Inhibitory Effect of Four Types of on the in vitro Digestion of Starch

Alyssa Gutierrez – University of Alabama Jiannan Feng – University of Alabama Libo Tan – University of Alabama Lingyan Kong – University of Alabama

Deposited 09/01/2020

Citation of published version:

Gutierrez, A., Feng, J., Tan, L., Kong, L. (2020): Inhibitory Effect of Four Types of Tea on the in vitro Digestion of Starch. Food Frontiers. DOI: https://doi.org/10.1002/fft2.39

© 2020 The Authors. Food Frontiers published by John Wiley & Sons Australia, Ltd and Nanchang University, Northwest University, Jiangsu University, Zhejiang University, Fujian Agriculture and Forestry University For published full text, send request to [email protected]

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2 Inhibitory Effect of Four Types of Tea on the in vitro Digestion of Starch

3

4 Alyssa Gutierrez1, Jiannan Feng2, Libo Tan2,*, Lingyan Kong2,*

5 6 1 Department of Biological Sciences, the University of Alabama, Tuscaloosa, AL 35487

7 2 Department of Human Nutrition and Hospitality Management, the University of Alabama,

8 Tuscaloosa, AL 35487

9 10 11 12 13 14 15 16 * Corresponding authors.

17 Address:

18 407 Russell Hall, 504 University Blvd, Tuscaloosa, AL 35487, USA (L. Tan)

19 482 Russell Hall, 504 University Blvd, Tuscaloosa, AL 35487, USA (L. Kong)

20 E-mail address:

21 [email protected] (L. Tan)

22 [email protected] (L. Kong)

23

24

1 25 Abstract

26 Phenolic compounds have been shown to decrease the rate of starch hydrolysis by inhibiting

27 digestive enzymes for starch. Tea, rich in phenolic compounds, has been widely reported for its

28 beneficial health effects. This study explored the inhibitory abilities of four types of tea, i.e.,

29 (GT), tea (OT), (BT), and (WT), on the in vitro enzymatic

30 digestion of potato starch (PS) and high-amylose maize starch (HAMS), respectively, using two

31 in vitro digestion methods. All significantly inhibited the digestion of both starches, with a

32 few exceptions at some time points. The resistant starch content was increased by all four teas in

33 HAMS, but only by BT and WT in PS. The total phenolic contents in the teas could not predict

34 the inhibititotry ability of the teas, possibly due to the structural diversity of phenolic compounds

35 and the presence of other compounds that could also alter enzymatic activity. Our results

36 suggested that tea, regardless of its type, can slow down the enzymatic digestion of starch and

37 therefore imply a potential benefit for controlling postprandial hyperglycemia.

38 Keywords

39 Starch; in vitro digestion; resistant starch; tea; total phenolic content

40

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41 1. Introduction

42 Starch is a polysaccharide comprising of a large number of glucose units linked by α-

43 glycosidic bonds. Abundant in staple foods, e.g., wheat, maize (corn), rice, and potatoes, starch

44 is the most common dietary carbohydrate and serves as a major source of energy in human diets.

45 In the gastrointestinal tract (GIT), starch is digested by a series of enzymes into glucose for

46 absorption. The kinetics of starch digestion and that of glucose release/absorption as the energy

47 source for the body determine the nutritional quality of starch and starch-containing foods

48 (Zhang & Hamaker, 2009). Rapid starch digestion may cause postprandial hyperglycemia and

49 hyperinsulinemia cycle, which is involved in a variety of metabolic diseases including metabolic

50 syndrome, obesity, type 2 diabetes, and cardiovascular diseases (Byrnes et al., 1995; Ludwig,

51 2002). Starch in certain foods, especially heavily processed foods (e.g., refined flour), can be

52 rapidly digested and results in the rapid elevation of blood glucose levels. On the contrary,

53 certain starch portions are slowly digested and even indigestible, and therefore cause a slower

54 and steadier rise in blood glucose concentration.

55 While the structure, processing, and storage conditions of starch greatly dictates its rate of

56 digestion (Jayawardena et al., 2017; Tamura et al., 2019), another critical factor is the activity of

57 starch digestive enzymes in the GIT. ⍺-Amylase is the major enzyme responsible for starch

58 digestion in the intraluminal phase. It is present in the mouth as salivary ⍺-amylase and in the

59 small intestine as pancreatic ⍺-amylase. Recent studies have demonstrated the ability of phenolic

60 compounds, a large group of plant secondary metabolites, to inhibit the activity of ⍺-amylase and

61 thus slow the rate of starch digestion (Sales et al., 2012; Xiao et al., 2013). A natural and

62 abundant source of phenolic compounds is tea, which is also known to confer other health

63 benefits such as antioxidant, anti-inflammatory, and anti-tumor properties (Pan et al., 2013; Su et

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64 al., 2016; Yen-Chen et al., 2018). Yet, previous studies that examined the effect of tea

65 on starch digestion showed inconsistent results. For instance, Sun et al. (2018)

66 showed that tea polyphenols inhibited pancreatic ⍺-amylase activity, and Kwon et al. (2008)

67 reported that black tea (BT), green tea (GT), and oolong tea (OT) had a mild inhibitory effect

68 (ranging from 35 to 40 percentage inhibition) on pancreatic ⍺-amylase. On the contrary, Yang

69 and Kong (2016) found the same teas to enhance the enzymatic activity. This same study,

70 however, also found that high concentrations of isolated tea polyphenols mildly inhibited ⍺-

71 amylase activity. Such findings indicate that while polyphenols inhibit ⍺-amylase activity,

72 another component of tea may instead enhance it.

73 Despite the invaluable foundation set by previous studies, discrepancies clearly exist

74 regarding the effect of tea and tea polyphenols on ⍺-amylase activity and starch digestion.

75 Therefore, this study further investigated the topic via a multifaceted approach to assess the

76 effects of different types of tea, including GT, BT, OT, and white tea (WT), on the digestion of

77 two types of starch, i.e., potato starch (PS) and high-amylose maize starch (HAMS). Two

78 simulated in vitro digestion methods that haven been widely adopted by different researchers

79 were utilized. One measured the total reducing sugar contents at 1 h and 2 h upon digestion using

80 a method outlined by Flores et al. (2014), while the other measured solely the glucose contents at

81 20 min and 120 min using a method described by Englyst et al. (1992). The former method aims

82 to obtain the percent inhibition by the inhibitors, i.e., the teas. The latter method established the

83 starch digestibility profiles, where the glucose content at 20 min represents rapidly digestible

84 starch (RDS), that at 120 min is slowly digestible starch (SDS), and the remaining starch is

85 regarded as resistant starch (RS) (Englyst et al., 1992). The total phenolic content in the teas

86 were determined to evaluate a potential correlation with their inhibitory effects. The two

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87 starches, with varied in their amylose contents (approximately 25% and 80% (w/w) in PS and

88 HAMS, respectively), were utilized to assess the potential outcomes of differing amylose content

89 in starch.

90 2. Materials and Methods

91 2.1 Materials

92 High amylose maize starch (HAMS; Gelose 80) was obtained from Ingredion (Bridgewater,

93 NJ, USA). Potato starch (PS, S2004), amyloglucosidase from Aspergillus niger (EC 3.2.1.3,

94 A7095, 300 U/mL), porcine pancreatin (EC 232-468-9, P7545, 8 x USP), invertase from baker’s

95 yeast (EC 3.2.1.26, I4504, 300 U/mg), guar gum (G4129), sodium acetate trihydrate (S7670),

96 maltose (M5885), and 3,5-dinitrosalicylic acid (128848), urea (U5378), lipase (L3126, EC

97 3.1.1.3), and porcine bile extract (B8631) were purchased from Sigma-Aldrich, Inc. (St. Louis,

98 MO, USA). Potassium sodium tartrate tetrahydrate (AAA10163-30), sodium phosphate

99 monobasic (97061-942), sodium phosphate dibasic (97061-588), sodium chloride (97061-904),

100 sodium bicarbonate (BDH9280) were purchased from VWR chemicals, LLC (VWR chemicals,

101 Solon, OH, USA). English breakfast black tea (BT) and green tea (GT) (Twinings@) were

102 purchased from a local grocery store. White tea (WT) and oolong tea (OT) were purchased from

103 grocery stores in Anxi and Anji (Fujian, China), respectively. D-glucose assay kit (GOPOD

104 format, K-GLUC) was purchased from Megazyme (Wicklow, Ireland).

105 2.2 Brewing of tea samples

106 Four types of tea leaves (1.0 g) were brewed with 40.0 mL of boiling deionized (DI) water

107 for 20 min, cooled to the room temperature (20 °C), filtered with Whiteman No. 4 filter paper,

108 and labeled as GT, BT, OT, and WT, respectively.

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109 2.3 Determination of total phenolic content

110 The fast blue salts BB assay was adopted to determine the total phenolic content in the

111 brewed tea samples according to Lester et al. (2012). In detail, 1.0 mL of 100-fold diluted tea

112 samples was mixed with 1.0 mL of 0.1% (w/v) Fast blue BB for 30 s, followed by adding 0.1 mL

113 of 5.0% (w/v) NaOH. The mixture was stirred with a vortex mixer and incubated for 90 min

114 under light at room temperature (20 °C). After that, absorbance was measured at 420 nm using a

115 Mettler Toledo UV5Bio UV-vis spectrophotometer (Mettler Toledo, Columbus, OH, USA).

116 standard solutions were used to establish the standard curve and results were

117 expressed as milligram of gallic acid equivalents (GAE) per liter of tea.

118 2.4 In vitro digestion – Flores method

119 In the first in vitro digestion study, two types of raw starch, PS and HAMS, were subject to

120 in vitro digestion with and without the presence of brewed teas according to the method outlined

121 by Flores et al. (2014). Specifically, 1.0 mL of each tea sample was added with 0.5 g of starch

122 sample in a 50 mL test tube, followed by adding 6.0 mL of simulated intestinal fluid and 3.0 mL

123 of bile fluid. The compositions of simulated digestive juices were shown in Table 1. The pH of

124 the simulated digestive juices was adjusted with 1 M HCl or 1 M NaOH. As a control, 1.0 mL of

125 DI water was added instead of brewed tea. In order to account for the interference in absorbance

126 measurements by particles other than sugar (e.g., digestive fluid components and tea residuals), a

127 blank that contained neither starch nor tea was used. The digestion trials were conducted in an

128 orbital shaking water bath at 37 °C with a shaking speed of 160 rpm for 2 h. At 1 h and 2h of

129 digestion, 0.5 mL aliquots were collected and immediately placed in a boiling water bath for 10

130 min to deactivate the enzymes and halt the reaction.

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131 Table 1. Compositions of simulated digestive juices. Simulated intestinal juice Bile juice 500 mL DI water 500 mL DI water 7.012 g NaCl 5.259 g NaCl 0.564 g KCl 0.376 g KCl 3.388 g NaHCO3 5.785 g NaHCO3 80.0 mg KH2PO4 0.25 g Urea 50.0 mg MgCl2 15.0 g Bile salts 0.1 g Urea 9.0 g Pancreatin 1.5 g Lipase pH 8.1 ± 0.2 6.9 ± 0.1 132 After the reaction was terminated, each sample was centrifuged at 2700 g for 3 min and the

133 supernatant was then diluted to a proper concentration. The reducing sugar content was then

134 determined using the 3, 5-dinitrosalicylic (DNS) colorimetric assay. For the assay, 0.2 mL of the

135 diluted samples was combined with 0.1 mL of DNS reagent. This solution was heated in a

136 boiling water bath for 8 min and allowed to cool to room temperature (20 °C). DI water (0.9 mL)

137 was then added to each sample for further dilution. Absorbance was measured at 540 nm using a

138 Mettler Toledo UV5Bio UV-vis spectrophotometer. Absorbance readings were subtracted by the

139 reading of the blank sample to eliminate the interference of foreign particles on light scattering.

140 The standard curve was established using maltose standard solutions and the results were

141 reported as maltose equivalent (mM).

142 2.5. In vitro digestion – Englyst method

143 The second in vitro starch digestion study was administered using the method reported by

144 Englyst et al. (1992) with a few modifications. Raw starch (80 mg) alone or raw starch mixed

145 with 1 mL of brewed tea were added into 20 mL round-bottom test tubes. A test tube with no

146 starch and inhibitors was used as the blank. Also mixed into the test tubes were 10 mg guar gum,

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147 6 mm glass beads, and 4 mL of 0.1 M acetate buffer. The reaction mixtures were placed in a

148 water bath at 37 °C. Immediately before the digestion reaction, an enzyme solution, containing

149 pancreatin (46.8 mg/mL prior to centrifugation), amyloglucosidase (13 U/mL), and invertase

150 (187.5 U/mL), was prepared by dissolving or diluting the enzymes in deionized water. Enzyme

151 solution (1 mL) was added into each tube at a certain time interval and this time interval was

152 accurately followed for sampling purpose. The test tubes were capped and placed into a shaking

153 water bath at 37 °C and 150 strokes per min. After incubating for 20 and 120 min, aliquots of

154 200 µL were taken from the reaction mixture and the reaction was stopped by mixing into 900

155 µL of 66% (v/v) ethanol vigorously. These digesta samples were kept at 4 °C overnight and then

156 centrifuged (2700 g, 3 min). The glucose concentrations in the supernatant were measured using

157 the glucose oxidase-peroxidase (GOPOD) method and used to calculate the amount of digested

158 starch. The starch contents digested during the first 20 min and between 20 and120 min were

159 regarded as the RDS and SDS proportions, respectively. The amount of RS was determined as

160 the amount of starch remaining after 120 min of digestion. Results were adjusted according to

161 the measured total starch content (81.10 ± 5.72% and 100 ± 2.80% in PS and HAMS,

162 respectively), and expressed as a percentage of total starch.

163 2.6 Statistical Analysis

164 All experiments were conducted in duplicates. Data were analyzed by one-way analysis of

165 variance (one-way ANOVA) followed by Tukey multiple comparison test using the GraphPad

166 Prism software (San Diego, CA). The letters a, b, and c indicate statistically significant

167 differences, p < 0.05 (a > b > c).

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168 3. Results and Discussion

169 3.1 Effect of teas on starch digestion

170 In order to evaluate the inhibitory effect of the four different teas on in vitro enzymatic

171 starch digestion, two types of starch, i.e., PS and HAMS, which vary in their amylose content,

172 were subject to simulated in vitro digestion with and without the presence of teas. Two simulated

173 in vitro digestion methods were used to evaluate starch digestion. In the first method, the extent

174 of starch enzymatic digestion after 1 and 2 h was measured by the amount of digestion product,

175 primarily reducing sugars, released from digestion and expressed as maltose equivalent (Figure

176 1). Samples without tea (NI) served as a control that demonstrated baseline starch digestion. All

177 four teas, when compared to the control, displayed significantly (p <0.05) lower values of

178 reducing sugar from the digestion of both starch types. An exception was noted for OT in PS

179 digestion at 2 h. These results suggested that tea has an inhibitory effect on the digestion of

180 starch by ⍺-amylase in the pancreatin.

181 The digestion of HAMS and PS proceeded to approximately the same extent by 2 h, but the

182 digestion at 1 h was significantly lower for PS. The difference in the digestion behavior of

183 HAMS and PS may be attributed to the regularity and compactness of molecular arrangement in

184 the two types of starch. Zhang et al. suggested that enzymatic resistance of native granular starch

185 was probably due to the densely packed molecular order in the surface region of starch granules

186 (Zhang et al., 2015). This difference in digestion may therefore be explained by the higher

187 amylopectin content of PS. Compared with amylose, amylopectin molecules are much bulkier,

188 highly branched and more densely packed in molecular arrangement. These properties make

189 amylopectin more difficult to hydrate and be accessed by enzymes in the aqueous phase. Hence,

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190 the presence of more amylopectin in PS could have delayed the binding of ⍺-amylase and its

191 substrates, and thus lower the enzymatic digestion rate in PS.

192 193 Figure 1. Extent of HAMS and PS digested, without inhibitors (NI) and with the presence of 194 green tea (GT), black tea (BT), oolong tea (OT), and white tea (WT), expressed as maltose 195 equivalent, with and without the presence of teas after 1 h and 2 h of digestion. Significant 196 differences among treatments within each figure are denoted by different letters (a > b > c, p < 197 0.05). 198 The percent inhibition values of the teas on starch digestion at 1 and 2 h were obtained and

199 compared in Figure 2. It shows that the inhibitory ability on α-amylolysis showed no significant

200 difference among the four teas, except for a lesser inhibition on HAMS digestion by OT at 2 h.

201 Furthermore, the inhibitory percentage ranged from 8.87% to 20.45% at 2 h of digestion. The

202 lower limit indicates a particularly mild inhibitory effect.

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203 204 Figure 2. Percent inhibition of teas on HAMS and PS digestion after 1 h and 2 h of digestion 205 using green tea (GT), black tea (BT), oolong tea (OT), and white tea (WT). Significant 206 differences among treatments within each figure are denoted by different letters (a > b, p < 0.05). 207 Starch digestion is executed primarily by a series of enzymes in the GIT, including ⍺-

208 amylase and maltase (e.g., ⍺-glucosidase). The first simulated in vitro digestion method only

209 involved the former enzyme. It is important to note, however, that the latter enzyme has been

210 reported to be potently inhibited by polyphenols. In fact, studies that utilized polyphenols found

211 in oregano (Gutiérrez-Grijalva et al., 2019) and olive oil (Figueiredo-González et al., 2019)

212 demonstrated significantly greater inhibition on the activity of ⍺-glucosidase than on that of ⍺-

213 amylase. Another study reported that green tea extracts were a stronger inhibitor of

214 ⍺-glucosidase than acarbose, which is a known ⍺-glucosidase inhibitor and common medication

215 prescribed to diabetics (Yan et al., 2018). Hence, the second simulated in vitro digestion study

216 was conducted to include the latter enzyme and obtain starch digestibility profiles as affected by

217 different teas.

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218 In the second digestive method, glucose contents of samples at 20 min and 120 min were

219 measured in order to determine starch digestibility profiles, i.e., the amounts of RDS, SDS, and

220 RS (Figure 3). Again, starches with no inhibitor (NI) were used as a control to demonstrate

221 baseline starch digestion. The RS content in HAMS was 63%, which was very close to other

222 reports in high amylose maize starches, e.g., the Hi-Maize 260 starch (Ingredion, 2019) and our

223 recent report (Gutierrez et al., 2020). The RS content in PS was notably low, yet the RDS content

224 was close to our previous reported value (Gutierrez et al., 2020). Both HAMS and PS are

225 regarded as the type 2 RS, or RS2. The resistance mechanism of RS2 was proposed to be due to

226 the densely packed molecular arrangement on the starch granule surface (Zhang et al., 2015). In

227 particular, no other RS except for high amylose starches have been classified as dietary fiber by

228 the United States Food and Drug Administration (Food and Drug Administration, 2018). Similar

229 to the results of the first digestion method, all four teas inhibited the digestion of HAMS, which

230 is expressed as a significantly increased proportion of RS and decreased RDS. The nominal RS

231 content of HAMS was increased to up to 88% by adding BT. Comparable to results of the first

232 digestion method, PS again had exceptions in exhibiting inhibitory effects, where GT and OT

233 had no appreciable effect on RDS and RS contents. When the percent inhibition was calculated

234 from these digestion data (Figure 4), the inhibitory effect of the teas was very different between

235 the two types of starches. In digesting HAMS, the extent of inhibition was much higher than that

236 observed in the first digestion method, but similarly, OT exerted the lowest inhibition upon

237 digestion for 2h. The protocols, including the enzymes and their concentrations, were different

238 between the two methods and could lead to different results. Indeed, upon digesting PS for 2 h,

239 although it agreed with the first method that there was no difference in the percent inhibition

240 values among the teas, the percent inhibition range was slightly lower than that in the first

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241 method, and GT showed the relatively low inhibition. Overall, these results were consistent in

242 that the inhibitory effect of tea is greater in the presence of starch containing higher proportions

243 of amylose. Meanwhile, the inhibitory effect on starches with lower amylose contents depends

244 on the type of tea.

245 246 Figure 3. Starch digestibility profiles, presented as rapidly digestible starch (RDS), slowly 247 digestible starch (SDS), and resistant starch (RS) contents, of raw high amylose maize starch 248 (HAMS) and raw potato starch (PS) without inhibitors (NI) and with the presence of green tea 249 (GT), black tea (BT), oolong tea (OT), and white tea (WT). Error bars show standard deviation; 250 n=2. Significant differences among treatments within each starch digestion fraction are denoted 251 by different letters (a > b > c, p < 0.05). 252

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253 254 Figure 4. Percent inhibition of teas on HAMS and PS digestion after 20 min and 2 h of digestion 255 using green tea (GT), black tea (BT), oolong tea (OT), and white tea (WT). Significant 256 differences among treatments within each figure are denoted by different letters (a > b, p < 0.05).

257 3.2 Phenolic content and inhibitory ability

258 Measurements for the total phenolic content of the teas demonstrate significant difference (p

259 < 0.05), except for between GT and OT (Figure 5). GT and OT contained the highest total

260 phenolic content, while WT had the lowest. These results are similar to that of Annunziata et al.,

261 who measured the total phenolic contents of WT, GT, and BT using the Folin-Ciocalteu method

262 (Annunziata et al., 2018). They found that WT had the lowest and GT had the highest phenolic

263 contents. The classification of tea into GT, BT, OT, and WT is based on the degree of

264 fermentation or oxidation. GT is unfermented or un-oxidized, WT is only slightly fermented, OT

265 tea is a semi-, while BT is a fully fermented or oxidized tea (Weerawatanakorn et

266 al., 2015). Fermentation is known to reduce the phenolic content in tea (Jayasekera et al., 2014),

267 yet there are many other factors determining the phenolic content in different tea samples. In the

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268 present study, the total phenolic content was not a predictive variable for the inhibitory ability on

269 α-amylolysis, as there was no significant difference in the activity among the four teas (Figure

270 2). The inhibitory ability of the teas did not increase with their total phenolic content. One

271 possible reason is the diversity of phenolic compounds and their variance in inhibitory ability.

272 Phenolic compounds are a large group of compounds derived from a phenol, and even within the

273 same sub-group of phenolic compounds, their inhibitory effect may vary. For instance, the

274 inhibitory effect of (Mkandawire et al., 2013) and proanthocyanins (Zhang et al., 2016)

275 both increased with molecular weight and degree of polymerization. Another study that

276 examined two major polyphenols of tea, i.e., (-)-epigallocatechin (EGC) and

277 (-)‑epigallocatechin‑3‑gallate (EGCG), found that the starch digestion rate was unaffected by

278 EGC but was significantly inhibited by EGCG (Xie et al., 2019). Despite the highly related

279 structures of EGC and EGCG, there is an evident disparity in their inhibitory abilities. The total

280 phenolic content determined cannot reveal the structural diversity of all phenolic compounds in

281 the teas. This could be regarded as a limitation of the present study. Quantification of individual

282 components of tea phenolics could help unravel which phenolic components may have a more

283 profound inhibitory effect on starch digestion. Secondly, the digestive processes may have

284 caused the varying results in inhibitory ability. Chen et al. reported that intestinal digestion was

285 able to degrade proanthocyandins from Acacia mearnsii bark (Chen et al., 2018). Subsequently,

286 its ⍺-glucosidase inhibitory activities were reduced by approximately 47% compared to the bark

287 extract after only gastric digestion. The various types of tea polyphenols may be degraded to

288 different extents, and their digestive products likely vary in their bioactivities. Interestingly, the

289 degradation of phenolic compounds can be beneficial. Annunziata et al. found that even though

290 WT had the lowest total phenolic content compared to GT and BT, the intestinal digestion of WT

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291 extract led to the highest duodenal and colonic bioaccessibility (Annunziata et al., 2018).

292 Enhanced bioaccessibility may yield more potent antioxidant effects. Thirdly, the lack of

293 correlation may also be attributed to other components in tea such as and ions. Caffeine

294 was reported to enhance ⍺-amylase activity (Kashani-Amin et al., 2013). Ions have been shown

295 to generate varying effects on ⍺-amylase activity. For instance, Aghajari et al. (2002) reported

296 chloride ions to enhance ⍺-amylase activity, while nitrate ions had no significant effect (Aghajari

297 et al., 2002). In relation to this theme, the content of chloride and nitrate ions in GT, BT, and WT

298 have been found to differ significantly (Mincă et al., 2013). Accordingly, it is reasonable to

299 speculate that the mild inhibitory effect in many cases was a net result of the inhibitory effect of

300 tea polyphenols and the augmenting effects of other components such as caffeine and ions.

301 Given that the composition of the other components may differ among the teas, their

302 compounding effects could also explain the lack of correlation between total phenolic content

303 and inhibition percentage.

304 305 Figure 5. Total phenolic content, expressed as mg of GAE per liter of tea, in green tea (GT), 306 black tea (BT), oolong tea (OT), and white tea (WT). Significant differences among samples are 307 denoted by different letters (a > b > c, p < 0.05).

308 4. Conclusion

309 In this study, the inhibitory effect of four types of teas, specifically GT, BT, OT, and WT, on

310 the enzymatic digestion of PS and HAMS was evaluated using two simulated in vitro digestion

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311 methods. All teas, regardless of type, were able to significantly decrease the extent of starch

312 digestion, with a few exceptions at some time points. In the first digestion method, the extent of

313 digestion in both starches was the same at 2 h, while the slower digestion of PS at 1 h may be a

314 result of its high amylopectin content. The second method further involved an important starch

315 digestive enzyme, ⍺-glucosidase, on which polyphenolic compounds might exhibit high

316 inhibitory activities. Regarding starch digestibility profiles, RS was increased by all four teas in

317 HAMS but only by BT and WT in PS. Since HAMS displayed consistent and significant

318 decreases in starch digestion by all four teas, but PS did not, this is evidence that the amylose-

319 amylopectin contents and their molecular arrangement might contribute to the extent of

320 inhibition on digestion. Accordingly, the beneficial inhibitory effects may be most applicable in

321 the digestion of high-amylose starches. The total phenolic contents in the teas were significantly

322 different, except for green and oolong teas, but could not predict the inhibitory ability of the teas.

323 It was possibly due to the structural diversity of phenolic compounds and the presence of other

324 compounds, such as caffeine and ions, which could also alter enzymatic activity. Our in vitro

325 method utilized porcine amylase and amyloglucosidase from Aspergillus niger, and therefore the

326 teas may exhibit different results when using human enzymes or in vivo methods. Nevertheless,

327 the net results of including tea in in vitro starch digestion appear to exhibit potential as a means

328 of delaying starch digestion and glucose absorption. In addition to many known health benefits

329 of tea polyphenols, the results of this study would promote tea consumption as a potential way of

330 modulating glycemic response.

331 Conflict of Interest

332 The authors confirm that they have no conflict of interest to declare for this publication.

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