Z-average and PDI of RBE, gelatin, and standard chemical compounds

Z-average and PDI of type A and type B gelatin at concentration of 1 and 2% (w/v) were presented in Figure 4.1. A 2% (w/v) of type B gelatin had the largest particle size (475.57 nm), followed by 1% (w/v) of type B gelatin (346.20 nm), and type A gelatin;

however, there was no significant difference between the Z-average of 1 and 2% (w/v) concentration (245.90, and 278.33 nm, respectively).

Figure 4.1 Z-average and PDI of gelatin

The size distribution of the particles in medium can be defined by PDI values. The PDI value ranges from 0.01 up to around 0.5 defines the monodispersed particles. On the other hand, PDI value greater than 0.7 is a characteristic of the polydispersed particles with broad size distribution (Donini et al., 2002). From the results, PDI values of 1 and 2% of type A gelatin, 1 and 2% (w/v) of type B gelatin were found to be 0.33, 0.32, 0.34, and 0.41,

104 respectively, which were lower than 0.5. These results revealed that all gelatin samples used in this study was monodispersed with narrow size distribution.

From Table 4.2, the RBE showed very large particle diameter (2451 nm) with broad size distribution (PDI = 0.97) because it contains various kinds of bioactive compounds, including phenolic acids, flavonoids, and anthocyanins which have different particle size. The Z-average values of major compounds contained in RBE ranged from 388.23−741.47 nm and the PDI values of all compounds were lower than 0.5.

Table 4.2 Z-average and PDI of RBE and standard chemical compounds

Sample Z-average (d.nm) PDI

RBE 2451 ± 40.31 0.97 ± 0.04

Phenolic acids

Protocatechuic acid 741.47 ± 28.68 0.12 ± 0.02

Vanillic acid 467.53 ± 39.56 0.16 ± 0.01

p-Coumaric acid 511.20 ± 13.35 0.24 ± 0.02

Ferulic acid 422.27 ± 17.86 0.22 ± 0.01

Sinapic acid 504.70 ± 46.24 0.22 ± 0.01

Flavonoids

Rutin 562.55 ± 9.12 0.46 ± 0.07

Myricetin 564.95 ± 41.65 0.39 ± 0.01

Quercetin 3-glucuronide 388.23 ± 19.82 0.28 ± 0.01 Anthocyanins

Cyanidin 3-glucoside 495.53 ± 34.89 0.31 ± 0.01

Peonidin 3-glucoside 460.80 ± 8.94 0.23 ± 0.01

Values are expressed as Mean ± S.D (n = 3).

105 4.3.3 Effect of simulated digestion on chemical properties of unencapsulated and encapsulated RBE

The percentage recovery of chemical properties (TPC, TFC, and antioxidant activity measured with FRAP assay) of the capsules under simulated gastrointestinal conditions are shown in Figure 4.2. The RBE encapsulated with 1% (w/v) of type A gelatin provided the highest percentage recovery of TPC, TFC, and FRAP after simulated saliva digestion (55.80, 81.23, and 86.79, respectively), as presented in Figure 4.2a. During in vitro gastric digestion (Figure 4.2b), there was no significant difference between percentage recovery of FRAP of RBE encapsulated with 1 and 2% (w/v) of type A gelatin (88.94, and 90.01%, respectively).

On the other hand, RBE encapsulated with 1% (w/v) of type A gelatin gave the highest percentage recovery of TPC (62.36%), whereas percentage recovery of TFC and FRAP were highest in RBE encapsulated with 2% (w/v) of type A gelatin (101.46, and 70.43%, respectively) during simulated intestinal digestion (Figure 4.2c). These FRAP results provide indirect evidence of structural inactivity of bioactive compounds after simulated digestion of capsules. During simulated digestion process, the structure of bioactive compounds might be changed from their original forms into the inactive forms, resulting in a low antioxidant activity value measured by FRAP assay. Therefore, capsules produced using 1% (w/v) of type A gelatin which provided the greatest value of percentage recovery of TPC, showed a lower value of percentage recovery of FRAP than that obtained using 2% (w/v) of type A gelatin.

106 Figure 4.2 Percentage recovery of chemical properties: TPC, TFC, and FRAP of unencapsulated (crude extract) and encapsulated RBE under simulated saliva (a), simulated gastric (b), and simulated intestinal (c) conditions

*1A = RBE encapsulated with 1% (w/v) of type A gelatin, 2A = RBE encapsulated with 2% (w/v) of type A gelatin, 1B = RBE encapsulated with 1% (w/v) of type B gelatin, and 2B = RBE encapsulated with 2%

(w/v) of type B gelatin

107 The chemical compositions of each capsule after in vitro gastrointestinal digestion were identified using HPLC. The results were calculated as percentage recovery of each compound (Table 4.3−4.5). From the results in Table 4.3, it was found that 1% (w/v) of type A gelatin had a higher capacity to protect total flavonoids (56.77%), whereas 2% (w/v) of gelatin showed a higher efficiency to preserve total anthocyanins (62.41%) from simulated saliva condition. In the case of simulated gastric digestion (Table 4.4), the RBE encapsulated with 2% (w/v) of type A gelatin provided a higher values of percentage recovery of total phenolic acids (38.22%), flavonoids, and anthocyanins (59.93%) than other samples. During in vitro intestinal digestion (Table 4.5), RBE encapsulated with 1% (w/v) of type A gelatin showed greater stability of total phenolic acids (30.12%) and anthocyanins (40.77%), while the highest percentage recovery of total flavonoids (29.63%) could be observed in those encapsulated with 2% (w/v) of type A gelatin. Nevertheless, vitamin E and γ-oryzanol could not be detected in all capsules due to the encapsulation technique. The encapsulation method used in this study is suitable for entrapping high-polarity compounds, including phenolic acids, and flavonoids. Therefore, low-polarity compounds including vitamin E and γ-oryzanol, could not be trapped in gelatin matrix using this method.

108

Table 4.3 Percentage recovery of bioactive compounds contained in RBE and capsules under simulated saliva condition CompoundsRBEType A gelatinType B gelatin 1%2%1%2% Phenolic acids Protocatechuic acid22.72c ± 1.6164.57a ± 4.5960.49ab ± 4.3054.12b ± 3.8525.92c ± 1.85 Vanillic acid25.12d ± 1.9263.59c ± 4.8581.09a ± 0.9570.45b ± 0.8326.83d ± 2.05 p-Coumaric acid80.96b ± 5.789.53e ± 0.67104.54a ± 2.2256.04c ± 4.0044.45d ± 3.18 Ferulic acid 20.85c ± 1.7346.74a ± 3.8418.19cd ± 1.5031.87b ± 2.6314.90d ± 0.01 Sinapic acid23.89d ± 0.4564.40b ± 1.2170.41a ± 1.1351.74c ± 1.03ND Total25.01c ± 1.4060.29a ± 3.3658.48a ± 0.7454.02b ± 0.6924.88c ± 1.41 Flavonoids Rutin21.32c ± 1.7361.88a ± 5.0313.14d ± 1.0712.84d ± 1.5230.32b ± 2.46 Myricetin68.17c ± 6.5166.56c ± 0.10101.08a ± 0.1084.83b ± 1.0032.66d ± 2.53 Quercetin 3-glucuronide17.94d ± 0.8151.22a ± 2.3243.36b ± 1.9746.37b ± 2.1021.46c ± 0.97 Total19.23d ± 1.6256.77a ± 2.6634.34b ± 1.6233.89b ± 1.5925.62c ± 1.20 Anthocyanins Cyanidin 3-glucoside12.56c ± 0.7823.07b ± 1.4336.71a ± 2.2823.25b ± 1.44ND Peonidin 3-glucoside21.16d ± 1.8940.96c ± 3.6573.88a ± 4.0753.38b ± 2.94ND Total17.48d ± 1.3233.32c ± 2.5262.41a ± 2.9042.00b ± 3.18ND Vitamin ENDNDNDNDND γ-oryzanol NDNDNDNDND a, b, row Values followed by different letters are significantly different (p ≤ 0.05). ND = not detectable.

109

Table 4.4 Percentage recovery of bioactive compounds contained in RBE and capsules under simulated gastric condition CompoundsRBEType A gelatinType B gelatin 1%2%1%2% Phenolic acids Protocatechuic acid20.23c ± 2.2532.80b ± 2.3313.81d ± 0.9843.99a ± 3.1323.35c ± 2.30 Vanillic acid16.23d ± 1.2441.71b ± 3.1862.45a ± 4.7715.10d ± 1.1534.67c ± 0.43 p-Coumaric acid39.58b ± 0.8277.58a ± 5.6127.37c ± 1.9834.06b ± 2.4327.78c ± 1.99 Ferulic acid18.83b ± 1.5518.87b ± 1.5654.12a ± 4.4716.71b ± 1.38 8.92c ± 0.74 Sinapic acid43.29c ± 0.8276.78a ± 1.4762.41b ± 1.0062.52b ± 1.2531.04d ± 0.62 Total19.56d ± 1.0933.54b ± 0.4438.22a ± 2.1330.58b ± 1.7124.14c ± 1.37 Flavonoids Rutin27.60c ± 2.24 45.84b ± 3.7362.41a ± 5.07 25.33c ± 2.055.44d ± 0.44 Myricetin65.06b ± 0.01109.33a ± 1.0048.34c ± 6.8979.77b ± 1.0620.73d ± 1.87 Quercetin 3-glucuronide23.82d ± 3.69 52.15b ± 2.3657.24a ± 2.6048.14c ± 2.189.20e ± 0.41 Total26.05d ± 1.22 53.07b ± 2.4959.93a ± 2.8140.21c ± 1.887.07e ± 1.01 Anthocyanins Cyanidin 3-glucoside45.57c ± 0.6682.59a ± 5.1165.13b ± 4.0462.14b ± 3.8529.19d ± 1.81 Peonidin 3-glucoside44.80c ± 2.4782.53a ± 7.3587.69a ± 4.8365.92b ± 5.8730.14d ± 1.66 Total44.05c ± 2.6885.73a ± 3.9783.63a ± 3.8766.80b ± 3.0930.57d ± 2.31 Vitamin ENDNDNDNDND γ-oryzanol NDNDNDNDND a, b, row Values followed by different letters are significantly different (p ≤ 0.05). ND = not detectable.

110

Table 4.5 Percentage recovery of bioactive compounds contained in RBE and capsules under simulated intestinal condition CompoundsRBEType A gelatinType B gelatin 1%2%1%2% Phenolic acids Protocatechuic acid23.80a ± 2.6515.21b ± 1.08 5.42c ± 0.3817.85b ± 1.27 4.26c ± 0.30 Vanillic acid25.87c ± 1.9753.84a ± 4.1144.19b ± 3.37 5.86d ± 0.0523.43c ± 1.79 p-Coumaric acid 77.96a ± 1.6262.98b ± 4.5214.35d ± 1.0413.77d ± 0.8021.74c ± 1.55 Ferulic acid17.52b ± 0.0123.31a ± 1.9213.97c ± 1.16 6.78e ± 0.5611.65d ± 0.01 Sinapic acidND53.95b ± 1.0357.61a ± 0.92 8.19d ± 0.1613.80c ± 0.28 Total23.40b ± 0.3030.12a ± 1.6819.44c ± 1.08 12.16d ± 0.6812.37d ± 0.70 Flavonoids RutinND16.47a ± 1.2718.42a ± 1.5010.28b ± 0.83 8.40b ± 0.68 MyricetinND40.82ab ± 5.7148.92a ± 6.9828.23bc ± 1.8020.10c ± 1.78 Quercetin 3-glucuronide19.12b ± 0.3516.95c ± 0.7639.87a ± 1.8119.53b ± 0.8920.13b ± 0.92 Total 9.56c ± 1.3715.34b ± 2.3829.63a ± 2.0315.17b ± 1.4615.63b ± 0.73 Anthocyanins Cyanidin 3-glucoside28.79a ± 1.7824.38b ± 1.5124.72b ± 1.53NDND Peonidin 3-glucosideND50.32a ± 4.4831.22b ± 2.78NDND Total12.29c ± 0.9340.77a ± 1.8928.43b ± 2.15NDND Vitamin ENDNDNDNDND γ-oryzanol NDNDNDNDND a, b, row Values followed by different letters are significantly different (p ≤ 0.05). ND = not detectable.

111 The overall results obviously exhibited that type A gelatin have a higher ability to protect bioactive compounds from RBE compared to type B gelatin. These results can be explained by the absolute values of ζ-potential and particle size of the RBE and gelatin, as described in section 4.3.1 and 4.3.2. The RBE had a negative surface charge because its major components are phenolic acids, flavonoids, and anthocyanins, which consist of negatively charged carboxyl and hydroxyl groups (Rawel et al., 2001). Therefore, they could provide stronger electrostatic interactions with the positively charged (type A) gelatin compared to negatively charged (type B) gelatin. From the result, it is clear that electrostatic interactions played an active role in an interaction between gelatin and bioactive compounds in RBE. Furthermore, gelatin can also interact with bioactive compounds through other bonds, namely hydrogen bondings, hydrophobic interactions, and covalent bondings (Aewsiri et al., 2009; Duconseille et al., 2015). Haroun and El Toumy (2010) studied the effect of polyphenols extract from Acacia nilotica bark on physicochemical properties of crosslinked gelatin-based polymeric biocomposite. They characterized the prepared films using Fourier transform infrared spectroscopy (FT-IR) and reported that hydrogen bonding and hydrophobic interactions were the major forces involved the stabilization of gelatin-based polymeric biocomposite film. Quiroz-Reyes et al. (2014) investigated the interactions among the gelatin protein matrix and polyphenolic extract from cocoa using FT-IR. They found that the C=O group of gelatin interacted with O-H groups of polyphenolic extract by hydrogen bonding.

In addition, the percentage recovery of chemical properties as well as chemical compounds of the RBE encapsulated with type A gelatin was also higher than those of unencapsulated RBE. The unencapsulated bioactive compounds have low stability under gastrointestinal digestion as described in many previous studies (Bermúdez-Soto, Tomás-Barberán, and García-Conesa, 2007; Correa-Betanzo et al., 2014; McDougall, Fyffe,

112 Dobson, and Stewart, 2007; Pérez-Vicente, Gil-Izquierdo, and García-Viguera, 2002; Saikia et al., 2015; Tagliazucchi et al., 2010). McDougall et al. (2007) examined the stability of anthocyanins from red cabbage under simulated gastrointestinal digestion. They reported that anthocyanins were effectively stable in the simulated gastric conditions but the total recovery after simulated intestinal digestion was low (around 25%) because anthocyanins broke down to form new phenolic components. Correa-Betanzo et al. (2014) have also studied the stability of wild blueberry polyphenols during simulated in vitro gastrointestinal digestion and found that total polyphenols and anthocyanins showed high stability during simulated gastric digestion, while polyphenol and anthocyanin contents decreased after simulated intestinal digestion.

Concentration of gelatin also has an influence on the formation of intra- and intermolecular crosslinks. Intermolecular crosslink is the interaction between functional group of two or more gelatin molecules, whereas intramolecular crosslink is the interaction within one molecule. At low concentration, intramolecular crosslinks are formed, while both intra- and intermolecular crosslinks occur at high concentration (Hernàndez-Balada, Taylor, Phillips, Marmer, and Brown, 2009; Yi, Kim, Bae, Whiteside, and Park, 2006), resulting in a high viscosity of gelatin solution. These phenomena may affect the Z-average values obtained by dynamic light scattering as well as the efficiency of capsules. In present study, it was found that the mean hydrodynamic diameter of 2% (w/v) of type B gelatin was significantly larger than 1% (w/v) concentration due to the formation of intermolecular crosslinks;

however, concentration seem to has no effect on the particle size of type A gelatin. These results were also observed in previous literature. Fruhner and Kretzschmar (1992) described that the viscosity of gelatin solution depends on the size and conformational changes of the molecules. The pH value applied in encapsulation process was around 5.8, which closed to the IEP of type B gelatin. Thus, gelatin might formed intra- and intermolecular crosslinks

113 formation and aggregated, resulting in the increase of viscosity. A high viscosity of the solution might prevent the internal interactions between gelatin and bioactive compounds from RBE, resulting in large and thick-walled capsules (Devi and Maji, 2010; Leo et al., 2000; Maji et al., 2007; Peanparkdee, Iwamoto, and Yamauchi, 2017). For this reason, 2%

(w/v) of Type B gelatin had a lower ability to entrap and protect bioactive compounds from RBE than 1% (w/v) concentration.