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The importance of an oral digestion step in evaluating simulated in vitro digestibility of starch from cooked rice grain
Accepted Manuscript The importance of an oral digestion step in evaluating simulated in vitro digestibility of starch from cooked rice grain
Masatsugu Tamura, Yumi Okazaki, Chisato Kumagai, Yukiharu Ogawa PII: DOI: Reference:
S0963-9969(17)30030-3 doi: 10.1016/j.foodres.2017.01.019 FRIN 6575
To appear in:
Food Research International
Received date: Revised date: Accepted date:
1 October 2016 23 January 2017 24 January 2017
Please cite this article as: Masatsugu Tamura, Yumi Okazaki, Chisato Kumagai, Yukiharu Ogawa , The importance of an oral digestion step in evaluating simulated in vitro digestibility of starch from cooked rice grain. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Frin(2017), doi: 10.1016/j.foodres.2017.01.019
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Title The importance of an oral digestion step in evaluating simulated in vitro digestibility of starch from cooked rice grain
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Running title
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Effect of mastication on cooked rice digestibility
Authors’ name
Faculty of Agriculture, Utsunomiya University, 350, Mine, Utsunomioya
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1
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Masatsugu Tamura1, Yumi Okazaki2, Chisato Kumagai3, Yukiharu Ogawa3
321-8505, Japan
Faculty of Horticulture, Chiba University, 648, Matsudo, Matsudo
3
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271-8510, Japan
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2
Graduate School of Horticulture, Chiba University, 648, Matsudo,
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Matsudo 271-8510, Japan
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Corresponding author TEL&FAX: +81-47-308-8848; EMAIL: [email protected]
Keywords Cooked rice, Mastication, Simulated digestion, Kinetic parameters, Tissue structure 1
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Highlights Oral digestion step plays important role in evaluation of the starch digestibility Structural changes in grain-scale impacts on starch digestibility of
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rice grain
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Food structure greater acts starch hydrolysis than salivary
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α-amylase concentration
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Abstract To examine the effect of oral digestion step in a simulated in vitro starch digestion model, the digestibility of intact, homogenized and actual chewed cooked rice grains was investigated and analyzed. The kinetics of
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starch digestibility were calculated from changes in the hydrolysis percent
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of starch that were achieved during simulated small intestinal digestion
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stage. Morphological and histological microscopic tissue structures were also examined. Compared with the trend of starch hydrolysis changes of
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the actual chewed grain, 1.3 U/ml of salivary α-amylase concentration treated for 60 min was regarded as a mimicked condition to the simulate in
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vitro oral digestion step in this study. The results showed that the equilibrium percent of starch hydrolysis for all of the samples ranged from
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84.2 % to 95.9 % with no significant differences observed regardless of
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whether the oral digestion step was included (p > 0.05). In contrast, the kinetic constant, which is one of the measure of starch digestion rate
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during small intestinal stage, significantly increased with the degree of grain homogenization increased: 120 s > actual chewed ≥ 1 s > intact, for
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both the gastrointestinal and oral plus gastrointestinal processes. These results indicated that the kinetic constant was influenced by the change of cooked rice grain structure in oral digestion step that would be related to increase in enzyme accessibility to rice starch. Thus, rice grain digestibility was affected by grain-scale structural changes, including grain tissue damages which were normally observed during the oral digestion step.
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1. Introduction A rapid postprandial increase in blood glucose levels is normally observed following the rapid ingestion of carbohydrate-rich foods. When this type of response is consistently induced, there is an
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increased risk of acquiring life-style related diseases such as type 2
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diabetes, hyperlipidemia, and cardiovascular disease (Jenkins et al.,
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2002; Lehmann and Robin, 2007). In contrast, a slow rate of ingestion of starchy foods may avoid potential problems and may
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have health benefits. Digestibility of the starches associated with carbohydrate-rich foods is also a relevant consideration.
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To investigate the digestibility of starchy foods, both in vivo animal models and in vitro simulated models have been developed to
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represent the gastrointestinal digestion system. Recently, the ease of
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using an in vitro simulated digestion process was demonstrated, as well as its flexibility regarding experimental conditions and its
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capacity for observing changes in food attributes (Tamura et al., 2016a, 2016b). Generally in vitro approaches consist of a two-stage
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simulated digestion system in which a gastric stage is established with a low pH and pepsin, and a subsequent small intestinal stage is established with a neutral pH and intestinal enzymes (Dartois et al., 2010). However, an oral digestion step, which is an intrinsic system for the human body, has typically not been considered for in vitro studies of starchy food digestibility because the food samples are often prepared as a powder or a slurry in order to reduce the 4
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experimental time (Englyst et al., 1992). Oral digestion as the first step of food digestion has two simultaneous actions: chewing to provide mechanical grinding and enzymatic starch hydrolysis that is mediated by the saliva. After food
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is crushed into small pieces and mixed with saliva with chewing, then
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amylase that is present in the saliva is able to act on the starch in the
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food. Hoebler et al. (1998) reported that approximately 25 % of the starch in pasta and 50 % of the starch in bread are hydrolyzed during
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oral starch digestion, thereby making a portion of the starch available for further enzymatic digestion in the gastrointestinal process.
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Bornhorst and Singh (2013) also demonstrated that α-amylase from saliva plays an important role in the breakdown kinetics of bread
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boluses in in vitro models. Meanwhile, Woolnough et al. (2010)
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reported that prolonged exposure to saliva during the chewing of starchy foods by volunteers did not contribute to the hydrolysis of
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starch during the gastrointestinal digestion process. Strahler et al. (2010) further reported that the concentration of salivary α-amylase
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in humans varies with age, psychosocial stress, and other factors, all of which were regarded as highly subjective according to individual conditions.
Rice (Oryza sativa L.) is a major source of carbohydrates, especially in Asian countries. Since rice is cooked and consumed as a whole grain, the structural attributes of cooked rice grain influence the digestibility of its starch components (Tamura et al., 2016a, 2016b). 5
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However, oral digestion approaches have not been considered and applied to the simulated digestion model for cooked rice grain. In this study, the effects of mimicked oral digestion conditions, including varying
degrees
of
sample
homogenization
and
varying
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concentrations of α-amylase in simulated salivary fluid, on a
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simulated in vitro starch digestion model were investigated and
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analyzed in relation to the digestibility of cooked rice starch.
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2. Materials and Methods 2.1. Materials
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Precooked steamed rice packaged as a ready-to-eat meal for the microwave oven (Wooke, Toyama, Japan) was purchased. The rice
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cultivar used in the product was “Koshihikari” and it was harvested in
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Nyuzen, Toyama, Japan in 2013. Alpha-amylase (porcine pancreas, type VI-B, ≥ 10 U/mg, solid, A3176-500KU), pepsin (P7000, porcine
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gastric mucosa, ≥ 250 U/mg, solid), pancreatin (hog pancreas, 4× USP), and invertase (from baker’s yeast, grade VII, ≥ 300 U/mg,
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solid) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Amyloglucosidase (3,260 U/ml) was purchased from Megazyme International Ireland (Wicklow, Ireland).
2.2. Sample preparation To prepare cooked grain samples, 60 g of pre-cooked steamed rice was put in a Ziplock bag and heated in a microwave oven 6
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(RE-SX10-W, Sharp, Osaka, Japan) at 500 W for 50 s. The heated sample was then cooled in an incubator (A0601-2V, Keepit, Nagano, Japan) at 30 °C for 30 min. To obtain mechanically crushed grain samples as an imitative
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chewed model, the cooked grain samples were homogenized using
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a household blender (THM310, Tescom, Tokyo, Japan) for 1 s or for
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120 s. In addition, an actual chewed grain model was prepared as previously described (Akerberg et al., 1998; Beer et al., 1997;
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Germaine et al., 2008; Woolnough et al., 2010). Two volunteers, who were early twenties and thirties and have normal dentition, were
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employed in this study. They brushed their teeth before chewing. Briefly, 20 g of a grain sample (approximately 6.8 g of starch content
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equivalent) was bitten 15 times in 15 s by each volunteer for two
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replicates. During actual chewing, grain sample was crushed and mixed with salivary α-amylase. The sample was then expectorated
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and left to sit as chewed boluses. A small portion of the cooked grain samples were freeze-dried
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(FDU-1100, Eyela, Tokyo, Japan), then ground using a household mixer (MR-280, Yamazen, Tokyo, Japan) and sieved using a 0.5 mm meshed sieve. The powdered material obtained was used to measure total starch (TS) and resistant starch (RS) contents using a resistant starch kit (K-RSTAR 08/11, Megazyme International).
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2.3. Moisture, amylose, and protein content The cooked grain samples were dried using a hot air oven (WFO-400, Eyela) at 135 °C, and after 24 h, the moisture content in wet basis (w.b.) was calculated. The sample grain was ground using a
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household mixer (MR-280, Yamazen) and was passed through a 0.5
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mm sieve to produce a sample powder. The apparent amylose
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content of the powder was then measured as previously described (Tamura et al., 2014b). Nitrogen content of the powder samples were
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measured by using a CN Corder (MT-700, Yanaco, Kyoto, Japan). Crude protein content was calculated from the measured nitrogen
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content with a nitrogen-protein conversion factor of 5.95. Hippuric acid (200-37032, Kishida Chemical, Osaka, Japan) was used as a
2.4. Microscopy
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standard for the nitrogen measurements.
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Grain shape was observed with a digital microscope (VH-Z-05 & VH-8000, Keyence, Osaka, Japan). Projected area was calculated
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from a number of pixels that represented grain portion after image binarization. Approximately 500 grains or grain fragments were randomly selected from the cooked grain sample and were manually arranged so that they did not touch each other on the observation stage of the microscope. To visualize the histological tissue structures of whole size rice grain, the autofluorescent imaging technique was applied (Ogawa et al., 8
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2003a; Tamura and Ogawa, 2012). To obtain morphological and histological images of the grains, a fluorescent stereomicroscope (MZ-FLIII, Leica, Wetzlar, Germany) equipped with a digital camera (DS-5M, Nikon, Tokyo, Japan) was used. The microscope had a 100
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W mercury arc lamp (ebq 100 dc, Leica), an ultraviolet fluorescence
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filter set (360/40 nm excitation filter, 420 nm barrier filter, Leica) for
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histological tissue observation, and a halogen lamp for morphological tissue examination. All imaging parameters, including focusing,
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lighting, and shuttering, were standardized for each sample. The captured images were processed and analyzed with Photoshop
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CC2014 software (Adobe, San Jose, CA, USA).
distribution
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Particle size
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2.5. Particle size
for
the
grain
samples
that
were
homogenized for 120 s was determined using a laser diffraction
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particle size analyzer (SALD-3100, Shimadzu, Kyoto, Japan). The
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applied relative refractive index was 1.70.
2.6. Simulated digestive fluid Simulated salivary fluid (SSF) was prepared as previously described (Beer et al., 1997; Tamura et al., 2013) and examined preliminarily. Briefly, 5 mg of α-amylase was dissolved in 1 ml of 0.036 mol/l calcium chlorite as a standard SSF in which the final α-amylase concentration was 0.3 U/ml. Two additional SSF standards were 9
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generated with 10 mg and 20 mg of α-amylase, resulting in final α-amylase concentrations of 0.7 U/ml and 1.3 U/ml, respectively. Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared according to the method of Dartois et al. (2010). The SGF
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contained pepcine. The SIF contained pancreatine, invertase, and
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amyloglucosidase.
2.7. Simulated in vitro gastrointestinal digestion
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A simulated in vitro gastrointestinal digestion model was established as previously described (Dartois et al., 2010). Briefly, 170 g of rice
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sample mixture with distilled water, which was prepared to contain 4 % of total starch, was added into a 500 ml jacketed glass reactor
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and continuously stirred with a magnetic stirrer (Color Squid, Ika,
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Staufen, Germany) at 350 rpm. When intact grain samples or grain samples homogenized for 1 s were placed in the reactor, they were
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placed in a polyethylene mesh to prevent direct contact between the samples and the magnetic bar stirring in the reactor. The reactor was
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connected to a circulatory water bath (NTT-20S, Tokyo Rikakikai, Tokyo, Japan) to maintain a temperature of 37 °C during simulated digestion. A pH meter (AS800, As One, Osaka, Japan) was used to adjust the pH of the liquid mixture to 1.20 ± 0.01. Addition of SGF started the simulated gastric stage and the pH during this stage was continuously maintained with the addition of 3 mol/l HCl solution. After 30 min, the pH was adjusted to 6.00 ± 0.01 with the addition of 10
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3 mol/l, 1 mol/l, and 0.2 mol/l NaOH solution to eliminate pepsin activity. For the simulated small intestinal stage, SIF was added to the reacted mixture. The pH was adjusted to, and then maintained at, 6.80 ± 0.01. Samples of the supernatant (0.5 ml each) were obtained
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at various time points for glucose analysis. During the simulated
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gastric stage, the collection time points were 5 and 30 min, while the
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time points for the small intestinal stage were 5, 10, 15, 30, 60, 90, 120, 150, 180, 210, 240 and 270 min. Each supernatant sample was
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mixed with 95% ethanol (3 ml) to stop the enzymatic activity.
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2.8. Oral digestion step
An in vitro simulated oral digestion step was performed as previously
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described (Beer et al., 1997; Tamura et al., 2013) with minor
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modifications. Briefly, crushed grains that were homogenized for 1 s or 120 s as imitative chewed samples were used to prepare samples
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containing approximately 6.8 g of total starch. These samples were placed in a polyethylene mesh and mixed with a 0.02 mol/l
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phosphate buffer solution containing 0.01 mol/l sodium chloride (pH 6.90 ± 0.01) up to a total mass of 170 g. The mixture was then poured into a jacketed glass reactor and was continuously agitated with magnetic stirrers at 350 rpm. The enzyme reaction was started when 0.4 ml SSF, which was varied in the concentrations for 0.3, 0.7 and 1.3 U/ml. The pH of the solution was adjusted and maintained at 6.90 ± 0.01 with the addition of 0.2 mol/l NaOH solution and/or 0.2 11
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mol/l HCL solution. To consider infiltration of SSF to sample tissue, the oral digestion step was extended over 60 min. Samples of the supernatant (0.5 ml each) were collected during the oral digestion step (e.g., at 0, 1, 5, 10, 15, 30, and 60 min) and each was mixed
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with 3 ml of 95 % ethanol to stop the enzyme reaction. The actual
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chewed grain model samples were also subjected to this protocol.
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To examine the effect of an oral digestion step on the digestibility of the starch in cooked rice, the oral digestion step was combined with
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the simulated in vitro gastrointestinal digestion process.
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2.9. Kinetics of starch hydrolysis
The ethanol mixture solutions were centrifuged at 1800 × g for 10
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min and then a 0.1 ml sample was collected from each supernatant.
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The samples were incubated at 37 °C for 10 min with amyloglucosidase and invertase (Monro et al., 2009) in order to
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convert all of the small sugars that were produced during hydrolysis into glucose. The concentration of glucose was then measured with a
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D-glucose assay kit (GOPOD Format K-GLUK 07/11, Megazyme International) and a spectrophotometer (V630BIO, Jasco, Tokyo, Japan). The results are expressed as percent starch hydrolysis as follows: %SH = 𝑆ℎ ⁄𝑆𝑖 = 0.9 × 𝐺𝑝 ⁄𝑆𝑖 where %SH is the percent starch hydrolysis, Sh is the amount of starch hydrolyzed, Si is the initial amount of starch, and Gp is the 12
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amount of glucose produced. A conversion factor of 0.9 was applied based on the molecular weight of a starch monomer divided by the molecular weight of glucose (162 / 180 = 0.9) (Goñi et al., 1997). According to Goñi et al. (1997), the kinetics of starch hydrolysis
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during simulated small intestinal digestion were also calculated as
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follows:
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C = C∞ (1 − exp(−kt))
where C, C∞, and k represent the percent hydrolysis, the percent
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2.10. Statistical analyses
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equilibrium hydrolysis, and the kinetic constant, respectively.
Results were calculated as the mean ± standard deviation (SD).
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Subsequently, Tukey’s test, in conjunction with analysis of variance
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(ANOVA), were used to determine significant differences among the mean data with an a priori significance level set at p< 0.05 using R
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software (R Core Team, 2014).
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3. Results and Discussion 3.1. Sample conditions The amount of water added for the cooking of rice is related to the moisture content of cooked rice and is one of the major factors that affect the texture of cooked rice (Juliano and Perez, 1983; Srisawas and Jindal, 2007). Normally, the moisture content of cooked japonica cultivars, including Koshihikari (which is generally cooked with a 13
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Japanese-style cooking method), is approximately 65 % (Tamura et al., 2014b). In the present study, the mean moisture content of the cooked grain samples was 65.7 ± 0.6 (% w.b.). The amylose and protein content of polished rice grain are also important factors for
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determining the texture of a cooked material (Champagne et al.,
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2009; Okadome et al., 1999). The mean apparent amylose content of
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the grain sample in this study was 28.1 ± 1.3 (% d.b.), which was a higher apparent amylose content than the same variety of polished
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grain reported in the previous studies (Ayabe et al., 2006; Kohyama et al., 2016; Tamura et al., 2014b). However, the value is consistent
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with the amylose content determined for 44 japonica rice cultivars by Mestres et al. (2011). In addition, the mean crude protein content of
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the grain samples used in the present study was 7.1 ± 0.1 (% d.b.).
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Previously, Chung et al. (2010) reported that the total and resistant starch content of cooked rice grains range from 86.7 % to 89.5 %
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and from 0.2 % to 1.1 %, respectively. In the present study, the mean total starch and resistant starch content values were 90.7 ± 1.4 (%
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d.b.) and 0.6 ± 0.0 (% d.b.), respectively. Thus, the cooked rice grain sample used in the present study represents a normal cooked rice. In Figure 1, representative images of the shape of the intact grains (Fig. 1a), the actual chewed grains (Fig. 1b), and the grain samples that were homogenized for 1 s (Fig. 1c) and 120 s (Fig. 1d) are shown. Projected area distributions for the first three types of sample grains are shown in Figure 1A, while the particle size distribution for 14
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the grain sample homogenized for 120 s is shown in Figure 1B. The intact cooked grain samples mostly maintained their shape (Fig. 1a). In contrast, many grain fragments were observed among a few intact grains in the actual chewed sample (Fig. 1b). The presence of
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fragments and intact grains are numerically represented in the
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projected area distribution data (Fig. 1A) by the two peaks which represent the intact grains, with areas ranging from 7–13 mm2
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(38.4 %) and 18–24 mm2 (38.4 %), and one peak (or mode value) for
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the actual chewed sample at 6–12 mm2 (43.3 %). The size of the fragments were smaller than the intact grains, yet were sufficiently
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larger than the cell size reported for cooked rice grain (Ogawa et al., 2003b; Tamura et al., 2016a). There were no intact grains observed
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in the samples that were homogenized for 1 s (Fig. 1c) or for 120 s
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(Fig. 1d). Moreover, the mode value in the projected area distribution data for the grains that were homogenized for 1 s was positioned at
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2–5 mm2 (39.5 %) (Fig. 1A), and two modes were positioned at 20– 57 μm (41.3 %) and 266–322 μm (9.0 %) for the grains that were
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homogenized for 120 s (Fig. 1B). In comparison, the sizes reported for rice starch granules have varied from 2–7 μm (Vandeputte and Delcour, 2004), and rice granules generally undergo swelling during gelatinization. Tamura et al. (2016a) also observed that starch granules in rice that was cooked and then homogenized ranged in size from 5–50 μm. Except for the starch granules, there are smaller particles such as three types of protein body, i.e. large spherical (1–2 15
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μm), small spherical (0.5–0.75 μm) and crystalline (2–3.5 μm), exist in the endosperm (Champagne et al., 2006). Smaller oil bodies (0.5– 1 μm) also present in rice embryo and aleurone layer (Wu et al., 1998). Thus, the particles in the sample that were homogenized for
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120 s appear to mostly represent gelatinized starch granules, while
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the larger particles potentially represent small fragments or
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segmented gelatinized starch granules. Accordingly, these results indicated that the grain homogenized for 1 s provided a better
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simulated sample for actual chewed cooked rice grain, although all of the prepared samples were employed and compared to investigate
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the effect of an oral digestion step on the digestibility of starch from
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cooked rice grain.
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3.2. Starch hydrolysis of intact and homogenized samples during the simulated gastrointestinal digestion process
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In general, starch hydrolysis is related to particle size and surface area of starchy foodstuffs during the oral and small intestinal
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digestion as previously demonstrated with barley and sorghum (Al-Rabadi et al., 2009), wheat, maize, and oat (Heaton et al., 1988). Read et al. (1986) further showed that thorough chewing of rice, potato, or sweet corn induced a postprandial increase in glucose, while swallowing of those foods without chewing significantly reduced the blood glucose response. In studies by Ranawana et al. (2010a, 2010b), particle size was found to be significantly related to 16
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the in vivo glycemic response to chewed cooked rice, although differences in individuals’ mastication may lead to differences in the glycemic response to rice. Figure 2 shows the changes in hydrolysis percent of starch of the intact grains and the samples that were
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homogenized for 1 s or 120 s during the simulated in vitro gastro
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small intestinal digestion process. Starch hydrolysis was initiated
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during the gastric stage and then increased during the small intestinal stage in the following order: samples homogenized for 120
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s, samples homogenized for 1 s, and intact rice grains. Therefore, starch hydrolysis was found to be related to the degree of
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homogenization that was achieved for the rice grains, and these results are consistent with those reported by Tamura et al. (2016a,
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2016b). Moreover, in the latter study, grain structure was found to be
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associated with the starch hydrolysis rate during simulated in vitro
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gastrointestinal digestion.
3.3 Condition of oral digestion step
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In general, salivary α-amylase is inactivated under low pH conditions such as those found in the stomach. Thus, oral starch digestibility due to salivary α-amylase has not been considered in most of the studies that have employed simulated in vitro digestion processes. However, in recent studies (Bornhorst et al., 2014; Mennah-Govela et al., 2015), salivary α-amylase was found to infiltrate a bolus after mastication and was able to maintain its activity during the gastric 17
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stage both in vitro and in vivo. Therefore, the effect of salivary α-amylase on starch digestibility from cooked rice samples should be investigated. Figure 3 shows the changes in starch hydrolysis (%) that were
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detected for intact grains (Fig. 3A) and for the samples homogenized
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for 120 s (Fig. 3B) in the presence of varying concentrations of SSF.
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The change in starch hydrolysis for the actual chewed cooked rice sample with actual saliva is also shown (Fig. 3C). In contrast, the
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absence of saliva conditions resulted in no change in starch hydrolysis for both the intact and homogenized samples. Meanwhile,
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starch hydrolysis observed under SSF conditions and actual chewed conditions increased with treatment time, yet the extent of the
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increase observed differed between the intact grain samples and the
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homogenized samples. For example, starch hydrolysis of the intact grains that were treated with 0.3, 0.7, and 1.3 U/ml of α-amylase in
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SSF for 60 min had similar percentage values of 12.0, 13.4, and 12.6, respectively (p > 0.05). In comparison, the percent hydrolysis values
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for the homogenized samples at 60 min were 12.5, 33.2, and 42.5, respectively (p < 0.05). These results indicated that the simulated starch hydrolysis of cooked rice was affected by the degree of homogenization that was more effective than salivary α-amylase concentration, while hydrolysis percent of starch was related to salivary α-amylase concentration when the grains were well homogenized. Meanwhile, the hydrolysis percent of starch was 18
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35.4 % when actual chewed samples were treated for 60 min (Fig. 3C). Although the duration was much longer than actual mastication, a similar trend was observed for the homogenized samples that were treated with concentrations of SSF that varied from 0.7 U/ml to 1.3
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U/ml. Therefore, treatment with 1.3 U/ml SSF for 60 min was
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digestion step and employed in this study.
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selected as a mimicked condition to simulate the in vitro oral
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3.4. Effect of an oral digestion step on starch hydrolysis from cooked rice samples
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Figure 4 shows the changes in hydrolysis percent of starch that were detected for the intact grain samples, the grains that were
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homogenized for 1 s or for 120 s, and actual chewed cooked rice
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samples after they were subjected to an in vitro digestion model that combined both an oral step (1.3 U/ml of SSF for 60 min) and a
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gastrointestinal process. For the oral step, the percent starch hydrolysis values were 12.6, 28.7, 42.5, and 35.4, respectively. After
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the simulated gastric process, no increase in starch hydrolysis was observed, similar to the results shown in Figure 2. The percent hydrolysis during the simulated small intestinal process increased and appeared to reach an equilibrium state for all of the samples. For example, there was a significant difference between the percent starch hydrolysis detected for the samples that were homogenized for 1 s and for 120 s after 90 min (p < 0.05). However, after 330 min, 19
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there was no significant difference between the same samples (p > 0.05). The absence of changes in hydrolysis percent of starch during the gastric stage are mainly attributed to inactivation of salivary amylase since its threshold value for activation is around pH 3 (Fried
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et al., 1987; Rosenblum et al., 1988). SGF at a low pH can also
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enhance texture degradation (Kong et al., 2011) and proteolysis
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(Fardet et al., 1998), which would influence starch hydrolysis in the small intestinal stage.
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Figure 5 shows images of the morphological and histological structures of an intact cooked grain sample prior to digestion (Fig. 5:
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A, B, a, b), a cooked grain sample after digestion by the gastrointestinal process (Fig. 5: C, D, c, d), and a cooked grain
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sample that was chewed by an individual and then digested by
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gastrointestinal process (Fig. 5: E, F, e, f). The histological tissue images (Fig. 5: B, D, F, b, d, f) showed cell wall arrangements
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(Ogawa et al., 2003a; Tamura and Ogawa, 2012) at the same position of the morphological images. Several morphological cracks
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were observed at the surface of the intact cooked grains prior to digestion (Fig. 5a), and these appear as darker regions in the corresponding histological image (Fig. 5b). In contrast, the cooked grains that underwent the simulated digestion process exhibited morphological deformations and destruction of the grains (Fig. 5, A & B vs. C-F). The morphological images of the digested samples also show a large number of cracks and honeycomb-like structures at 20
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their center (Fig 5c, e), and these data are consistent with the corresponding histological images (Fig. 5, d, f). Previously, Tamura et al. (2014a) mainly observed cracks and voids in polished rice grains with cooking, while the cracks observed in the present study were at
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the grain surface of the post-digested samples (Fig. 5 D, F). It was
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also previously demonstrated that despite the disappearance of
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starchy materials from rice grains during an in vitro hydrolysis process, part of the grain tissue remained in the digested residues,
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and this tissue mainly consisted of indigestible cell wall materials (Alminger et al., 2012; Tamura et al., 2016a, 2016b). Distinct
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histological structures (Fig. 5 d, f) were observed in the digested grain samples in the present study, and the structures appeared to
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be thin materials. However, no differences were observed between
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the morphological and histological grain surface tissue structures for the post-digested and actual chewed samples.
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To evaluate starch hydrolysis behavior among the different structural states of the cooked rice samples examined, kinetic parameters
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during the simulated small intestinal stage were calculated according to a first-order equation model (Goñi et al., 1997) with a non-linear curve fitting method. Table 1 lists the equilibrium percentages for starch hydrolysis and the kinetic constants for the different samples during both the simulated gastrointestinal digestion process and the combined
oral
and
gastrointestinal
digestion
process.
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equilibrium starch hydrolysis percentages varied from 84.2 % to 21
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95.9 % and there was no significant difference among the values (p > 0.05), regardless of the type of digestion step. In contrast, the kinetic constants significantly increased as the degree of homogenization increased: 120 s > actual chewed ≥ 1 s > intact, for both the
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gastrointestinal and combined processes. These results indicated
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that the kinetic constants during the small intestinal stage were
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influenced by the oral digestion step and the degree of homogenization. In fact, degree of homogenization major impacted
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on the kinetic constant in starch digestibility to salivary α-amylase. These results also correspond with a previous observation that
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kinetic constant was affected on increase in enzyme accessibility to rice related to the extent that a grain’s structure is changed (Tamura
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et al., 2016a, 2016b). As shown in the combined process (see Fig. 4),
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a larger starch hydrolysis percentage was observed at the gastric stage compared with the gastrointestinal process (see Fig. 2). The
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inclusion of the oral digestion step in the combined process also resulted in higher kinetic constant values for the samples examined,
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except for the sample that was homogenized for 120 s. When Germaine et al. (2008) investigated glycemic index (GI) and estimated glycemic index (eGI; calculated from kinetic constant and equilibrium hydrolysis percent) values of minced cereals that were treated with simulated salivary amylase, they concluded that the addition of simulated salivary amylase did not affect the GI value, and thus, could be omitted. However, the results of the present study 22
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reveal that the degree of homogenization that is achieved with the simulated oral digestion step influences the kinetic constant of starch digestibility in cooked rice. Moreover, a significant difference in starch digestibility was observed for the combined process except for the
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sample that was only homogenized for 1 s.
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4. Conclusions
Our initial hypothesis was that rice grain digestibility would be
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affected by grain-scale changes rather than cell-scale structural changes, and these would be demonstrated in an oral digestion step.
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We observed that the effect of salivary α-amylase on the simulated oral digestion step influenced both the equilibrium percentage of
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starch hydrolysis as well as the kinetic constant during the combined
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in vitro digestion process, and these would potentially affect the increases of postprandial blood glucose levels in humans. Thus, the
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simulated oral digestion step with an appropriate condition should be included in a simulated in vitro digestion process to evaluate the
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starch digestibility of foods containing structural carbohydrates, such as cooked rice grain.
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Figure Captions Fig. 1 Representative images of intact cooked rice grains (a), actual chewed grains (b), grains homogenized for 1 s (c), and grains homogenized for 120 s (d). The corresponding projected area distributions for panels a-c are presented in panel A, and
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the particle size distribution for the grains homogenized for 120 s is shown in panel B.
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Scale bar (a-d), 10 mm.
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Fig. 2 Changes in hydrolysis percent of starch during a simulated gastrointestinal digestion process for intact cooked grain versus cooked grains that were homogenized
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for 1 s or for 120 s. Error bars represent SD values (n = 3–4). Fig. 3 Changes in hydrolysis percent of starch for intact cooked grains (A), cooked
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grains that were homogenized for 120 s during treatment with SSF containing various concentrations of α-amylase (B) and actual chewed cooked rice with actual saliva (C).
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Error bars represent SD values (n = 3–4).
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Fig. 4 Changes in hydrolysis percent of starch for intact cooked grains, cooked grains homogenized for 1 s, cooked grains homogenized for 120 s, and actual chewed cooked
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rice samples during a simulated in vitro digestion process that combined oral and gastrointestinal processes. Error bars represent SD values (n = 3–4).
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Fig. 5 Morphological and histological images of an intact cooked grain sample (A, B, a, b), a cooked grain sample after digestion by the gastrointestinal process (C, D, c, d), and a cooked grain sample that was chewed and then digested by gastrointestinal process (E, F, e, f). Panels A, C, E, a, c, and e represent the morphological images and panels B, D, F, b, d, and f represent the histological images. Scale bars indicate 5 mm (A-F) and 1 mm (a-f).
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Table 1. Kinetic parameters of simulated in vitro starch digestion from intact cooked grain. C∞ (%)
k ×10-2 (min-1)
Intact
91.3 ± 2.6 a
1.1 ± 0.1 c
1s
95.9 ± 7.8 a
2.1 ± 0.9 c
120 s
88.2 ± 3.5 a
22.1 ± 0.5 a
Combined oral and
Intact
93.8 ± 9.4 a
gastrointestinal process
1s
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Gastrointestinal
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Simulated digestion process
87.5 ± 6.2 a
6.9 ± 1.7 b
84.2 ± 2.3 a
20.9 ± 0.5 a
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120 s
Actual chewed
2.0 ± 0.3 c
90.2 ± 4.6 a
9.4 ± 2.1 b
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1 s / 120 s: samples were homogenized for 1 s or 120 s with a blender. Data are presented as
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the mean ± SD (n = 3–4). The superscripted letters indicate significant differences (p < 0.05).
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Graphical abstract
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Masatsugu Tamura, Yumi Okazaki, Chisato Kumagai, Yukiharu Ogawa PII: DOI: Reference:
S0963-9969(17)30030-3 doi: 10.1016/j.foodres.2017.01.019 FRIN 6575
To appear in:
Food Research International
Received date: Revised date: Accepted date:
1 October 2016 23 January 2017 24 January 2017
Please cite this article as: Masatsugu Tamura, Yumi Okazaki, Chisato Kumagai, Yukiharu Ogawa , The importance of an oral digestion step in evaluating simulated in vitro digestibility of starch from cooked rice grain. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Frin(2017), doi: 10.1016/j.foodres.2017.01.019
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Title The importance of an oral digestion step in evaluating simulated in vitro digestibility of starch from cooked rice grain
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Running title
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Effect of mastication on cooked rice digestibility
Authors’ name
Faculty of Agriculture, Utsunomiya University, 350, Mine, Utsunomioya
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1
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Masatsugu Tamura1, Yumi Okazaki2, Chisato Kumagai3, Yukiharu Ogawa3
321-8505, Japan
Faculty of Horticulture, Chiba University, 648, Matsudo, Matsudo
3
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271-8510, Japan
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2
Graduate School of Horticulture, Chiba University, 648, Matsudo,
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Matsudo 271-8510, Japan
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Corresponding author TEL&FAX: +81-47-308-8848; EMAIL: [email protected]
Keywords Cooked rice, Mastication, Simulated digestion, Kinetic parameters, Tissue structure 1
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Highlights Oral digestion step plays important role in evaluation of the starch digestibility Structural changes in grain-scale impacts on starch digestibility of
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rice grain
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Food structure greater acts starch hydrolysis than salivary
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α-amylase concentration
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Abstract To examine the effect of oral digestion step in a simulated in vitro starch digestion model, the digestibility of intact, homogenized and actual chewed cooked rice grains was investigated and analyzed. The kinetics of
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starch digestibility were calculated from changes in the hydrolysis percent
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of starch that were achieved during simulated small intestinal digestion
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stage. Morphological and histological microscopic tissue structures were also examined. Compared with the trend of starch hydrolysis changes of
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the actual chewed grain, 1.3 U/ml of salivary α-amylase concentration treated for 60 min was regarded as a mimicked condition to the simulate in
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vitro oral digestion step in this study. The results showed that the equilibrium percent of starch hydrolysis for all of the samples ranged from
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84.2 % to 95.9 % with no significant differences observed regardless of
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whether the oral digestion step was included (p > 0.05). In contrast, the kinetic constant, which is one of the measure of starch digestion rate
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during small intestinal stage, significantly increased with the degree of grain homogenization increased: 120 s > actual chewed ≥ 1 s > intact, for
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both the gastrointestinal and oral plus gastrointestinal processes. These results indicated that the kinetic constant was influenced by the change of cooked rice grain structure in oral digestion step that would be related to increase in enzyme accessibility to rice starch. Thus, rice grain digestibility was affected by grain-scale structural changes, including grain tissue damages which were normally observed during the oral digestion step.
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1. Introduction A rapid postprandial increase in blood glucose levels is normally observed following the rapid ingestion of carbohydrate-rich foods. When this type of response is consistently induced, there is an
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increased risk of acquiring life-style related diseases such as type 2
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diabetes, hyperlipidemia, and cardiovascular disease (Jenkins et al.,
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2002; Lehmann and Robin, 2007). In contrast, a slow rate of ingestion of starchy foods may avoid potential problems and may
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have health benefits. Digestibility of the starches associated with carbohydrate-rich foods is also a relevant consideration.
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To investigate the digestibility of starchy foods, both in vivo animal models and in vitro simulated models have been developed to
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represent the gastrointestinal digestion system. Recently, the ease of
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using an in vitro simulated digestion process was demonstrated, as well as its flexibility regarding experimental conditions and its
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capacity for observing changes in food attributes (Tamura et al., 2016a, 2016b). Generally in vitro approaches consist of a two-stage
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simulated digestion system in which a gastric stage is established with a low pH and pepsin, and a subsequent small intestinal stage is established with a neutral pH and intestinal enzymes (Dartois et al., 2010). However, an oral digestion step, which is an intrinsic system for the human body, has typically not been considered for in vitro studies of starchy food digestibility because the food samples are often prepared as a powder or a slurry in order to reduce the 4
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experimental time (Englyst et al., 1992). Oral digestion as the first step of food digestion has two simultaneous actions: chewing to provide mechanical grinding and enzymatic starch hydrolysis that is mediated by the saliva. After food
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is crushed into small pieces and mixed with saliva with chewing, then
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amylase that is present in the saliva is able to act on the starch in the
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food. Hoebler et al. (1998) reported that approximately 25 % of the starch in pasta and 50 % of the starch in bread are hydrolyzed during
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oral starch digestion, thereby making a portion of the starch available for further enzymatic digestion in the gastrointestinal process.
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Bornhorst and Singh (2013) also demonstrated that α-amylase from saliva plays an important role in the breakdown kinetics of bread
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boluses in in vitro models. Meanwhile, Woolnough et al. (2010)
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reported that prolonged exposure to saliva during the chewing of starchy foods by volunteers did not contribute to the hydrolysis of
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starch during the gastrointestinal digestion process. Strahler et al. (2010) further reported that the concentration of salivary α-amylase
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in humans varies with age, psychosocial stress, and other factors, all of which were regarded as highly subjective according to individual conditions.
Rice (Oryza sativa L.) is a major source of carbohydrates, especially in Asian countries. Since rice is cooked and consumed as a whole grain, the structural attributes of cooked rice grain influence the digestibility of its starch components (Tamura et al., 2016a, 2016b). 5
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However, oral digestion approaches have not been considered and applied to the simulated digestion model for cooked rice grain. In this study, the effects of mimicked oral digestion conditions, including varying
degrees
of
sample
homogenization
and
varying
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concentrations of α-amylase in simulated salivary fluid, on a
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simulated in vitro starch digestion model were investigated and
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analyzed in relation to the digestibility of cooked rice starch.
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2. Materials and Methods 2.1. Materials
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Precooked steamed rice packaged as a ready-to-eat meal for the microwave oven (Wooke, Toyama, Japan) was purchased. The rice
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cultivar used in the product was “Koshihikari” and it was harvested in
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Nyuzen, Toyama, Japan in 2013. Alpha-amylase (porcine pancreas, type VI-B, ≥ 10 U/mg, solid, A3176-500KU), pepsin (P7000, porcine
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gastric mucosa, ≥ 250 U/mg, solid), pancreatin (hog pancreas, 4× USP), and invertase (from baker’s yeast, grade VII, ≥ 300 U/mg,
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solid) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Amyloglucosidase (3,260 U/ml) was purchased from Megazyme International Ireland (Wicklow, Ireland).
2.2. Sample preparation To prepare cooked grain samples, 60 g of pre-cooked steamed rice was put in a Ziplock bag and heated in a microwave oven 6
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(RE-SX10-W, Sharp, Osaka, Japan) at 500 W for 50 s. The heated sample was then cooled in an incubator (A0601-2V, Keepit, Nagano, Japan) at 30 °C for 30 min. To obtain mechanically crushed grain samples as an imitative
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chewed model, the cooked grain samples were homogenized using
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a household blender (THM310, Tescom, Tokyo, Japan) for 1 s or for
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120 s. In addition, an actual chewed grain model was prepared as previously described (Akerberg et al., 1998; Beer et al., 1997;
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Germaine et al., 2008; Woolnough et al., 2010). Two volunteers, who were early twenties and thirties and have normal dentition, were
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employed in this study. They brushed their teeth before chewing. Briefly, 20 g of a grain sample (approximately 6.8 g of starch content
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equivalent) was bitten 15 times in 15 s by each volunteer for two
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replicates. During actual chewing, grain sample was crushed and mixed with salivary α-amylase. The sample was then expectorated
CE
and left to sit as chewed boluses. A small portion of the cooked grain samples were freeze-dried
AC
(FDU-1100, Eyela, Tokyo, Japan), then ground using a household mixer (MR-280, Yamazen, Tokyo, Japan) and sieved using a 0.5 mm meshed sieve. The powdered material obtained was used to measure total starch (TS) and resistant starch (RS) contents using a resistant starch kit (K-RSTAR 08/11, Megazyme International).
7
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2.3. Moisture, amylose, and protein content The cooked grain samples were dried using a hot air oven (WFO-400, Eyela) at 135 °C, and after 24 h, the moisture content in wet basis (w.b.) was calculated. The sample grain was ground using a
PT
household mixer (MR-280, Yamazen) and was passed through a 0.5
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mm sieve to produce a sample powder. The apparent amylose
SC
content of the powder was then measured as previously described (Tamura et al., 2014b). Nitrogen content of the powder samples were
NU
measured by using a CN Corder (MT-700, Yanaco, Kyoto, Japan). Crude protein content was calculated from the measured nitrogen
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content with a nitrogen-protein conversion factor of 5.95. Hippuric acid (200-37032, Kishida Chemical, Osaka, Japan) was used as a
2.4. Microscopy
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D
standard for the nitrogen measurements.
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Grain shape was observed with a digital microscope (VH-Z-05 & VH-8000, Keyence, Osaka, Japan). Projected area was calculated
AC
from a number of pixels that represented grain portion after image binarization. Approximately 500 grains or grain fragments were randomly selected from the cooked grain sample and were manually arranged so that they did not touch each other on the observation stage of the microscope. To visualize the histological tissue structures of whole size rice grain, the autofluorescent imaging technique was applied (Ogawa et al., 8
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2003a; Tamura and Ogawa, 2012). To obtain morphological and histological images of the grains, a fluorescent stereomicroscope (MZ-FLIII, Leica, Wetzlar, Germany) equipped with a digital camera (DS-5M, Nikon, Tokyo, Japan) was used. The microscope had a 100
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W mercury arc lamp (ebq 100 dc, Leica), an ultraviolet fluorescence
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filter set (360/40 nm excitation filter, 420 nm barrier filter, Leica) for
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histological tissue observation, and a halogen lamp for morphological tissue examination. All imaging parameters, including focusing,
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lighting, and shuttering, were standardized for each sample. The captured images were processed and analyzed with Photoshop
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CC2014 software (Adobe, San Jose, CA, USA).
distribution
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Particle size
D
2.5. Particle size
for
the
grain
samples
that
were
homogenized for 120 s was determined using a laser diffraction
CE
particle size analyzer (SALD-3100, Shimadzu, Kyoto, Japan). The
AC
applied relative refractive index was 1.70.
2.6. Simulated digestive fluid Simulated salivary fluid (SSF) was prepared as previously described (Beer et al., 1997; Tamura et al., 2013) and examined preliminarily. Briefly, 5 mg of α-amylase was dissolved in 1 ml of 0.036 mol/l calcium chlorite as a standard SSF in which the final α-amylase concentration was 0.3 U/ml. Two additional SSF standards were 9
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generated with 10 mg and 20 mg of α-amylase, resulting in final α-amylase concentrations of 0.7 U/ml and 1.3 U/ml, respectively. Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared according to the method of Dartois et al. (2010). The SGF
PT
contained pepcine. The SIF contained pancreatine, invertase, and
SC
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amyloglucosidase.
2.7. Simulated in vitro gastrointestinal digestion
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A simulated in vitro gastrointestinal digestion model was established as previously described (Dartois et al., 2010). Briefly, 170 g of rice
MA
sample mixture with distilled water, which was prepared to contain 4 % of total starch, was added into a 500 ml jacketed glass reactor
D
and continuously stirred with a magnetic stirrer (Color Squid, Ika,
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Staufen, Germany) at 350 rpm. When intact grain samples or grain samples homogenized for 1 s were placed in the reactor, they were
CE
placed in a polyethylene mesh to prevent direct contact between the samples and the magnetic bar stirring in the reactor. The reactor was
AC
connected to a circulatory water bath (NTT-20S, Tokyo Rikakikai, Tokyo, Japan) to maintain a temperature of 37 °C during simulated digestion. A pH meter (AS800, As One, Osaka, Japan) was used to adjust the pH of the liquid mixture to 1.20 ± 0.01. Addition of SGF started the simulated gastric stage and the pH during this stage was continuously maintained with the addition of 3 mol/l HCl solution. After 30 min, the pH was adjusted to 6.00 ± 0.01 with the addition of 10
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3 mol/l, 1 mol/l, and 0.2 mol/l NaOH solution to eliminate pepsin activity. For the simulated small intestinal stage, SIF was added to the reacted mixture. The pH was adjusted to, and then maintained at, 6.80 ± 0.01. Samples of the supernatant (0.5 ml each) were obtained
PT
at various time points for glucose analysis. During the simulated
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gastric stage, the collection time points were 5 and 30 min, while the
SC
time points for the small intestinal stage were 5, 10, 15, 30, 60, 90, 120, 150, 180, 210, 240 and 270 min. Each supernatant sample was
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mixed with 95% ethanol (3 ml) to stop the enzymatic activity.
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2.8. Oral digestion step
An in vitro simulated oral digestion step was performed as previously
D
described (Beer et al., 1997; Tamura et al., 2013) with minor
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modifications. Briefly, crushed grains that were homogenized for 1 s or 120 s as imitative chewed samples were used to prepare samples
CE
containing approximately 6.8 g of total starch. These samples were placed in a polyethylene mesh and mixed with a 0.02 mol/l
AC
phosphate buffer solution containing 0.01 mol/l sodium chloride (pH 6.90 ± 0.01) up to a total mass of 170 g. The mixture was then poured into a jacketed glass reactor and was continuously agitated with magnetic stirrers at 350 rpm. The enzyme reaction was started when 0.4 ml SSF, which was varied in the concentrations for 0.3, 0.7 and 1.3 U/ml. The pH of the solution was adjusted and maintained at 6.90 ± 0.01 with the addition of 0.2 mol/l NaOH solution and/or 0.2 11
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mol/l HCL solution. To consider infiltration of SSF to sample tissue, the oral digestion step was extended over 60 min. Samples of the supernatant (0.5 ml each) were collected during the oral digestion step (e.g., at 0, 1, 5, 10, 15, 30, and 60 min) and each was mixed
PT
with 3 ml of 95 % ethanol to stop the enzyme reaction. The actual
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chewed grain model samples were also subjected to this protocol.
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To examine the effect of an oral digestion step on the digestibility of the starch in cooked rice, the oral digestion step was combined with
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the simulated in vitro gastrointestinal digestion process.
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2.9. Kinetics of starch hydrolysis
The ethanol mixture solutions were centrifuged at 1800 × g for 10
D
min and then a 0.1 ml sample was collected from each supernatant.
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The samples were incubated at 37 °C for 10 min with amyloglucosidase and invertase (Monro et al., 2009) in order to
CE
convert all of the small sugars that were produced during hydrolysis into glucose. The concentration of glucose was then measured with a
AC
D-glucose assay kit (GOPOD Format K-GLUK 07/11, Megazyme International) and a spectrophotometer (V630BIO, Jasco, Tokyo, Japan). The results are expressed as percent starch hydrolysis as follows: %SH = 𝑆ℎ ⁄𝑆𝑖 = 0.9 × 𝐺𝑝 ⁄𝑆𝑖 where %SH is the percent starch hydrolysis, Sh is the amount of starch hydrolyzed, Si is the initial amount of starch, and Gp is the 12
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amount of glucose produced. A conversion factor of 0.9 was applied based on the molecular weight of a starch monomer divided by the molecular weight of glucose (162 / 180 = 0.9) (Goñi et al., 1997). According to Goñi et al. (1997), the kinetics of starch hydrolysis
PT
during simulated small intestinal digestion were also calculated as
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follows:
SC
C = C∞ (1 − exp(−kt))
where C, C∞, and k represent the percent hydrolysis, the percent
MA
2.10. Statistical analyses
NU
equilibrium hydrolysis, and the kinetic constant, respectively.
Results were calculated as the mean ± standard deviation (SD).
D
Subsequently, Tukey’s test, in conjunction with analysis of variance
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(ANOVA), were used to determine significant differences among the mean data with an a priori significance level set at p< 0.05 using R
CE
software (R Core Team, 2014).
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3. Results and Discussion 3.1. Sample conditions The amount of water added for the cooking of rice is related to the moisture content of cooked rice and is one of the major factors that affect the texture of cooked rice (Juliano and Perez, 1983; Srisawas and Jindal, 2007). Normally, the moisture content of cooked japonica cultivars, including Koshihikari (which is generally cooked with a 13
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Japanese-style cooking method), is approximately 65 % (Tamura et al., 2014b). In the present study, the mean moisture content of the cooked grain samples was 65.7 ± 0.6 (% w.b.). The amylose and protein content of polished rice grain are also important factors for
PT
determining the texture of a cooked material (Champagne et al.,
RI
2009; Okadome et al., 1999). The mean apparent amylose content of
SC
the grain sample in this study was 28.1 ± 1.3 (% d.b.), which was a higher apparent amylose content than the same variety of polished
NU
grain reported in the previous studies (Ayabe et al., 2006; Kohyama et al., 2016; Tamura et al., 2014b). However, the value is consistent
MA
with the amylose content determined for 44 japonica rice cultivars by Mestres et al. (2011). In addition, the mean crude protein content of
D
the grain samples used in the present study was 7.1 ± 0.1 (% d.b.).
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Previously, Chung et al. (2010) reported that the total and resistant starch content of cooked rice grains range from 86.7 % to 89.5 %
CE
and from 0.2 % to 1.1 %, respectively. In the present study, the mean total starch and resistant starch content values were 90.7 ± 1.4 (%
AC
d.b.) and 0.6 ± 0.0 (% d.b.), respectively. Thus, the cooked rice grain sample used in the present study represents a normal cooked rice. In Figure 1, representative images of the shape of the intact grains (Fig. 1a), the actual chewed grains (Fig. 1b), and the grain samples that were homogenized for 1 s (Fig. 1c) and 120 s (Fig. 1d) are shown. Projected area distributions for the first three types of sample grains are shown in Figure 1A, while the particle size distribution for 14
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the grain sample homogenized for 120 s is shown in Figure 1B. The intact cooked grain samples mostly maintained their shape (Fig. 1a). In contrast, many grain fragments were observed among a few intact grains in the actual chewed sample (Fig. 1b). The presence of
PT
fragments and intact grains are numerically represented in the
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projected area distribution data (Fig. 1A) by the two peaks which represent the intact grains, with areas ranging from 7–13 mm2
SC
(38.4 %) and 18–24 mm2 (38.4 %), and one peak (or mode value) for
NU
the actual chewed sample at 6–12 mm2 (43.3 %). The size of the fragments were smaller than the intact grains, yet were sufficiently
MA
larger than the cell size reported for cooked rice grain (Ogawa et al., 2003b; Tamura et al., 2016a). There were no intact grains observed
D
in the samples that were homogenized for 1 s (Fig. 1c) or for 120 s
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(Fig. 1d). Moreover, the mode value in the projected area distribution data for the grains that were homogenized for 1 s was positioned at
CE
2–5 mm2 (39.5 %) (Fig. 1A), and two modes were positioned at 20– 57 μm (41.3 %) and 266–322 μm (9.0 %) for the grains that were
AC
homogenized for 120 s (Fig. 1B). In comparison, the sizes reported for rice starch granules have varied from 2–7 μm (Vandeputte and Delcour, 2004), and rice granules generally undergo swelling during gelatinization. Tamura et al. (2016a) also observed that starch granules in rice that was cooked and then homogenized ranged in size from 5–50 μm. Except for the starch granules, there are smaller particles such as three types of protein body, i.e. large spherical (1–2 15
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μm), small spherical (0.5–0.75 μm) and crystalline (2–3.5 μm), exist in the endosperm (Champagne et al., 2006). Smaller oil bodies (0.5– 1 μm) also present in rice embryo and aleurone layer (Wu et al., 1998). Thus, the particles in the sample that were homogenized for
PT
120 s appear to mostly represent gelatinized starch granules, while
RI
the larger particles potentially represent small fragments or
SC
segmented gelatinized starch granules. Accordingly, these results indicated that the grain homogenized for 1 s provided a better
NU
simulated sample for actual chewed cooked rice grain, although all of the prepared samples were employed and compared to investigate
MA
the effect of an oral digestion step on the digestibility of starch from
D
cooked rice grain.
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3.2. Starch hydrolysis of intact and homogenized samples during the simulated gastrointestinal digestion process
CE
In general, starch hydrolysis is related to particle size and surface area of starchy foodstuffs during the oral and small intestinal
AC
digestion as previously demonstrated with barley and sorghum (Al-Rabadi et al., 2009), wheat, maize, and oat (Heaton et al., 1988). Read et al. (1986) further showed that thorough chewing of rice, potato, or sweet corn induced a postprandial increase in glucose, while swallowing of those foods without chewing significantly reduced the blood glucose response. In studies by Ranawana et al. (2010a, 2010b), particle size was found to be significantly related to 16
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the in vivo glycemic response to chewed cooked rice, although differences in individuals’ mastication may lead to differences in the glycemic response to rice. Figure 2 shows the changes in hydrolysis percent of starch of the intact grains and the samples that were
PT
homogenized for 1 s or 120 s during the simulated in vitro gastro
RI
small intestinal digestion process. Starch hydrolysis was initiated
SC
during the gastric stage and then increased during the small intestinal stage in the following order: samples homogenized for 120
NU
s, samples homogenized for 1 s, and intact rice grains. Therefore, starch hydrolysis was found to be related to the degree of
MA
homogenization that was achieved for the rice grains, and these results are consistent with those reported by Tamura et al. (2016a,
D
2016b). Moreover, in the latter study, grain structure was found to be
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associated with the starch hydrolysis rate during simulated in vitro
CE
gastrointestinal digestion.
3.3 Condition of oral digestion step
AC
In general, salivary α-amylase is inactivated under low pH conditions such as those found in the stomach. Thus, oral starch digestibility due to salivary α-amylase has not been considered in most of the studies that have employed simulated in vitro digestion processes. However, in recent studies (Bornhorst et al., 2014; Mennah-Govela et al., 2015), salivary α-amylase was found to infiltrate a bolus after mastication and was able to maintain its activity during the gastric 17
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stage both in vitro and in vivo. Therefore, the effect of salivary α-amylase on starch digestibility from cooked rice samples should be investigated. Figure 3 shows the changes in starch hydrolysis (%) that were
PT
detected for intact grains (Fig. 3A) and for the samples homogenized
RI
for 120 s (Fig. 3B) in the presence of varying concentrations of SSF.
SC
The change in starch hydrolysis for the actual chewed cooked rice sample with actual saliva is also shown (Fig. 3C). In contrast, the
NU
absence of saliva conditions resulted in no change in starch hydrolysis for both the intact and homogenized samples. Meanwhile,
MA
starch hydrolysis observed under SSF conditions and actual chewed conditions increased with treatment time, yet the extent of the
D
increase observed differed between the intact grain samples and the
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homogenized samples. For example, starch hydrolysis of the intact grains that were treated with 0.3, 0.7, and 1.3 U/ml of α-amylase in
CE
SSF for 60 min had similar percentage values of 12.0, 13.4, and 12.6, respectively (p > 0.05). In comparison, the percent hydrolysis values
AC
for the homogenized samples at 60 min were 12.5, 33.2, and 42.5, respectively (p < 0.05). These results indicated that the simulated starch hydrolysis of cooked rice was affected by the degree of homogenization that was more effective than salivary α-amylase concentration, while hydrolysis percent of starch was related to salivary α-amylase concentration when the grains were well homogenized. Meanwhile, the hydrolysis percent of starch was 18
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35.4 % when actual chewed samples were treated for 60 min (Fig. 3C). Although the duration was much longer than actual mastication, a similar trend was observed for the homogenized samples that were treated with concentrations of SSF that varied from 0.7 U/ml to 1.3
PT
U/ml. Therefore, treatment with 1.3 U/ml SSF for 60 min was
SC
digestion step and employed in this study.
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selected as a mimicked condition to simulate the in vitro oral
NU
3.4. Effect of an oral digestion step on starch hydrolysis from cooked rice samples
MA
Figure 4 shows the changes in hydrolysis percent of starch that were detected for the intact grain samples, the grains that were
D
homogenized for 1 s or for 120 s, and actual chewed cooked rice
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samples after they were subjected to an in vitro digestion model that combined both an oral step (1.3 U/ml of SSF for 60 min) and a
CE
gastrointestinal process. For the oral step, the percent starch hydrolysis values were 12.6, 28.7, 42.5, and 35.4, respectively. After
AC
the simulated gastric process, no increase in starch hydrolysis was observed, similar to the results shown in Figure 2. The percent hydrolysis during the simulated small intestinal process increased and appeared to reach an equilibrium state for all of the samples. For example, there was a significant difference between the percent starch hydrolysis detected for the samples that were homogenized for 1 s and for 120 s after 90 min (p < 0.05). However, after 330 min, 19
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there was no significant difference between the same samples (p > 0.05). The absence of changes in hydrolysis percent of starch during the gastric stage are mainly attributed to inactivation of salivary amylase since its threshold value for activation is around pH 3 (Fried
PT
et al., 1987; Rosenblum et al., 1988). SGF at a low pH can also
RI
enhance texture degradation (Kong et al., 2011) and proteolysis
SC
(Fardet et al., 1998), which would influence starch hydrolysis in the small intestinal stage.
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Figure 5 shows images of the morphological and histological structures of an intact cooked grain sample prior to digestion (Fig. 5:
MA
A, B, a, b), a cooked grain sample after digestion by the gastrointestinal process (Fig. 5: C, D, c, d), and a cooked grain
D
sample that was chewed by an individual and then digested by
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gastrointestinal process (Fig. 5: E, F, e, f). The histological tissue images (Fig. 5: B, D, F, b, d, f) showed cell wall arrangements
CE
(Ogawa et al., 2003a; Tamura and Ogawa, 2012) at the same position of the morphological images. Several morphological cracks
AC
were observed at the surface of the intact cooked grains prior to digestion (Fig. 5a), and these appear as darker regions in the corresponding histological image (Fig. 5b). In contrast, the cooked grains that underwent the simulated digestion process exhibited morphological deformations and destruction of the grains (Fig. 5, A & B vs. C-F). The morphological images of the digested samples also show a large number of cracks and honeycomb-like structures at 20
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their center (Fig 5c, e), and these data are consistent with the corresponding histological images (Fig. 5, d, f). Previously, Tamura et al. (2014a) mainly observed cracks and voids in polished rice grains with cooking, while the cracks observed in the present study were at
PT
the grain surface of the post-digested samples (Fig. 5 D, F). It was
RI
also previously demonstrated that despite the disappearance of
SC
starchy materials from rice grains during an in vitro hydrolysis process, part of the grain tissue remained in the digested residues,
NU
and this tissue mainly consisted of indigestible cell wall materials (Alminger et al., 2012; Tamura et al., 2016a, 2016b). Distinct
MA
histological structures (Fig. 5 d, f) were observed in the digested grain samples in the present study, and the structures appeared to
D
be thin materials. However, no differences were observed between
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the morphological and histological grain surface tissue structures for the post-digested and actual chewed samples.
CE
To evaluate starch hydrolysis behavior among the different structural states of the cooked rice samples examined, kinetic parameters
AC
during the simulated small intestinal stage were calculated according to a first-order equation model (Goñi et al., 1997) with a non-linear curve fitting method. Table 1 lists the equilibrium percentages for starch hydrolysis and the kinetic constants for the different samples during both the simulated gastrointestinal digestion process and the combined
oral
and
gastrointestinal
digestion
process.
The
equilibrium starch hydrolysis percentages varied from 84.2 % to 21
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95.9 % and there was no significant difference among the values (p > 0.05), regardless of the type of digestion step. In contrast, the kinetic constants significantly increased as the degree of homogenization increased: 120 s > actual chewed ≥ 1 s > intact, for both the
PT
gastrointestinal and combined processes. These results indicated
RI
that the kinetic constants during the small intestinal stage were
SC
influenced by the oral digestion step and the degree of homogenization. In fact, degree of homogenization major impacted
NU
on the kinetic constant in starch digestibility to salivary α-amylase. These results also correspond with a previous observation that
MA
kinetic constant was affected on increase in enzyme accessibility to rice related to the extent that a grain’s structure is changed (Tamura
D
et al., 2016a, 2016b). As shown in the combined process (see Fig. 4),
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a larger starch hydrolysis percentage was observed at the gastric stage compared with the gastrointestinal process (see Fig. 2). The
CE
inclusion of the oral digestion step in the combined process also resulted in higher kinetic constant values for the samples examined,
AC
except for the sample that was homogenized for 120 s. When Germaine et al. (2008) investigated glycemic index (GI) and estimated glycemic index (eGI; calculated from kinetic constant and equilibrium hydrolysis percent) values of minced cereals that were treated with simulated salivary amylase, they concluded that the addition of simulated salivary amylase did not affect the GI value, and thus, could be omitted. However, the results of the present study 22
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reveal that the degree of homogenization that is achieved with the simulated oral digestion step influences the kinetic constant of starch digestibility in cooked rice. Moreover, a significant difference in starch digestibility was observed for the combined process except for the
RI
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sample that was only homogenized for 1 s.
SC
4. Conclusions
Our initial hypothesis was that rice grain digestibility would be
NU
affected by grain-scale changes rather than cell-scale structural changes, and these would be demonstrated in an oral digestion step.
MA
We observed that the effect of salivary α-amylase on the simulated oral digestion step influenced both the equilibrium percentage of
D
starch hydrolysis as well as the kinetic constant during the combined
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in vitro digestion process, and these would potentially affect the increases of postprandial blood glucose levels in humans. Thus, the
CE
simulated oral digestion step with an appropriate condition should be included in a simulated in vitro digestion process to evaluate the
AC
starch digestibility of foods containing structural carbohydrates, such as cooked rice grain.
23
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Figure Captions Fig. 1 Representative images of intact cooked rice grains (a), actual chewed grains (b), grains homogenized for 1 s (c), and grains homogenized for 120 s (d). The corresponding projected area distributions for panels a-c are presented in panel A, and
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the particle size distribution for the grains homogenized for 120 s is shown in panel B.
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Scale bar (a-d), 10 mm.
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Fig. 2 Changes in hydrolysis percent of starch during a simulated gastrointestinal digestion process for intact cooked grain versus cooked grains that were homogenized
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for 1 s or for 120 s. Error bars represent SD values (n = 3–4). Fig. 3 Changes in hydrolysis percent of starch for intact cooked grains (A), cooked
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grains that were homogenized for 120 s during treatment with SSF containing various concentrations of α-amylase (B) and actual chewed cooked rice with actual saliva (C).
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Error bars represent SD values (n = 3–4).
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Fig. 4 Changes in hydrolysis percent of starch for intact cooked grains, cooked grains homogenized for 1 s, cooked grains homogenized for 120 s, and actual chewed cooked
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rice samples during a simulated in vitro digestion process that combined oral and gastrointestinal processes. Error bars represent SD values (n = 3–4).
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Fig. 5 Morphological and histological images of an intact cooked grain sample (A, B, a, b), a cooked grain sample after digestion by the gastrointestinal process (C, D, c, d), and a cooked grain sample that was chewed and then digested by gastrointestinal process (E, F, e, f). Panels A, C, E, a, c, and e represent the morphological images and panels B, D, F, b, d, and f represent the histological images. Scale bars indicate 5 mm (A-F) and 1 mm (a-f).
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Table 1. Kinetic parameters of simulated in vitro starch digestion from intact cooked grain. C∞ (%)
k ×10-2 (min-1)
Intact
91.3 ± 2.6 a
1.1 ± 0.1 c
1s
95.9 ± 7.8 a
2.1 ± 0.9 c
120 s
88.2 ± 3.5 a
22.1 ± 0.5 a
Combined oral and
Intact
93.8 ± 9.4 a
gastrointestinal process
1s
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87.5 ± 6.2 a
6.9 ± 1.7 b
84.2 ± 2.3 a
20.9 ± 0.5 a
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120 s
Actual chewed
2.0 ± 0.3 c
90.2 ± 4.6 a
9.4 ± 2.1 b
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1 s / 120 s: samples were homogenized for 1 s or 120 s with a blender. Data are presented as
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the mean ± SD (n = 3–4). The superscripted letters indicate significant differences (p < 0.05).
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