Impact of in-vitro gastro-pancreatic digestion on polyphenols and cinnamaldehyde bioaccessibility and antioxidant activity in stirred cinnamon- fortified yogurt

Abstract In this study, cinnamon powder was supplemented into yogurt as a functional ingredient. The total phenolic compounds, individual phytochemicals and radical scavenging activity of the yogurts were measured and compared with a cinnamon water extract treated in the same way as the fortified yogurt. Cinnamon-fortified yogurt displayed higher total phenolic content (P


19
Developing of functional foods with health promoting natural ingredients has increased in the 20 past decade (Granato, Nunes, & Barba, 2017). The development of new products with 21 potentially positive effect on health using traditional herbs and food, which are known to be safe 22 from the toxicological standpoint, is generally desirable since there is an increasing interest 23 among consumers to look for healthier and natural food (Granato et al., 2017). Traditional herbs 24 and food used to improve the functionality of food are normally chosen because rich in phenolic 25 compounds, which possess strong antioxidant activity and show protective effects against 26 chronic diseases including diabetes, cardiovascular diseases and cancer (Del Rio et al., 2013). In 27 the Middle East and Arab countries, cinnamon powder is a well-known and commonly used 28 food and traditional herbal medicine. Cinnamon showed several beneficial health properties 29 such as anti-tumoural, cardiovascular, cholesterol lowering, and antioxidant activities 30 (Gruenwald, Freder, & Armbruester 2010;Hlebowicz, Darwiche, Bjorgell, & Almer, 2007;31 Hlebowicz et al., 2009). Cinnamon polyphenols mainly consist of condensed tannins 32 (oligomeric and polymeric procyanidins) and monomeric phenolic compounds such as flavonols 33 and phenolic acids (Gu et al., 2004;Helal, Tagliazucchi, Verzelloni & Conte, 2014). 34 Cinnamaldehyde is also a major component in cinnamon bark, which exhibits several biological 35 effects such as anti-tumoural, pro-apoptotic and anti-inflammatory activities (Chao, et al., 2008; tannins and individual free phytochemicals as well as for the radical scavenging activity 111 analysis. 112 113 2.5 Determination of total phenolic content total tannins content 114 Quantification of total phenolic compounds was carried out with the Folin-Ciocalteau assay as 115 reported by Singleton, Orthofer, & Lamuela-Raventós (1999). The clear supernatant, obtained as 116 described in section 2.4, was diluted at least three times to reduce the interferences due to the 117 digestive enzymes, bile salts and sucrose (Helal et al., 2014). In a 1.5 mL Eppendorf tube 118 790 μL of distilled water, 10 μL of diluted sample and 50 μL of the Folin-Ciocalteu reagent 119 were added and mixed. After exactly 1 min, 150 μL of 20% aqueous sodium carbonate was 120 added, the mixture was mixed and left to stand at room temperature in the dark for 120 min.

121
Detection was achieved at 760 nm.

122
Total tannins were determined according to Hagerman and Butler (1978) on the clear 123 supernatant of the sample containing cinnamon (cinnamon water extract, cinnamon-fortified 124 yogurt and the corresponding digested samples). Briefly, 1 mL of three times diluted sample 125 was added to 2 mL of standard protein solution (bovine serum albumin dissolved at a 126 concentration of 1 mg/mL in 0.2 mol/L acetate buffer, pH 5, containing 0.17 mol/L sodium 127 chloride). The solutions were mixed and allowed to stand at room temperature for 15 min and For the analysis of milk proteins-tannin interaction (Helal et al., 2014), 15 mg of cinnamon 136 powder was added to 1 mL of reconstituted milk so that the final concentration of milk proteins 137 in the assay was 3.5% (w/v) and of cinnamon powder was 1.5% (w/v). Samples were 138 immediately centrifuged after mixing and the pellets analysed for tannins content as reported 139 above. Two different methods were used to determine the radical scavenging activity, namely ABTS 143 and DPPH assays. ABTS radical scavenging activity was carried out according to Re et al. 144 (1999) and results expressed as mg ascorbic acid/100 g of yogurt or cinnamon water extract.

145
Three times diluted sample (40 μL) was added to 1960 μL of the resulting blue-green ABTS 146 radical cation. The mixture was incubated at 37°C for 10 min and the decrease in absorbance 147 measured at 734 nm.

148
DPPH method was carried out according to Behrad, Yusof, Goh, & Baba (2009) with slight 149 modification as reported by Illupapalayam et al. (2014). To 3 ml of 60 μmol/L DPPH in 150 methanol, 250 μL of three times diluted sample was added. Samples were incubated in the dark 151 and after 20 min, the absorbance was measured at 517 nm. Results were expressed as mg of 152 trolox/100 g of yogurt or cinnamon water extract.   Water extract of cinnamon bark powder, prepared using the same protocol as for yogurt 180 production but without milk (Figure 1), was characterized for its content in total and individual 181 phenolic compounds, total tannins and antioxidant activity. The total amount of phenolic 182 compounds extracted from cinnamon bark was 76.6 ± 4.2 mg of catechin/100 g of water extract. total phenolic/g of cinnamon powder and 33.6 mg of tannin/g of cinnamon powder (Helal et al.,192 2014) which is in agreement with the data found in this study.  this study have been already described in Cinnamon cassia in amount lower than that found in 211 this study (Helal et al., 2014;Klejdus & Kováĉic, 2016). Wide variation of phytochemical 212 concentration were found in Cinnamon cassia bark between single bark sticks, even within the 213 sticks of a package and also within bark samples originating from the same tree (Woehrlin,Fry,214 Abraham, & Preiss-Weigert, 2010). Quercetin-3-rhamnoside, kaempferol and quercetin have 215 been already reported in Cinnamon cassia at concentration similar or lower than that found in 216 this study (Prasad et al., 2009;Helal et al., 2014). Solvent used in the extraction procedure as 217 well as the provenience of the samples and other parameters (age, bark thickness, duration of 218 storage) certainly affect chemical composition of cinnamon bark. The amount of 219 cinnamaldehyde found in this study was in the range already reported (from about 9 to more 220 than 50 mg/g) for Cinnamon cassia (Shan et al., 2005;Woehrlin et al., 2010;Helal et al., 2014).

221
The total antioxidant activity of cinnamon extract was 129.1 ± 5.6 mg of ascorbic acid/100 g of 222 cinnamon water extract when the ABTS assay was applied. In the DPPH assay, the antioxidant bind and precipitate cinnamon polyphenols. In a previous study, Helal et al. (2014) found that 247 the addition of 25% milk to a cinnamon beverage determined a decrease of about 28% in total 248 polyphenols content and this decrease is a result of the formation of insoluble complexes 249 between cinnamon tannins and milk proteins. Indeed, the acidic pH, as that found in yogurt because of fermentation, may enhance the binding affinity between phenolic compounds and 251 milk proteins. Hala Mohamed et al. (2015) found that the optimum pH of the interactions 252 between tannins and milk caseins was at pH 5. In general, the formation of insoluble complexes 253 between proteins and tannins is maximum at pH values near the isoelectric point of the protein 254 (Hagerman & Butler, 1978). To gain more information, the interaction of milk proteins with 255 cinnamon tannins was investigated by precipitation assay. Milk proteins at concentration of 256 3.5% (w/v) were able to precipitate 27.4 ± 0.6 mg of catechin/100 g. This amount of precipitated 257 tannins explain more than 77% of polyphenols lost during yogurt preparation.

258
The most representative cinnamon monomeric phenolic compounds and cinnamaldehyde were 259 identified and quantified using HPLC in the supernatant of cinnamon-fortified yogurt ( Table 1). 260 As found in the cinnamon water extract, phenolic acids were present in higher concentration  Table 1). The recovery yield was different among 266 the different monomeric compounds. In the case of syringic acid, ferulic acid, quercetin and 267 quercetin-3-rhamnoside the recovery was higher than 50%, whereas coumaric acid and 268 especially kaempferol showed the lowest recovery. The addition of 7.5% sucrose had no 269 significant effect on monomeric phenolic content in the prepared yogurt mixture ( Table 1). This   extract before the digestion. The bioaccessibility index in the cinnamon-fortified yogurt was 318 86.7%, which was significantly higher (P<0.05) than that calculated for the cinnamon water 319 extract. The protective effect of yogurt matrix can be due to the initial binding between milk 320 proteins and tannins, which make them no longer available for the interaction with pepsin. As 321 the digestion proceeds, milk proteins are hydrolysed and tannins can be released from milk 322 proteins resulting in an increased total polyphenols bioaccessibility. Sucrose addition to 323 cinnamon-fortified yogurt did not induce any significant effect on bioaccessibility of polyphenols (Figure 2). These results clearly showed that yogurt matrix enhanced the gastro-325 intestinal stability and the bioaccessibility of cinnamon polyphenols.

326
The behaviour of monomeric phenolic compounds in fortified yogurt and cinnamon water 327 extract during the in-vitro digestion was investigated and the results shown in Table 2 found a decrease of 78% of syringic acid after digestion of a cinnamon tea. Quercetin-3-338 rhamnoside was found to be more stable than the corresponding aglycone ( Table 2). The 339 presence of the sugar moiety may increase the stability of the phenolic compounds as suggested 340 by Boyer et al. (2005). Coumaric acid content decrease of about 50% during digestion. Similar 341 behavior of coumaric acid during in-vitro digestion was already reported by other authors using 342 different food sources and cooking methods (Helal et al., 2014;Juaniz et al., 2017).

343
Ferulic acid and kaempferol showed the lowest decrease during pancreatic stage with a 344 bioaccessibility index of 89.3% and 84.5%, respectively. These results confirmed previously 345 reported data (Helal et al., 2014;Zaupa et al., 2014). Similarly, cinnamaldehyde was found to be 346 especially stable under in-vitro digestive condition as already suggested by Helal et al. (2014).

347
In-vitro gastro-intestinal digestion of the cinnamon-fortified yogurt resulted in a significant 348 higher concentration of phenolic acids and flavonols at the end of the pancreatic phase of digestion compared to the digested cinnamon water extract ( Table 2). As reported above, the 350 presence of yogurt matrix determined an initial low recovery yield of the individual phenolic.

351
However, as the digestion proceeded, low molecular weight phenolic compounds were released 352 from the food matrix to the gastro-intestinal fluids. The hydrolysis of caseins during digestion, 353 especially during the pancreatic phase, allowed the release of the bound compounds, resulting in 354 a higher bioaccessibility index respect to the cinnamon water extract. Previous studies showed 355 that the presence of dairy matrices significantly improved the total polyphenols recovery during 356 the digestion, as the interaction between polyphenols and milk proteins exhibited a protective 357 effect (Green, Murphy, Schulz, Watkins, & Ferruzzi, 2007). This interaction may provide a 358 physical trapping and increase the polyphenols stability during the digestion (Hasni et al., 2011).

359
During in-vitro digestion of the cinnamon water extract, cinnamaldehyde was quite stable with a 360 bioaccessibility index of 90.6%. In the case of cinnamon-fortified yogurt, the cinnamaldehyde 361 concentration significantly increased during peptic digestion (P<0.05). A further but not 362 significant increase was found also at the end of the pancreatic digestion. However, differently 363 from the monomeric phenolic compounds, the bioaccessibility index of cinnamaldehyde was 364 lower (P<0.05) in the cinnamon-fortified yogurt compared to the cinnamon water extract (Table   365 2). The presence of sucrose had no significant effect on phenolic acids, flavonols and 366 cinnamaldehyde bioaccessibility ( Table 2). 367 Changes in radical scavenging activity were also evaluated during the in-vitro digestion, and the 368 data are presented in Figure 3. The radical scavenging activity of plain yogurt progressively 369 increased in both the assays during digestion as a result of the further release of antioxidant 370 peptides and amino acids encrypted in the milk proteins sequences .

371
On the contrary, no significant changes in the radical scavenging activity of the cinnamon water 372 extract were found during the in-vitro digestion with both the assays. At the end of the 373 pancreatic digestion, the cinnamon-fortified yogurt showed the highest radical scavenging activity values with both the assays. The presence of sucrose had no significant effect on radical 375 scavenging activity values ( Table 2).

377
Cinnamon powder was successfully employed for the production of cinnamon-fortified yogurt.

378
The supplemented samples contained cinnamon polyphenols in amounts lower than those 379 present in the cinnamon water extract but contained more total phenolics and exhibited higher  powder and added at 12% (w/v) concentration. Cinnamon powder was added at 1.5% (w/v) concentration. Sucrose was added at 7.5% (w/v) concentration. Cinnamon water extract was formulated in the same way as the cinnamon-fortified yogurt omitting milk powder from the preparation. After water addition, all the treatments were heat-treated at 95°C for 5 min followed by cooling to 45°C and then inoculated with starter culture and incubated at 45°C until the pH reached 4.4 (~8 h). Abbreviations: HPLC, high performance liquid chromatography.  results are shown. Note that the radical scavenging activity in cinnamon water extract (black columns) is referred to 100 g of cinnamon water extract. Values are means of three independent digestions ± standard deviation (SD). Different letters indicate significantly different values (P <  Table 1. Monomeric phenolic compounds and cinnamaldehyde content in cinnamon water extract and cinnamon-fortified yoghurts supernatant determined by HPLC. Results are expressed as μg or mg of individual compound in 100 g of water extract or yoghurt. a The recovery yield was defined as the percentage ratio between the concentration in the cinnamon-fortified yogurt and the concentration in the cinnamon water extract. Values represent means ± standard deviation of triplicate determination; different superscript letters within the same row indicate that the values are significantly different (P < 0.05).  Table 2. Effect of in vitro digestion on cinnamon monomeric phenolic compounds and cinnamaldehyde in cinnamon water extract and cinnamon-fortified yoghurts. Results are expressed as μg or mg of individual compound in 100g of water extract or yoghurt.

Cinnamon water extract
*Bioaccessibility index (BI%) of monomeric component is the percentage ratio between the post pancreatic concentration and the concentration before the digestion in the cinnamon water extract. Data are means ± SD (n=3). a-e Significant differences within the same column are shown by different letters (Tukey's test, P < 0.05).