J Food Bioact

Journal of Food Bioactives, ISSN 2637-8752 print, 2637-8779 online

Journal website www.isnff-jfb.com

Original Research

Volume 11, September 2020, pages 84-94

Effects of an herbal extract composed of white tea, roasted yerba mate and fermented rooibos on the antioxidant activity and sensory properties of popsicles manufactured with different protein sources

Figure 1. Total phenolic content (a) and condensed tannins (b) of popsicles manufactured with different sources of proteins, with or without the addition of the lyophilized herbal extract (LME). ¹Probability value obtained by the paired Student-t test; ²Probability value obtained by one-way ANOVA; Different lower case letters indicate a significant difference between popsicle manufactured using the same protein source, with and without LME; Different capital letters indicate a significant difference among all popsicle formulations; +extract = popsicle with addition of 1 g/100 g of lyophilized mixed extract; −extract = popsicle without adding 1 g/100 g of lyophilized herbal extract.

Figure 2. In vitro antioxidant activity (FRAP, A; DPPH, B; TRC, C) of popsicles manufactured with different protein sources, with or without the addition of the lyophilized herbal extract (LME). ¹Probability value obtained by the paired Student-t test; ²Probability value obtained by one-way ANOVA; Different lower case letters indicate a significant difference between popsicle manufactured using the same protein source, with and without LME; Different capital letters indicate a significant difference among all popsicle formulations; +extract = popsicle with addition of 1 g/100 g of lyophilized mixed extract; −extract = popsicle without adding 1 g/100 g of lyophilized herbal extract.

Figure 3. Overall sensory acceptance of popsicle formulations manufactured with different protein sources with or without the addition of the lyophilized herbal extract (LME). Note: The probability values are based on the paired Student-t test.

**Table 1.** Formulation of popsicles according to the protein source and addition of an herbal extract containing antioxidants
Formulations	Bovine milk whey protein (g/100 g)	Rice protein (g/100 g)	Lyophilized herbal extract (g/100 g)¹
¹Herbal extract composed of 82.9% white tea + 10.9% mate tea + 6.2% fermented rooibos tea.
F1	5	0	0
F2	5	0	1
F3	0	5	0
F4	0	5	1
F5	2.5	2.5	0
F6	2.5	2.5	1

Table 1. Formulation of popsicles according to the protein source and addition of an herbal extract containing antioxidants

Formulations

Bovine milk whey protein (g/100 g)

Rice protein (g/100 g)

Lyophilized herbal extract (g/100 g)¹

¹Herbal extract composed of 82.9% white tea + 10.9% mate tea + 6.2% fermented rooibos tea.

2.5

**Table 2.** Phenolic compounds in the lyophilized herbal extract characterized by UHPLC-QTOF MS in negative mode and quantified by HPLC/UHPLC methods
Phenolic compounds	UHPLC-QTOF MS: [M-H]⁻ (calculated mass)	MS²	Amount (mg/100 g)
Gallic acid (C₇H₆O₅)	169.0123 (169.0140)	125.053	815 ± 2
3-Caffeoylquinic acid (C₁₆H₁₈O₉)	353.0857 (353.0873)	191.051, 179.031	170 ± 3
5-Caffeoylquinic acid (C₁₆H₁₈O₉)	353.0852 (353.0873)	191.053, 179.035	207 ± 5
4-Caffeoylquinic acid (C₁₆H₁₈O₉)	353.0865 (353.0873)	179.031, 191.052	61 ± 4
Dicaffeoylquinic acid 1 (C₂₅H₂₄O₁₂)	515.1176 (515.1190)	353.086, 191.052, 179.031	<LOQ
Dicaffeoylquinic acid 2 (C₂₅H₂₄O₁₂)	515.1199 (515.1190)	353.086, 191.053, 179.031	138 ± 2
Dicaffeoylquinic acid 3 (C₂₅H₂₄O₁₂)	515.1198 (515.1190)	353.086, 179.031, 191.053	136 ± 2
Total phenolic acids			1,527 ± 19
Catechin (C₁₅H₁₄O₆)	289.0675 (289.0712)		19 ± 1
Epicatechin (C₁₅H₁₄O₆)	289.0704 (289.0712)		233 ± 12
Gallocatechin (C₁₅H₁₄O₇)	305.0638 (305.0661)		19 ± 1
Epigallocatechin (C₁₅H₁₄O₇)	305.0648 (305.0661)		1,060 ± 80
Epigallocatechin gallate (C₂₂H₁₈O₁₁)	457.0769 (457.0771)	305.064, 169.010	877 ± 18
Procyanidin B2 (C₃₀H₂₆O₁₂)	577.1340 (577.1350)	425.086, 289.070	18 ± 1
Gallolyated dimeric procyanidin (C₃₇H₃₀O₁₆)	729.1453 (729.1456)	577.098, 289.067, 169.006	310 ± 29
Total flavan-3-ols			2,536 ± 141
Myricetin-hexoside (C₂₁H₂₀O₁₃)	479.0827 (479.0826)	317.026	323 ± 38 (myricetin, total amount)
Myricetin-rhamnoside-hexoside (C₂₇H₃₀O₁₇)	625.1410 (625.1405)	479.081, 317.026
Quercetin-hexoside (C₂₁H₂₀O₁₂)	463.0879 (463.0877)	301.032	6,340 ± 310 (quercetin, total amount)
Quercetin-pentoside (C₂₀H₁₈O₁₁)	433.0750 (433.0771)	301.031
Quercetin-rhamnoside-hexoside 1 (C₂₇H₃₀O₁₆)	609.1467 (609.1456)	301.032
Quercetin-rhamnoside-hexoside 2 (C₂₇H₃₀O₁₆)	609.1471 (609.1456)	301.032
Quercetin-rhamnoside-dihexoside 1 (C₃₃H₄₀O₂₁)	771.1996 (771.1984)	301.033
Quercetin-rhamnoside-dihexoside 2 (C₃₃H₄₀O₂₁)	771.2012 (771.1984)	301.033
Quercetin-coumroyl-rhamnoside-hexoside	755.1804 (755.1823)	301.032
Kaempferol-hexoside	447.0905 (447.0927	285.039
Kaempferol-rhamnoside-hexoside (C₂₇H₃₀O₁₅)	593.1507 (593.1506)	285.083	1,010 ± 80 (kaempferol, total amount)
Kaempferol-rhamnoside-dihexoside (C₃₃H₄₀O₂₀)	755.2042 (755.2035)	285.038
Kaempferol-dirhamnoside-hexoside (C₃₃H₄₀O₁₉)	739.2075 (739.2086)	285.037
Apigenin-C,C-dihexoside 1 (C₂₇H₃₀O₁₅)	593.1493 (593.1506)	353.063	<LOQ (apigenin, total amount)
Apigenin-C,C-dihexoside 2 (C₂₇H₃₀O₁₅)	593.1512 (593.1506)	413.085
Apigenin-C,C-pentoside-hexoside (C₂₆H₂₈O₁₄)	563.1412 (563.1401)	353.063
Total flavonols			7,673 ± 428
Total phenolic content quantified			11,736

Table 2. Phenolic compounds in the lyophilized herbal extract characterized by UHPLC-QTOF MS in negative mode and quantified by HPLC/UHPLC methods

Phenolic compounds

UHPLC-QTOF MS: [M-H]⁻ (calculated mass)

MS²

Amount (mg/100 g)

Gallic acid (C₇H₆O₅)

169.0123 (169.0140)

125.053

815 ± 2

3-Caffeoylquinic acid (C₁₆H₁₈O₉)

353.0857 (353.0873)

191.051, 179.031

170 ± 3

5-Caffeoylquinic acid (C₁₆H₁₈O₉)

353.0852 (353.0873)

191.053, 179.035

207 ± 5

4-Caffeoylquinic acid (C₁₆H₁₈O₉)

353.0865 (353.0873)

179.031, 191.052

61 ± 4

Dicaffeoylquinic acid 1 (C₂₅H₂₄O₁₂)

515.1176 (515.1190)

353.086, 191.052, 179.031

<LOQ

Dicaffeoylquinic acid 2 (C₂₅H₂₄O₁₂)

515.1199 (515.1190)

353.086, 191.053, 179.031

138 ± 2

Dicaffeoylquinic acid 3 (C₂₅H₂₄O₁₂)

515.1198 (515.1190)

353.086, 179.031, 191.053

136 ± 2

Total phenolic acids

1,527 ± 19

Catechin (C₁₅H₁₄O₆)

289.0675 (289.0712)

19 ± 1

Epicatechin (C₁₅H₁₄O₆)

289.0704 (289.0712)

233 ± 12

Gallocatechin (C₁₅H₁₄O₇)

305.0638 (305.0661)

19 ± 1

Epigallocatechin (C₁₅H₁₄O₇)

305.0648 (305.0661)

1,060 ± 80

Epigallocatechin gallate (C₂₂H₁₈O₁₁)

457.0769 (457.0771)

305.064, 169.010

877 ± 18

Procyanidin B2 (C₃₀H₂₆O₁₂)

577.1340 (577.1350)

425.086, 289.070

18 ± 1

Gallolyated dimeric procyanidin (C₃₇H₃₀O₁₆)

729.1453 (729.1456)

577.098, 289.067, 169.006

310 ± 29

Total flavan-3-ols

2,536 ± 141

Myricetin-hexoside (C₂₁H₂₀O₁₃)

479.0827 (479.0826)

317.026

323 ± 38 (myricetin, total amount)

Myricetin-rhamnoside-hexoside (C₂₇H₃₀O₁₇)

625.1410 (625.1405)

479.081, 317.026

Quercetin-hexoside (C₂₁H₂₀O₁₂)

463.0879 (463.0877)

301.032

6,340 ± 310 (quercetin, total amount)

Quercetin-pentoside (C₂₀H₁₈O₁₁)

433.0750 (433.0771)

301.031

Quercetin-rhamnoside-hexoside 1 (C₂₇H₃₀O₁₆)

609.1467 (609.1456)

301.032

Quercetin-rhamnoside-hexoside 2 (C₂₇H₃₀O₁₆)

609.1471 (609.1456)

301.032

Quercetin-rhamnoside-dihexoside 1 (C₃₃H₄₀O₂₁)

771.1996 (771.1984)

301.033

Quercetin-rhamnoside-dihexoside 2 (C₃₃H₄₀O₂₁)

771.2012 (771.1984)

301.033

Quercetin-coumroyl-rhamnoside-hexoside

755.1804 (755.1823)

301.032

Kaempferol-hexoside

447.0905 (447.0927

285.039

Kaempferol-rhamnoside-hexoside (C₂₇H₃₀O₁₅)

593.1507 (593.1506)

285.083

1,010 ± 80 (kaempferol, total amount)

Kaempferol-rhamnoside-dihexoside (C₃₃H₄₀O₂₀)

755.2042 (755.2035)

285.038

Kaempferol-dirhamnoside-hexoside (C₃₃H₄₀O₁₉)

739.2075 (739.2086)

285.037

Apigenin-C,C-dihexoside 1 (C₂₇H₃₀O₁₅)

593.1493 (593.1506)

353.063

<LOQ (apigenin, total amount)

Apigenin-C,C-dihexoside 2 (C₂₇H₃₀O₁₅)

593.1512 (593.1506)

413.085

Apigenin-C,C-pentoside-hexoside (C₂₆H₂₈O₁₄)

563.1412 (563.1401)

353.063

Total flavonols

7,673 ± 428

Total phenolic content quantified

11,736

**Table 3.** Proximal composition and physicochemical properties of popsicles manufactured with different protein sources, with or without the addition of the lyophilized herbal extract (LME) containing antioxidants
Evaluated parameters	Bovine whey protein + LME	Rice protein + LME	Bovine whey protein + rice protein + LME
Different letters in the same line represent a statistical difference between popsicles (p < 0.05).
Proximal composition
Lipid (g/100 g)	1.02 ± 0.04^a	1.02 ± 0.03^a	1.00 ± 0.05^a
Ash (g/100 g)	0.71 ± 0.07^a	0.62 ± 0.03^a	0.47 ± 0.02^b
Protein (N × 6.38; g/100 g)	7.41 ± 0.47^a	7.54 ± 0.02^a	6.11 ± 0.13^b
Moisture (g/100 g)	82.51 ± 0.14^a	76.95 ± 0.32^b	82.90 ± 1.94^a
Carbohydrates + fibers (g/100 g)	8.35 ± 0.57^b	13.87 ± 0.28^a	9.52 ± 2.02^ab
Mean energy value (kCal/100 g)	72	95	71
Physicochemical properties
pH	6.29 ± 0.03^a	6.71 ± 0.01^a	6.61 ± 0.01^a
Titratable acidity (g/100 g)	0.63 ± 0.03^a	0.18 ± 0.01^c	0.28 ± 0.01^b
Total soluble solids (°Brix)	24	14	18

Table 3. Proximal composition and physicochemical properties of popsicles manufactured with different protein sources, with or without the addition of the lyophilized herbal extract (LME) containing antioxidants

Evaluated parameters

Bovine whey protein + LME

Rice protein + LME

Bovine whey protein + rice protein + LME

Different letters in the same line represent a statistical difference between popsicles (p < 0.05).

Proximal composition

Lipid (g/100 g)

1.02 ± 0.04^a

1.02 ± 0.03^a

1.00 ± 0.05^a

Ash (g/100 g)

0.71 ± 0.07^a

0.62 ± 0.03^a

0.47 ± 0.02^b

Protein (N × 6.38; g/100 g)

7.41 ± 0.47^a

7.54 ± 0.02^a

6.11 ± 0.13^b

Moisture (g/100 g)

82.51 ± 0.14^a

76.95 ± 0.32^b

82.90 ± 1.94^a

Carbohydrates + fibers (g/100 g)

8.35 ± 0.57^b

13.87 ± 0.28^a

9.52 ± 2.02^ab

Mean energy value (kCal/100 g)

Physicochemical properties

6.29 ± 0.03^a

6.71 ± 0.01^a

6.61 ± 0.01^a

Titratable acidity (g/100 g)

0.63 ± 0.03^a

0.18 ± 0.01^c

0.28 ± 0.01^b

Total soluble solids (°Brix)

**Table 4.** : Sensory data of popsicles manufactured with different protein sources, with or without the addition of an herbal extract containing antioxidants.
Formulations	Odor	Taste	Consistency	Color
¹Probability values obtained by one-way ANOVA; Different letters in the same column represent statistical difference between popsicle formulations.
Bovine whey protein – herbal extract	7.1 ± 1.5^ab	8.0 ± 1.3^a	7.9 ± 1.3^a	8.2 ± 1.0^a
Bovine whey protein + herbal extract	7.4 ± 1.6^a	7.7 ± 1.3^ab	7.6 ± 1.4^ab	8.2 ± 1.2^a
Rice protein – herbal extract	6.8 ± 1.8^bc	6.6 ± 1.9^c	6.5 ± 1.8^c	6.9 ± 1.7^b
Rice protein + herbal extract	6.4 ± 1.9^c	5.8 ± 2.0^d	6.3 ± 2.2^c	7.3 ± 1.7^b
Bovine whey protein + rice protein – herbal extract	7.5 ± 1.6^a	7.4 ± 1.5^b	7.3 ± 1.6^b	8.2 ± 1.3^a
Bovine whey protein + rice protein + herbal extract	7.1 ± 1.6^ab	6.8 ± 1.9^c	6.7 ± 1.9^c	8.3 ± 1.2^a
p-Value¹	0.001	<0.001	<0.001	<0.001

Table 4. : Sensory data of popsicles manufactured with different protein sources, with or without the addition of an herbal extract containing antioxidants.

Formulations

Odor

Taste

Consistency

Color

¹Probability values obtained by one-way ANOVA; Different letters in the same column represent statistical difference between popsicle formulations.

Bovine whey protein – herbal extract

7.1 ± 1.5^ab

8.0 ± 1.3^a

7.9 ± 1.3^a

8.2 ± 1.0^a

Bovine whey protein + herbal extract

7.4 ± 1.6^a

7.7 ± 1.3^ab

7.6 ± 1.4^ab

8.2 ± 1.2^a

Rice protein – herbal extract

6.8 ± 1.8^bc

6.6 ± 1.9^c

6.5 ± 1.8^c

6.9 ± 1.7^b

Rice protein + herbal extract

6.4 ± 1.9^c

5.8 ± 2.0^d

6.3 ± 2.2^c

7.3 ± 1.7^b

Bovine whey protein + rice protein – herbal extract

7.5 ± 1.6^a

7.4 ± 1.5^b

7.3 ± 1.6^b

8.2 ± 1.3^a

Bovine whey protein + rice protein + herbal extract

7.1 ± 1.6^ab

6.8 ± 1.9^c

6.7 ± 1.9^c

8.3 ± 1.2^a

p-Value¹

0.001

<0.001