitro
AMBGR
AMileo
0
5
10
15
20
25
30
35
40
45
50
Car
bohy
drat
es (g
) per
50g
tCH
O p
ortio
n of
Pro
duct
In vitroIn vivo BGRIn vivo Ileostomy
HM HY AM HM HY AMTreatment
Fiber Available Carbohydrates
A) Bars represent the mean fiber and available carbohydrate content of each treatment by each method B) Abbreviations: HM – Hi-maize® 260, HY – Hylon® VII, AM – Amioca
73
For AR001, there were significant differences between control and HM, HM and HY and HM
and AM (p≤0.0001) and control and AM (p≤0.05). No significant differences were observed for
AR002 and AR003 among the treatments and no significant differences were observed for all 3
subjects after each treatment.
Table 19 – Total Bacterial Count of Ileal Effluent:
Treatment Subject Control HM HY AM Mean±SD AR001 1.8x1010a 7.7x109b 2.0x1010ad 2.05x1010cd 1.64x1010±6.0x109
Mean±SD 6.2x109±9.8x109 2.9x109±4.1x109 7.3x109±1.1x1010 7.6x109±1.1x1010 Values are expressed as CFU/g of ileal effluent Values with different letters differ significantly Abbreviations: HM – Hi-maize® 260, HY – Hylon® VII, AM – Amioca 5.3.8.1: Comparison of Log Transformed Values of Total Bacterial Count
The log transformed values of TBC for each subject after each treatment are shown in Table 20
below. A two-way RM ANOVA was performed on the data with subjects being the largest
source of variation (93.1%) (p<0.0001). There was a significant difference between log
transformed values of control and HM for Subject 1 (AR001) (p≤0.01), control and HY for
Subject 2 (AR002) (p≤0.01) and Subject 3 (AR003) (p≤0.05) and control and AM for Subject 2
(AR002) and Subject 3 (AR003) (p≤0.01). No significant differences were found among the log
transformed values after each treatment (F=0.84, p= 0.54).
74
Table 20 – Comparison of Log Transformed Values of Total Bacterial Count:
Values expressed as log count/g of ileal effluent Values with different letters differ significantly Abbreviations: HM – Hi-maize® 260, HY – Hylon® VII, AM – Amioca
5.3.8.2: Correlations Between Total Bacterial Count and Carbohydrate Measure
5.3.8.2.1: Correlation Between Total Bacterial Count and Resistant Starch
The correlation between TBC and RS is shown in Figure 11 below. Two correlations were
performed; one for the treatments that contained a higher content of RS (HM and HY) and the
other for the treatments that contained minimal or no RS (AM and control). For HM and HY,
the Pearson correlation coefficient r was 0.9646 (95% CI = 0.7049 to 0.9963 and R2 = 0.9305)
indicating a strong positive association between TBC, HM and HY. The association was
significant at two-tailed (p=0.002) and TBC accounted for 93% of the variation in the RS
measure for HM and HY ((0.9646)2 x 100% = 93%). The linear regression equation was
y=3.829x – 29.81 and the slope had a significant deviation from zero (F=53.56, p=0.002).
For AM and control, the Pearson correlation coefficient r was 0.9216 (95% CI = 0.4363 to
0.9915 and R2 = 0.8493) indicating a strong positive association between TBC, control and AM.
The association was significant at two-tailed (p=0.009) and TBC accounted for 85% of the
variation in the RS measure for AM and control ((0.9216)2 x 100% = 85%). The linear
regression equation was y=0.4134x – 3.235 and the slope had a significant deviation from zero
(F=22.54, p=0.009).
Figure 11 – Correlation Between Total Bacterial Count and Resistant Starch:
5.3.8.2.2: Correlation Between Total Bacterial Count and Carbohydrates by Difference
The correlation between TBC and CHOD is shown in Figure 12 below. Two correlations were
performed; one for the treatments that contained a higher content of dietary fiber (HM and HY)
and the other for the treatments that contained minimal or no dietary fiber (AM and control). For
HM and HY, the Pearson correlation coefficient r was 0.4955 (95% CI = -0.5286 to 0.9322 and
R2 = 0.2455) indicating a moderate positive association between TBC, HM and HY. The
association was not significant at two-tailed (p=0.32) and TBC accounted for 25% of the
variation in the CHOD measure for HM and HY ((0.4955)2 x 100% = 25%). The linear
regression equation was y=2.768x + 5.019 and the slope was not significantly deviated from zero
(F=1.3, p=0.32).
RS values are unadjusted Abbreviations: HM – Hi-maize® 260, HY – Hylon® VII, AM – Amioca
7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.00
1
2
3
4
5
6
7
8
9
10
Total Bacteria (log count/g of ileal effluent)
Res
ista
nt S
tarc
h (g
)
1
1
1
12
22
2
33
33
Subject
r=0.9216
1 - AR0012 - AR0023 - AR003
Treatment
HMHYAM
r=0.9646
Control
76
For AM and control, the Pearson correlation coefficient r was 0.8068 (95% CI = -0.0139 to
0.9780 and R2 = 0.6509) indicating a strong positive association between TBC, control and AM.
The association was not significant at two-tailed (p=0.052) and TBC accounted for 65% of the
variation in the CHOD measure for AM and control ((0.8068)2 x 100% = 65%). The linear
regression equation was y=4.426x – 29.71 and the slope was not significantly deviated from zero
zero (F=7.5, p=0.052).
Figure 12 – Correlation Between Total Bacterial Count and Carbohydrates by Difference:
CHOD values are unadjusted Abbreviations: HM – Hi-maize® 260, HY – Hylon® VII, AM – Amioca
7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.00
5
10
15
20
25
30
35
40
Total Bacteria (log count/g of ileal effluent)
Car
bohy
drat
es b
y D
iffer
ence
(g)
r=0.8068
1
11
1
2
2
2
2
3 3
3
r=0.4955
3
Treatment
Subject1 - AR0012 - AR0023 - AR003
HMHYAM
Control
77
5.4: Discussion
Our results showed that dietary fiber content determined in vitro did not reflect the dietary fiber
content in vivo based on the results from the BGR and ileostomy studies. Even though results
from the ileostomy study were different from the BGR study, the conclusion remained consistent
that in vitro analysis underestimated fiber in HY and AM and overestimated fiber in HM.
The amount of ileal effluent collected from each subject was quite variable but a consistent
pattern was observed (Table 12). Subject 1 (AR001) had the highest output for both the control
and starch test days, followed by Subject 3 (AR003) and Subject 2 (AR002) had the lowest
output. Generally, all the subjects experienced lower output than usual due to prolonged GI
transit time but one subject experienced this more than the others. Subject 2 (AR002) initially
described her daily output as “very active”; however, repeatedly mentioned that her output
during the visits was very low due to the fact that her usual diet had three times more fiber than
what was provided in her meal plan. This likely caused a disturbance in her small intestinal
motility and subsequently, a great increase in her GI transit time. To clarify, Subject 2’s
(AR002) first visit consisted of AM as the treatment that she consumed at 8:30am. Due to low
output, her first collection was at 5:00 pm and we managed to collect 103 g of effluent in a 10
hour period compared with 690 g from AM for Subject 1 (AR001) and 231 g for Subject 3
(AR003). The mean±SD amount of ileal effluent collected for the control test days was
404.0±282.3 g, well within the range for healthy ileostomates reported previously by Englyst &
Cummings (1985), Englyst et al. (1996) and McNeil et al. (1982).
The mean percent dry matter produced in 10 hours (Table 14) for the control test days was
previously reported to be between 7.5-9% (Englyst & Cummings 1986; Englyst et al., 1996;
Silvester et al., 1995) and for the high-RS treatment, it was reported to be between 10-12%
78
(Englyst et al., 1996; Langkilde et al., 2002; Silvester et al., 1995). The mean percent dry matter
produced in 10 hours for the control test days was 9.2% and 15.7% for the high-RS starch (HM),
values higher than those previously reported. One explanation for the higher mean percent dry
matter values is because the control diets in this study had a mean of 8 g of fiber compared to 0 g
of fiber reported for the control diets in previous ileostomy studies (Englyst & Cummings, 1985;
1986; 1987). Hence, at baseline, the fiber values for the control diet were higher, leading to a
higher percentage of dry matter for all treatments. Although the control diets were required to be
polysaccharide-free, the source of fiber was from the prepackaged food items available on the
menu for the subjects’ meal plans. Great efforts were made by the study coordinator to provide
menu items with 0 g fiber; however, this proved to be difficult as prepackaged food items were
necessary for this study to reduce food handling.
As for proximate analysis (Table 15), some of the results of the protein and fat during the starch
test days were less than that from the control day whereby an adjustment results in negative
values. This can be largely due to the subjects’ prolongation in GI transit time caused by the fine
particle size of RS as previously reported by Cummings et al. (1996); hence, suggesting
macronutrient hyperabsorption in the small intestines.
Englyst et al. (1996), Schweizer et al. (1990) and Silvester et al. (1995) reported that the mean
RS recoveries in ileal effluent were 95-100% of what was fed to the subjects. When comparing
the amount of RS fed (in vitro value determined by AOAC 991.43) to the adjusted amount of RS
recovered (in vivo value determined by AOAC 2002.02), 13.4% and 43.3% of the RS in HM and
HY respectively were recovered in ileal effluent and AM contained more RS (0.14 g) than
accounted for in vitro (0 g) (Table 16). Our percentages were much less than what was
previously reported indicative that the in vitro method AOAC 991.43 did not accurately reflect
the true RS content of the investigational products. The comparison would have been much
79
more meaningful had the initial in vitro method been specific for RS content, i.e.: comparing in
vitro and in vivo values of RS both determined by AOAC 2002.02 method. Similarly, when
comparing the amount of CHOD fed to the adjusted amount of CHOD recovered, 57% and 187%
of the CHOD in HM and HY respectively were recovered in ileal effluent and AM contained
more CHOD (1.72 g) than accounted for in vitro (0 g) (Table 17). As claimed by the sponsor,
the dietary fiber component of the RS products is entirely from an RS source. If this claim is
true, the CHOD values and RS values for each treatment should be equal, i.e.: RS entirely
constitutes the dietary fiber component of the investigational products. Comparing CHOD and
RS values showed that HM and HY contained 4.2 and 4.3 times more CHOD than RS in the
products. A possible explanation for this phenomenon is that the RS in the products was partly
digested in the small intestine so was no longer analyzed as RS but was still considered CHOD.
This sheds light on the importance of measuring carbohydrates in novel starch products using a
combination of general and specific methods to detect any differences in total carbohydrates vs.
RS fraction and thus, reducing the risk of inaccurate fiber labeling.
Table 18 gives the final fiber and avCHO values for each treatment for each method (in vitro, in
vivo BGR study and in vivo ileostomy study). No one single method produced the highest fiber
or avCHO values for the treatments. The fiber values from the BGR study were not in line with
the fiber values from the ileostomy study. For example, the fiber value for HM from the
ileostomy study was 51% higher than the value from the BGR study. In terms of variation, the
%CV of the fiber values from the BGR and ileostomy studies were as follows: 29% for HM,
44% for HY and 81% for AM. As described in the discussion of Chapter 4 (section 4.4),
accurate knowledge of the product’s GI was necessary to produce accurate avCHO values which
in terms produce accurate fiber values. A slight increase or decrease in the product GI
calculation can result in big differences in the avCHO content of the products, affecting fiber
80
values. This is one possible scenario that can explain the variation in the fiber values of the BGR
and ileostomy studies.
There was an interest in the ileostomy study to determine the quantity of total bacteria in the
ileum of the subjects. It was reasoned that a strong negative correlation between TBC, CHOD
and RS may provide an explanation to lower amounts of recovered CHOD and RS in effluent
due to bacterial fermentation. The mean TBC after each treatment was 109 CFU/g of ileal
effluent and for the subjects, Subject 1 (AR001) had the highest TBC, 1010 CFU/g of ileal
effluent followed by Subject 3 (AR003), 109 CFU/g of ileal effluent and last, Subject 2 (AR002)
with 108 CFU/g of ileal effluent. TBC calculated for each treatment and for each subject
individually all warrant a diagnosis of small intestinal bacterial overgrowth (SIBO), defined as
>105 CFU/ml of intestinal fluid (DiBaise, 2008) and the subjects’ TBC were all substantially
higher than those of healthy patients (<104 CFU/ml of intestinal) (Quigley & Quera, 2006).
The TBC was transformed into log values and there was no consistent pattern observed with the
log transformed values, i.e.: they did not increase or decrease in the same manner in response to
a single starch treatment. All correlations between TBC, CHOD and RS were positive
confirming that SIBO was not a confounding factor in this study, i.e.: TBC had no effect on the
recovered CHOD and RS values.
The end of this discussion raises an important question: How reliable are in vitro methods for
determining the carbohydrate content of manufactured starch products? The answer is not simply
‘reliable’ or ‘not reliable.’ The answer needs to take into account which type of in vitro method
is employed to measure the carbohydrate fraction of interest. If the in vitro method takes into
account all dietary fiber components, ex: AOAC 2009.01 ITDF method, and is complemented
with a secondary method that measures the specific carbohydrate fraction of interest, ex: AOAC
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2002.02 RS method, then the methods can be considered reliable and the food manufacturer need
not pursue further in vivo investigations. However, if the in vitro method has been proven to be
incompetent for the measurement of the specific carbohydrate fraction like RS, relevant in vitro
methods or in vivo methods can be used in order to produce an accurate measure of the fraction
of interest. In this case, AOAC 991.43 was not the relevant in vitro method to measure the TDF
content of the 3 investigational starch products.
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Chapter 6.0 Conclusion and Implications
6.0: Conclusion and Implications
The first hypothesis of this project was that the AOAC Official Method 991.43 initially used in
vitro to determine the total dietary fiber content of the three investigational starch products will
underestimate the total dietary fiber content of the products by underestimating RS content. It is
concluded that the in vitro AOAC 991.43 method greatly underestimated the fiber content in HY,
marginally underestimated the fiber content in AM; however, overestimated the fiber content in
HM. Our first hypothesis was true for HY and AM but not for HM. The first hypothesis also
stated that the results from the BGR study will be accurately reflected in the ileostomy study, but
the results from the BGR study were not in line with the results from the ileostomy study due to
the sensitivity of the BGR method to slight changes in GI.
The second hypothesis stated that ileostomates will exhibit a higher quantity of total bacteria in
their ileum and it is concluded that ileostomates exhibited a higher than normal quantity of
bacteria in their ileum compared to data from healthy subjects in the literature.
The third hypothesis stated that a strong negative association between TBC, CHOD and RS will
provide an explanation for an underestimation in these components and it is concluded that since
the associations were positive, high TBC in the ileum of the ileostomates did not have an effect
on these components.
The main implication of this project reflects the importance of accurate dietary fiber labeling of
novel RS products. For manufacturers, it is crucial to identify the relevant general (ex: AOAC
2009.01 ITDF method) and specific (ex: AOAC 2002.02 RS method) in vitro methods used to
accurately determine the amount of RS in novel RS products. Accurate dietary fiber labeling for
83
RS products is not only important for the food ingredient itself, but for accurate labeling of the
food products which incorporate the RS products. For example, in order to obtain a significant
reduction in calories or an increase in dietary fiber per serving of cookies, 25-50% of refined
flour must be replaced by an RS ingredient (Haynes et al., 2013) such as HM. If one cookie
accounts for one serving and the average size of the cookie is 12g, 4g of HM (33%) is required to
satisfy a “high source of fiber” claim (Food and Drug Regulations (C.R.C., c.870, page 52),
2014). If the same HM product used in this study was used as an ingredient in these cookies, 4g
of HM would not have provided 4g of fiber in the form of RS because data from the ileostomy
study showed that only 13.4% (Table 17) of the RS fed was recovered. This would mean that
only 0.5g of the 4g of HM used could actually be considered as RS. Hence, since these cookies
do not satisfy the requirements for a “high source of fiber” claim, they are considered
mislabeled, i.e.: if a label is false or misleading in any particular way such as in product
composition (Consumer Packaging and Labelling Act (R.S.C.. 1985, c. C-38, page 3), 2014).
Accurate dietary fiber labeling is also essential for the health of people who suffer from chronic
diseases such as type 1 diabetes. If an individual with type 1 diabetes were to purchase a high-
RS containing product that really contains more avCHO than fiber, it may affect the amount of
insulin they need to inject or the amount of carbohydrates they need to consume along with the
RS product, i.e.: disturbing the delicate balance between carbohydrate counting and type and
amount of injected insulin units (National Diabetes Information Clearinghouse, 2014).
There is also a need for tighter regulations on a global basis on the methods used to determine
the total amount of dietary fiber in RS containing products for labeling purposes. Fortunately in
Canada, Health Canada in consultation with the Canadian Food Inspection Agency (CFIA) has
made it mandatory that the TDF content on nutrition labels needs to be determined by AOAC
84
2009.01 method because it takes into account not only the RS fraction in its entirety, but all the
other dietary fiber fractions as well (Health Canada, 2012).
The overall conclusions and implications of this project can be summed up in the following
points:
1) The new in vivo BGR method seems promising but might be carbohydrate specific, i.e.:
produces accurate results with some sources of carbohydrates and not others
2) Difficulty in finding conventional ileostomates for this project further emphasizes the
need for a new in vivo method for the determination of the avCHO and fiber content of
RS products
3) Manufacturers need to identify the relevant general and specific methods used to measure
the dietary fiber fraction of interest in their products
4) Wider recognition from health authorities that RS is one type of dietary fiber will call for
stricter regulations on the methodology used for determining the fiber content for
products high in RS content; thus, benefitting all consumers
85
Chapter 7.0 Limitations and Future Directions
7.0: Limitations and Future Directions
One of the limitations of study 1 was the sensitivity of the BGR method to slight changes in GI.
For the future, the method needs development in such a way where slight changes in GI won’t
produce great changes in avCHO content of the products of interest.
As for study 2, it was true that the sample size was small but it was sufficient to detect
statistically significant differences among the treatments as described in the results section 5.3.
Another limitation of study 2 was providing prepackaged food items as part of the study meal
plan in order to minimize food handling. As a result, finding prepackaged food items with 0g
fiber to satisfy the ‘polysaccharide-free control diet’ requirement was difficult. This limitation
could have been avoided had the study been conducted in a metabolic kitchen where
macronutrient content can be strictly regulated. The last limitation of study 2 was the time at
which the effluent was collected. We initially planned to collect effluent every 2 hours;
however, as a result of pooling the samples in the end, we decided to collect effluent when the
pouch was one third full. A change in diet as well as the addition of starch to the ileostomates
diet greatly increased their GI transit time and the first effluent collection was usually around 1
pm (starch was consumed at 8:30am). The ileostomates mentioned that by 1 pm on regular days,
they would have had to empty their pouches at least once if not more. This essentially means
that the collection of ileal effluent every 2 hours to prevent further enzymatic breakdown and
fermentation of the organic matter in the pouch wouldn’t have even been possible due to low
output.
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The starch products had their own limitations too. It would have been best if a combination of
the relevant general (AOAC 2009.01) and specific (AOAC 2002.02) methods were used to
measure the dietary fiber and resistant starch fraction, respectively, of the products. Since we
used the AOAC 2002.02 method to measure the RS fraction in ileal effluent, it would have been
interesting to compare this value with the value obtained in vitro had this method been initially
used to determine RS content of the starch products. For study 2, we compared the RS value
obtained in vivo, and measured by AOAC 2002.02 method, with the fiber value obtained in vitro,
and measured by AOAC 991.43 method. Thus, we were comparing a specific method for a
dietary fiber fraction with a general method for the total measurement of dietary fiber and
whether this comparison is valid or not remains questionable.
As for future directions, it would be interesting to compare RS values when the chemical
analysis remains constant, i.e.: AOAC 2002.02, and the method of starch digestibility is variable,
i.e. in vitro vs. in vivo. For example, AOAC 2002.02 is used to measure the RS fraction in a
food product in vitro and this value is compared to the RS value recovered in vivo from ileal
effluent, also measured by AOAC 2002.02. Moreover, feedback from the ileostomates focused
on an interest in the role of RS, by its ability to prolong GI transit time, in the enhancement of
the athletic performance of athletes with an ileostomy and this can be extended to ileostomates in
the general public who are active outdoors (ex: camping, hiking, swimming and horseback
riding). This can also be applied to ileostomates who enjoy traveling and are required to be on
long plane rides where they can sleep comfortably without stressing about their pouches getting
too full and leaking. Increasing the confidence and quality of life of ileostomates by adding RS
to their daily diets is a great example of knowledge translation and customer benefits.
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Appendix 1
Ileostomy Study Menu
Subjects can choose from a variety of food items on this menu. Subjects can choose from 1, 2 or all categories of each meal as desired.
Food items chosen and the amount desired by the subjects will remain constant for all 4 test days. Please feel free to ask the study staff questions about any of the food items listed below.
Meal Category Food Item* Amount Energy (cal)
Carbs (g)
Fiber (g)
Starch (g)
Sugars (g)
Protein (g)
Fat (g)
Dinnera Frozen Entréesb
Meat Options
Vegetarian
Options
Healthy Choice Gourmet Steamers: Grilled Balsamic
Chicken
Per entrée 283g
320 41 4 N/Ac 10 21 8
Blue Menu Italian Lasagna
Per entrée 320g
340 46 4 N/A 9 20 8
Blue Menu Roasted Vegetable
Lasagna
Per entrée 300g
310 46 5 N/A 10 15 7
Blue Menu Macaroni & 3
Cheeses
Per entrée 300g
360 50 5 N/A 4 20 9
Dessertb President’s Choice Banana Cake with Chocolate Chips
Per 1 cake 30g
130 16 1 N/A 10 2 6
President’s Choice Brownies with Dark
Chocolate Chips
Per 1 brownie 35g
150 23 1 N/A 16 2 5
Fruit Juiceb Minute Maid 100% Apple Juice
from Concentrate
1 package 200ml
90 23 N/A N/A 21 0.3 0
Minute Maid 100% Orange Juice
from Concentrate
1 package 200ml
100 23 N/A N/A 20 1 0
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Meal Category Food Item* Amount Energy (cal)
Carbs (g)
Fiber (g)
Starch (g)
Sugars (g)
Protein (g)
Fat (g)
Breakfast Test meal is consumed during breakfast by mixing it with waterd, milke, yogurt, pudding, fruit juice or the meal-replacement
Breakfast Fruit Juiceb Minute Maid 100% Apple Juice
from Concentrate
1 package 200ml
90 23 N/A N/A 21 0.3 0
Minute Maid 100% Orange Juice
from Concentrate
1 package 200ml
100 23 N/A N/A 20 1 0
Meal-Replacementb
Ensure Nutrition Shake
Milk Chocolate
1 bottle 240ml
250 40 1 N/A 22 9 6
Ensure Nutrition Shake
Vanilla
1 bottle 240ml
250 40 0 N/A 23 9 6
Mid-morning Snack: Subject can choose to snack from any of the following categories: cheese, yogurt or pudding along with fruit juice. They also have the option of choosing a meal-replacement.
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Meal Category Food Item* Amount Energy (cal)
Carbs (g)
Fiber (g)
Starch (g)
Sugars (g)
Protein (g)
Fat (g)
Lunch Sandwichh
Deli Meat Optionsb
Wonder Classic Thin Sandwich Soft White
Bread
2 slices 140 27 1 N/A 5 4 1.5
Blue Menu Oven Roasted Turkey
Breast Fully Cooked
(175g)
3 slices 66g
70 2 0 N/A 0 14 1
Blue Menu Extra Lean Stone
Roasted Ham (175g)
3 slices 53g
60 2 0 N/A 1 11 1
Cheese included in this sandwich is the same cheese or cheese-alternative the subject has chosen for breakfast (8-slice package).
Fruit Juiceb Minute Maid 100% Apple Juice from
Concentrate
1 package 200ml
90 23 N/A N/A 21 0.3 0
Minute Maid 100% Orange Juice from
Concentrate
1 package 200ml
100 23 N/A N/A 20 1 0
* All food items are from Loblaws (60 Carlton Street, Toronto, ON M5B 1L1). a Dinner meal consumed the night before the test day. b Subject will choose 1 option only. c N/A – Information not available. d Water can be consumed as much as needed throughout the day. e Subjects are responsible for providing their own milk, tea, coffee, sugar or sweetener. These items can be consumed as desired throughout the day. f Fruits may vary depending on the season. g Product did not contain a nutrition label and was not found in the Canadian Nutrient File, version 2010. Nutritional information was found in DailyBurn Tracker Food Database (Source: No Author. (2013). Loblaws Fruit Salad. DailyBurn Tracker. Retrieved May 26, 2013, from http://tracker.dailyburn.com/recipes/loblaws_fruit_salad). h Subjects can choose to have one or two sandwiches.
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Appendix 2 Control Diet of Subject 1 (AR001)
Subject 1 (AR001) preferred the inclusion of animal products (i.e.: meat and cow’s
milk) in her meal plan because of the satiating effect of animal proteins. Her meal
plan is outlined in the table below.
Meal Food Itema Amount Energy (cal)
tCHOb (g)
Fiber (g)
Protein (g)
Fat (g)
Dinner Italian Lasagna 320 g 340 46 4 20 8 Apple Juice 200 ml 90 23 N/Ac 0.3 0
Diet Gingerale 355 ml 0 0 0 0 0 Breakfast Cheddar Cheese 78.5 g 353.3 3.9 0 19.6 27.5
Vanilla Pudding 90 g 109.1 18.2 0 0.9 3.2 Coffeed 340 ml 2 0 0 0.4 0.06 2% milk 160 ml 83.2 7.6 0 5.8 3.2
Sugar 15 g 60 15 0 0 0 Snack Orange Juice 200 ml 100 23 N/Ac 1 0 Lunch Thin White
Bread 54 g 140 27 1 4 1.5
Oven Roasted Turkey Breast
48 g 50.9 1.5 0 10.2 0.7
Orange Juice 200 ml 100 23 N/Ac 1 0 Other Coffeed 170 ml 1 0 0 0.2 0.03
2% milk 80 ml 41.6 3.8 0 2.9 1.6 Sugar 7.5 g 30 7.5 0 0 0
TOTAL 1,501 200 5 66 46 a Food item’s nutrient composition taken from nutrition label unless otherwise stated b Total Carbohydrates
c N/A: Not Available d Coffee, brewed / Café infuse (Food Code: 2873). Canadian Nutrient File, 2010 <http://webprod3.hc-sc.gc.ca/cnf-fce/report-rapport.do?lang=eng>.
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Appendix 3 Control Diet of Subject 2 (AR002)
Due to Subject 2’s (AR002) religious beliefs, all food items in her meal plan were
carefully checked and selected by her. Her meal plan is outlined in the table below.
Meal Food Itema Amount Energy (cal)
tCHOb (g)
Fiber (g)
Protein (g)
Fat (g)
Dinner Roasted Vegetable Lasagna
300 g 310 46 5 15 7
Apple Juice 200 ml 90 23 N/Ac 0.3 0 Breakfast Swiss Cheese 38.5 g 121.6 0 0 14.2 6.1
Raspberry Vanilla Yoghurt
90 g 72 10.8 0 4.5 1.8
Apple Strudel 70 g 246.5 27.6 0.99 2.96 13.8 Coffeed 170 ml 1 0 0 0.2 0.03 2% milk 80 ml 41.6 3.8 0 2.9 1.6
Snack Swiss Cheese 52.5 g 165.8 0 0 19.3 8.3 Coffeed 170 ml 1 0 0 0.2 0.03 2% milk 80 ml 41.6 3.8 0 2.9 1.6
Lunch Thin White Bread
54 g 140 27 1 4 1.5
Swiss Cheese 38.5 g 121.6 0 0 14.2 6.1 Apple Juice 200 ml 90 23 N/Ac 0.3 0
Other Coffeed 170 ml 1 0 0 0.2 0.03 2% milk 80 ml 41.6 3.8 0 2.9 1.6
Thin White Bread
54 g 140 27 1 4 1.5
Swiss Cheese 18 g 56.8 0 0 6.6 2.8 TOTAL 1,682 196 8 95 54
a Food item’s nutrient composition taken from nutrition label unless otherwise stated b Total Carbohydrates
c N/A: Not Available d Coffee, brewed / Café infuse (Food Code: 2873). Canadian Nutrient File, 2010 <http://webprod3.hc-sc.gc.ca/cnf-fce/report-rapport.do?lang=eng>.
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Appendix 4 Control Diet of Subject 3 (AR003)
Subject 3 (AR003) suffers from eosinophilic esophagitis and restricts soy, mushroom,
red peppers among other food items in her diet. All the ingredients in her meal plan
were meticulously checked and approved by her before the commencement of her
visits. Her meal plan is outlined in the table below.
Meal Food Itema Amount Energy (cal)
tCHOb (g)
Fiber (g)
Protein (g)
Fat (g)
Dinner Macaroni & 3 Cheese
300 g 360 50 5 20 9
Apple Juice 200 ml 90 23 N/Ac 0.3 0 Breakfast Cheddar Cheese 38.5 g 173.3 1.9 0 9.6 13.5
Chocolate Pudding
90 g 109.1 19.1 0.91 1.8 3.2
Apple Strudel 70 g 246.5 27.6 0.99 2.96 13.8 Blueberriesd 50 g 28.5 7.3 1.3 0.37 0.17 Red Grapese 50 g 34.5 9 0.5 0.5 0 Apple Juice 200 ml 90 23 N/Ac 0.3 0
Snack Cheddar Cheese 38.5 g 173.3 1.9 0 9.6 13.5 Blueberriesd 25 g 14.3 3.7 0.7 0.19 0.09 Red Grapese 25 g 17.3 4.5 0.25 0.25 0 Apple Juice 200 ml 90 23 N/Ac 0.3 0
Lunch Thick White Bread
54 g 135 26.3 1.5 4.5 1.2
Oven Roasted Turkey Breast
48 g 50.9 1.5 0 10.2 0.7
Cheddar Cheese 38.5 g 173.3 1.9 0 9.6 13.5 Apple Juice 200 ml 90 23 N/Ac 0.3 0
TOTAL 1,876 247 11 71 69 a Food item’s nutrient composition taken from nutrition label unless otherwise stated b Total Carbohydrates
c N/A: Not Available d Blueberry, raw / Bleuet, cru (Food Code: 1705). Canadian Nutrient File, 2010 <http://webprod3.hc-sc.gc.ca/cnf-fce/report-rapport.do?lang=eng>. e Grape, red or green (European type, such as Thompson seedless), adherent skin, raw / Raisin, rouge ou vert (Type Européen, comme Thompson sans grains), peau adhérente, cru (Food Code: 1718). Canadian Nutrient File, 2010 <http://webprod3.hc-sc.gc.ca/cnf-fce/report-rapport.do?lang=eng>.