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1
*
Human Nutrition Unit and
Department of Agricultural Chemistry and Soil Science, University of Sydney, NSW, Australia 2006
1To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-amylase (EC
3.2.1.1) and amyloglucosidase (EC 3.2.1.33). Lauric, myristic, palmitic
and oleic acids and lysolecithin inhibited enzymic hydrolysis of
amylose by ~35% (P < 0.05). Stearic acid and
cholesterol had no effect on the enzymic breakdown of amylose.
Retrograded amylose was hydrolyzed less readily (P
< 0.05) than solubilized amylose, but the breakdown was not
further inhibited in the presence of lauric acid. Fatty acids had no
effect on the enzymic hydrolysis of amylopectin, whereas inhibition by
fatty acids of the breakdown of whole starch was consistent with only
the amylose fraction being affected. The possibility that interactions
between starch and fatty acids in the digestive tract could contribute
to the formation of resistant starch is considered.
KEY WORDS: • resistant starch • fatty acids • amylose • amylolysis of starch
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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[1
4]-linked glucan, and
amylopectin, which contains glucose units in [14] and
[16] links. Unbranched [14] glucan chains such as
amylose have a helical conformation and can form inclusion complexes
with a variety of small hydrophobic molecules, including certain types
of lipids. The formation of such complexes may result in significant
changes in the properties of the glucan, including decreased
solubility, increased gelatinization temperature and retarded
retrogradation during storage (Eliasson et al. 1981
, Krog 1971
).
Ingested starch was previously considered to be completely hydrolyzed
to glucose in the upper gut by digestive enzymes. However, it is now
known that a starch fraction, termed resistant starch, makes an
important contribution to dietary fiber because it passes through the
small intestine and is fermented in the hind gut by resident microflora
yielding short-chain fatty acids (SCFA), which can have beneficial
actions on the colon (Annison and Topping 1994
,
Baghurst et al. 1996
, Brown 1996
,
Englyst and Cummings 1982
, Muir et al. 1994
). The amount and form of resistant starch in foods vary
depending on the botanical source of the starch, the molecular
structure of the starch and interactions that may occur between the
starch and other food constituents during processing, cooking and
eating (Englyst and Cummings 1982
, Englyst et al. 1996
, Muir and O’Dea 1992
). One such
interaction that can change the behavior of starch is the formation of
complexes with lipids. Complex formation has been suggested to occur in
situ in the digestive tract (Holm et al. 1983
) and is
thought to decrease the digestibility of starch and modulate the
glycemic response to ingested carbohydrate (Murray et al. 1998
). In this study, we demonstrate that certain fatty acids
inhibit the enzymic hydrolysis of the amylose component of starch. This
inhibition results in the increased availability of [14] glucan
substrates for fermentation in the hind gut.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Potato starch, potato amylose, potato amylopectin, fatty acids,
lysolecithin (egg yolk; predominantly palmitic and stearic acids) and
cholesterol were from Sigma (St Louis, MO). Porcine pancreatic
-amylase (EC 3.2.1.1) was from Boehringer Mannheim (Mannheim,
Germany) and amyloglucosidase (EC 3.2.1.33) was from Megazyme
International (Bray, Ireland).
Enzymic digestions.
Amylose, amylopectin and starch (25 mg) were dissolved in 5 mL of 0.1
mol/L NaOH by heating for 10 min in a boiling water bath with mixing.
The resulting solution was cooled to room temperature and freed of NaOH
with an Econo-Pac 10 DG column (Bio Rad, Hercules, CA) according to
the supplier’s instructions. Neutralized glucan solution (100 µL
containing 0.36 mg of glucan), 400 µL of 0.2 mol/L sodium acetate (pH
4.5) and 10 µL of a methanolic solution of the lipid were incubated
in microcentifuge tubes for 10 min at 37°C before initiating enzymic
digestions by the addition of -amylase (1.4 U) and
amyloglucosidase (0.03 U). The final volume of the reaction
mixtures was 1 mL. Reactions were stopped by heating in a boiling water
bath for 10 min, and free glucose was determined colorimetrically using
the glucose oxidase–peroxidase method as described by Blakeney and Matheson (1984)
. The amylose and amylopectin content of the
starch were determined by iodine binding according to the method of
Chang (1979)
. Significant differences between two
measurements were determined using paired t tests.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). More than 90% of the amylose was converted to glucose in 6 h.
In the presence of lauric acid, 45% of the amylose was hydrolyzed
after 1 h, 50% after 2 h and ~60% after 6 h (Fig. 1)
. In a control experiment, the addition of 10 µL of methanol to the
reaction mixture had no effect on the enzymic breakdown of amylose
(data not shown). Based on results shown in Figure 1
and taking into
account the definition of resistant starch as being that portion of
starch not hydrolyzed after 2 h in an in vitro assay
(Englyst et al. 1996
), we chose an incubation time of
2 h in subsequent experiments to monitor the formation of
enzymically resistant starch.
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). However, neither stearic acid nor cholesterol inhibited the enzymic
breakdown of amylose (Table 1)
, and there was no effect of stearic acid
when the temperature of the enzymic digestion was increased to 50°C
(data not shown).
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). The extent of hydrolysis of retrograded amylose was ~50% after
2 h, and the addition of lauric acid had no further inhibitory
effect (Fig. 2)
. Lauric acid had no effect on the enzymic breakdown of
amylopectin, whereas the breakdown of whole starch was inhibited 12%
by lauric acid (results not shown). This value was consistent with
lauric acid affecting the hydrolysis of the amylose fraction only,
which was determined by iodine binding to be 31% of the starch.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,
Guraya et al. 1997
, Hanna and Lelievre 1975
, Hoover and Hadziyev 1981
, Krog 1971
). Complex formation with monoglycerides has been shown to
inhibit digestion of amylose (Carlsson et al. 1979
,
Guraya et al. 1997
), but information on the effect of
free fatty acids is limited. In this study, complexes were formed by
incubating mixtures of glucans (2.2 µmol glucose units) and fatty
acids (0.04–0.05 µmol) for 10 min at 37°C. Previous studies on
interactions between the saturated 12-carbon lauric acid and amylose
have shown that complexes can form rapidly at 37°C under conditions
resembling those that occur physiologically (Seligman et al. 1998
). Amylose binds one lauric acid molecule per ~20 glucose
units in the glucan chain, but in contrast, very little lauric acid
binds under the same conditions to amylopectin and other branched
glucans (Seligman et al. 1998
). The experiments were
performed at a pH of 4.5 to reflect the moderately acidic pH of
partially digested food as it enters the duodenum and because of the
acidic pH optima of the enzymes used.
The initial rate of glucose release was rapid from amylose alone and
from amylose that was complexed with lauric acid. However after 1 h, hydrolysis of the complexed amylose had slowed down significantly
compared with amylose alone. After 6 h, only 60% of the amylose
complexed with lauric acid had converted to glucose, whereas in the
same time >90% of the uncomplexed amylose had completely hydrolyzed.
Myristic, palmitic and oleic acids and lysolecithin were similar to
lauric acid in their inhibitory effect on the enzymic breakdown of
amylose. However, stearic acid did not inhibit amylose hydrolysis,
which may be because it did not form a complex under the conditions of
our experiments. The hydrolysis of amylopectin was not
inhibited by fatty acids, which is consistent with fatty acids binding
poorly to amylopectin (Guraya et al. 1997
,
Seligman et al. 1998
). Our observations on the
retardation of amylose breakdown by free fatty acids may have
physiological relevance. The duration of our experiments was comparable
to the time frame for the transit of food through the small intestine,
which is considered to be ~6 h (Holgate and Read 1983
).
Starch-containing foods that digest slowly and release glucose for
absorption along the length of the small intestine result in reduced
postprandial glucose and insulin responses (Jenkins et al. 1982
), which could be beneficial for glycemic control
particularly for type 2 diabetics. The amylose content is an important
factor that determines the digestibility of starch, and therefore,
foods that contain starches with a high amylose content are considered
to be effective in producing a lower glycemic response
(Björck et al. 1994
, Goddard et al. 1984
). The susceptibility of amylose and amylopectin to enzymic
attack will be determined by the structural characteristics of these
molecules and may be influenced by retrogradation and complex formation
with small molecules. Amylose has a greater tendency to retrograde than
amylopectin and thereby to regain a semicrystalline structure that is
more resistant to enzyme attack (Berry 1986
,
Biliaderis 1991
). Our results suggest that
retrogradation also reduced the capacity of amylose to bind fatty
acids. Further, amylose forms inclusion complexes more readily than
amylopectin, which can also reduce digestibility. Foods may contain
small amounts of free fatty acids that could complex with amylose
during eating. Complexes could also form in the small intestine by the
interaction of linear [14] glucan fragments from partially
digested amylose and amylopectin with free fatty acids released from
triglycerides by the action of lipases. The apparent digestibility of
linear glucose oligomers, produced by debranching of amylopection, is
decreased in ileal-canulated dogs by mixing with monoglycerides of
stearate and palmitate (Murray et al. 1998
). We suggest
that glucan-fatty acid complexes with increased resistance to
enzymic hydrolysis can form rapidly at 37°C and the breakdown of
glucan in these complexes could be retarded sufficiently for it to
contribute to the resistant starch component of dietary fiber.
Manuscript received December 1, 1999. Revision accepted March 30, 2000.
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REFERENCES |
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1 h.