Bioactive components. Total phenolic contents varied from 85.9 to 107 mg GAE/g across different ultrasonic processing conditions (Table 1). Amplitude had a significant effect (P ≤ 0.05) on total phenolic content during 10 to 15 min extraction time. The highest total phenolic content of 107 ± 1.0 mg GAE/g was obtained using the highest amplitude (50%) for 10 min. In contrast, the 30% and 40% amplitude (vs. 50%) for 10 min yielded significantly lower phenolic content (85.9 ± 1.7 and 91.5 ± 2.2 mg GAE/g respectively). A higher amplitude creates higher thermal energy to break the plant cellular structure. Increased permeability of cell walls and membranes and the breakdown of secondary metabolites from matrix interactions (polyphenols with lipoproteins) caused enhancement of polyphenols solubility and mass transfer. Thus, a higher ultrasonic amplitude increased extraction efficiency and yielded greater amounts of bioactive compounds 19,20. In this study, increased extraction time from 10 to 20 min at 50% amplitude resulted in lower phenolic content. Longer exposure time could increase solvent temperature beyond optimal levels, resulting in the degradation of thermo-sensitive compounds presented in the GSP samples. Evidently, the extraction condition of 10 min at 50% amplitude was deemed optimal for phenolic content.
Flavonoids have been shown to improve blood lipid profiles, enhance immunity, and have antioxidant, antibacterial and antitumor properties 21. Table 1 shows a significant (P ≤ 0.05) time × amplitude interaction. Increasing extraction time from 10 to 20 min increased flavonoid content only at 30% amplitude, while at 40% and 50% amplitude, total flavonoid increased up to 15 min before declining at 20 min. When considering the effect of amplitude, a higher amplitude resulted in a higher flavonoid content only in the 10-min extraction groups. In contrast, the 50% amplitude mitigated flavonoid content significantly (P ≤ 0.05) in the 20-min extraction groups. Loss of flavonoids at higher amplitude or longer extraction time was due to overheating by the ultrasound treatment, which especially affected the heat-sensitive flavonoids 22. Moreover, flavonoids (e.g., rutin) were more sensitive to thermal degradation than phenolic acids. The concentration of rutin from olive leaves using ultrasound-assisted extraction was 2.11 ± 0.1 mg/g during longer extraction time of 21 min which was lower than the extraction time of 7 min (2.22 ± 0.1 mg/g) 23. From the results, the total flavonoid contents of the extract increased with increasing time from 10 to 15 min, after which the values reduced slightly. According to Bi et al. 24, the gradual increase in the bioactivity of the extract with time may be attributed to the fact that polyphenols, and other bioactive compounds were still bound within the cell matrices during the early stage of extraction. The sufficient time was thus required to allow for their release. The subsequent decrease in bioactivity might be due to the longer time of exposure to ultrasonic conditions, inducing the degradation or oxidation of these bioactive compounds. Based on these findings, the extraction condition of 15 min at 50% amplitude was deemed optimal for flavonoid content. Therefore, the suitable range of extraction time for the subsequent optimization process was chosen to be from 10 to 15 min.
Table 1. Total phenolic (mg GAE/g) and flavonoid (mg CAE/g) content and antioxidant activity based on DPPH and FRAP (%) of ultrasound-assisted green soybean pod extracts as a function of time and the ultrasonic amplitude level.
Time (min)
|
Amplitude
(%)
|
Total phenolic content
(mg GAE/g)
|
Total flavonoid content
(mg CAE/g)
|
Antioxidant activities
(µmol Trolox/g)
|
DPPH
|
FRAP
|
10
|
30
|
85.9 ± 1.7 d
|
6.19 ± 0.2 e
|
24.4 ± 0.1 a
|
45.4 ± 0.1 a
|
40
|
91.5 ± 2.2 c
|
6.56 ± 0.7 d
|
24.3 ± 0.3 ab
|
45.3 ± 0.1 a
|
50
|
107 ± 1.0 a
|
7.75 ± 0.9 c
|
24.1 ± 0.3 abc
|
45.6 ± 0.1 a
|
15
|
30
|
90.9 ± 2.2 c
|
8.50 ± 0.3 abc
|
23.6 ± 0.3 bcd
|
45.4 ± 0.1 a
|
40
|
93.2 ± 2.0 c
|
8.69 ± 0.6 ab
|
23.6 ± 0.7 abcd
|
45.5 ± 0.1 a
|
50
|
102.7 ± 1.0 b
|
8.94 ± 0.3 a
|
24.1 ± 0.1 abc
|
45.4 ± 0.1 a
|
20
|
30
|
90.6 ± 2.0 c
|
8.81 ± 0.3 ab
|
23.3 ± 0.3 d
|
45.4 ± 0.1 a
|
40
|
91.3 ± 2.3 c
|
7.94 ± 0.9 bc
|
23.4 ± 0.4 cd
|
45.5 ± 0.1 a
|
50
|
90.2 ± 2.3 c
|
5.50 ± 0.5 e
|
24.0 ± 0.9 abcd
|
45.5 ± 0.1 a
|
Data are expressed as means ± standard deviation (n = 3). Different letters (a-e) in the same column represent statistically significant difference (p ≤ 0.05). DPPH = 2,2-diphenyl-1-picryl-hydrazyl radical; FRAP = ferric reducing antioxidant power.
Antioxidant activity. DPPH assay had been used widely and was a popular technique to assess the free radical scavenging activity of different plant extracts. DPPH free radical reduction was determined by the decrease in its absorption at 517 nm when the color of the DPPH assay solution changed from purple to light yellow. The scavenging potential of plant extract antioxidants corresponds to the degree of the discoloration 25. The effect of different conditions on DPPH radical scavenging activity of GSP extracts (Table 1) revealed high antioxidant activity of all sample extracts in the range of 23.3 - 24.4 µmol Trolox/g. Moreover, the 10 min, 30% amplitude has the highest DPPH scavenging activity (24.4 ± 0.1 µmol Trolox/g). Amplitude did not significantly influence DPPH activity at any extraction time, while longer extraction time seemed to decrease DPPH activity at the 30% and the 40% amplitude. It was observed that the longer of the extraction time than 15 min had significant influence on the antioxidant extraction (P ≤ 0.05). This might be due to the small particle diameter of the green soybean pod powder, as the result indicated that 10-15 min was sufficient to reach the maximum efficiency of the extraction. These results were consistent with an earlier report by Wang et al. 26, who found no increase in total content of phenolic and flavonoid with extraction time beyond 15 min when extracting blueberry leaves using ultrasonic extraction. Moreover, Ćujić et al. 27 reported no difference between the total phenolic content from dried chokeberries obtained after 30 and 60 min ultrasonic extraction in 50% ethanol. It was noteworthy that longer extraction time in water could lead to the decrease in antioxidant activity. It was evident that with some plant materials, excessive extraction duration in water may cause degradation of some target compounds resulting in reduced contents. In contrast to the DPPH results, FRAP antioxidant activity did not differ (p ˃ 0.05) between treatment conditions. The highest FRAP activity (45.6 ± 0.1 µmol Trolox/g) was exhibited by samples extracted with 50% amplitude with extraction time of 10 min. Moreover, the variation trend of FRAP values was consistent with the total phenolic contents. These results were in accordance with Hassan et al. 28 who observed that phenolics contents of brown seaweed extract using UAE with a working frequency fixed at 42 kHz and a power of 100 W had a close correlation with FRAP antioxidant. In addition, there was no significant difference (p < 0.05) of total phenolic contents and FRAP antioxidant power of brown seaweeds extract for extraction time of 20 min (4.23 ± 0.1 mg GAE/g and 4.83 ± 0.1%) and 30 min (4.34 ± 0.2 mg GAE/g and 5.09 ± 0.1%), respectively.
The correlation between UAE conditions and response variables could be fit with quadratic and linear models, as follows:
Total phenolics = 28.63 + 7.31T + 0.13A – 0.11T2 + 0.03A2 – 0.11TA (p < 0.0001, R2 = 0.90)
Total flavonoids = -20.17 + 2.93T + 0.34A – 0.06T2 -0.02TA (p < 0.0001, R2 = 0.77)
DPPH = 27.21 – 0.26T – 0.06A + 4.86TA (p = 0.0005, R2 = 0.42)
FRAP = 45.14 + 9.22T + 3.56A (p = 0.0102, R2 = 0.24)
where T was extraction temperature and A was ultrasonic amplitude. However, the R2 for for DPPH and FRAP models were less than 0.5. Therefore, optimization criteria was set for maximum total phenolics and total flavonoids. The response surfaces of these variables are shown in Figure 1. The highest values occurred with 50% amplitude and extraction time of 12.5 min which yielded the total phenolics value of 104 mg GAE/g and total flavonoids of 8.61 mg CAE/g.
Evaluation of antioxidant and sensory properties of green soybean milk fortified with GSP Extracts. Bioactive compounds comprised an excellent pool of molecules for the production of nutraceuticals, functional foods, and food additives 29. The pod of green soybean waste was collected from shelling process before the seed was grounded for milk production. The GSP extracts produced from the optimized UAE were used as natural antioxidants in term of food additive for improving the oxidative stability in green soybean milk. Phenolic and flavonoid contents as well as antioxidant activity of GSP-fortified green soybean milk are shown in Table 2. Among all samples, the 3% fortified milk sample had the highest phenolic (136 ± 0.5 mg GAE/g) and flavonoid (109 ± 0.5 mg GAE/g) content and the highest DPPH (176 ± 1.9 µmol Trolox/g) and FRAP (248 ± 0.3 µmol Trolox/g) antioxidant activity. In a similar study, Dabija et al. 30 revealed that the fortification of yogurt with hawthorn (Crataegus monogyna) extracted in the increasing concentration levels (0.25, 0.50, 0.75, and 1% (w/w)) could promote the higher total phenolic content (3.46, 3.88, 4.22, 4.34 mg GAE/ mL, respectively) and DPPH activity (19.23, 21.60, 32.02, and 33.38%, respectively). Lee et al. 31 also found that the increase in concentration level of Inula britannica flower extract for cheese fortification from 0.25 to 1.0% (w/v) caused an increase of total phenolic content and DPPH of from 54.8 to 70.8 mg of GAE/g and from 53.3 to 79.1%, respectively. It was thus evident that the bioactive compounds and antioxidant activities of green soybean milk could be enhanced by the pod extracted fortification compared to milk alone.
Table 2. The content of phenolic and flavonoid, antioxidant activities of green soybean milk fortified with pod extracted.
Pod extracted in green soybean milk (%)
|
Total phenolic content
(mg GAE/g)
|
Total flavonoid content
(mg CAE/g)
|
Antioxidant activities
(µmol Trolox/g)
|
DPPH
|
FRAP
|
0 (Control)
|
81.3 ± 0.8c
|
42.0 ± 0.9c
|
53.2 ± 1.2d
|
239 ± 0.4b
|
1
|
115 ± 2.5b
|
85.3 ± 1.2b
|
125 ± 1.1c
|
240 ± 0.2b
|
2
|
114 ± 2.8b
|
85.7 ± 1.1b
|
132 ± 1.9b
|
240 ± 0.1b
|
3
|
136 ± 0.5a
|
109 ± 0.5a
|
176 ± 1.9a
|
248 ± 0.3a
|
Data are expressed as means ± standard deviation (n = 3). Different letters (a-c) in the same column represent statistically significant difference (p ≤ 0.05). NS = non-significant.
Sensory evaluation of fortified green soybean milk was conducted by 100 untrained panelists on a 9-point structured scale, with 9 being the best and 1 the worst quality. All sensory attributes were in the range of 5 - 8 indicating that all formulae were at least moderately acceptable. Addition of 3% (v/v) GSP extracts resulted in higher aroma (6.24 ± 1.6), sweetness (5.88 ± 1.6) and saltiness (5.94 ± 1.7) ratings, and a lower color (7.28 ± 1.2) rating, compared to the control formula (Table 3). The lower appearance rating may be attributed to the intense green color of the product due to addition of the GSP extract, which increased the green color, but reduced the luminosity of the milk. More intense green color was not generally well accepted by consumer. Tamer et al. 32 reported that lemonade with 5% (v/v) green tea was rated lower in terms of color compared to control samples (0%). In addition, Farhan et al. 33 found that yogurt fortified with mint leave extracts had a lower color score than the control. Although color was directly related to consumer acceptability of the product 34, overall acceptability scores did not differ between the 3% formula and the control. In fact, the color of the 3% (v/v) GSP-fortified milk, which was supposedly the greenest, was accepted equally to the color of the control. The panelists preferred the 3% formula the most, even more than the 2% formula. Based on the favorable sensory and antioxidant results, the 3% (v/v) GSP-fortified milk was selected for quantification of phytochemicals by HPLC.
Table 3. Sensory analysis of green soybean milk fortified with GSP extracts. Product preference was evaluated using a 9-point hedonic scale.
Green soybean pod fortification (%)
|
Color
|
Texture
|
Aroma
|
Sweetness
|
Saltiness
|
Overall
|
0 (Control)
|
7.39 ± 1.2a
|
6.75 ± 1.5a
|
6.01 ± 2.0a
|
5.74 ± 1.8b
|
5.34 ± 2.0b
|
6.46 ± 1.7ab
|
1
|
7.24 ± 1.1ab
|
6.67 ± 1.5a
|
6.20 ± 1.6a
|
6.07 ± 1.7ab
|
5.90 ± 1.7a
|
6.46 ± 1.5ab
|
2
|
7.16 ± 1.4b
|
6.37 ± 1.5b
|
6.01 ± 1.7a
|
6.12 ± 1.7a
|
5.76 ± 1.8a
|
6.17 ± 1.5b
|
3
|
7.28 ± 1.2ab
|
6.96 ± 1.7a
|
6.24 ± 1.6a
|
5.88 ± 1.6ab
|
5.94 ± 1.7a
|
6.54 ± 1.4a
|
Data are expressed as means ± standard deviation (n = 100). Different letters (a-c) in the same column represent statistically significant difference (p ≤ 0.05). NS = Non-significant.
Quantitative analysis of phytochemicals composition. Quantification of phytochemical contents (procyanidins, quercetin, glycitein, daidzein, genistin, and linalool) in GSP extracts and green soybean milk with and without addition of GSP were determined using HPLC (Figure 2). The most abundant phytochemicals in GSP were procyanidins (0.72 ± 0.01 mg/100 g) followed by linalool (0.69 ± 0.11 mg/100 g) and quercetin (0.47 ± 0.02 mg/100 g). The procyanidin content in the GSP extracts in the present study was higher than lentils (0.5 mg/100 g) 35. Compared to GSP, greater amounts of phytochemicals were observed in green soybean seed, especially procyanidins (3.89 ± 0.04 mg/100 g), linalool (2.79 ± 0.01 mg/100 g), glycitein (1.36 ± 0.01 mg/100 g) and quercetin (1.14 ± 0.01 mg/100 g).
These results were not surprising because bean seeds are nutrient- and antioxidant-rich 36. Hence, the green soybean milk had more content of these phytochemical group than the pod extracted sample. Nonetheless, the GSP extracts contained greater amounts of daidzein and genistein than the green soybean milk, according to Avanza et al. 8 who compared the content of polyphenols from cowpea seeds and pods in the extracts of water by pressurized liquid extraction. Although the result showed the higher polyphenol content in pods than in seeds, there were remarkable differences between the analyzed of flavonoids group. Cowpea seed extracts exhibited higher content on quercetin, procyanidin, and other tetrahydroxylated flavonoids than pod extracts. Regarding pod extract, the presence of gallic, ferulic acids, and o-hydroxybenzoic acid were in greater abundant. These results might be due to the different groups of polyphenol compounds (flavonoid and phenolic acid) existed naturally in legume pods and seeds. Procyanidins were a subclass of flavonoids found in commonly consumed foods such as fruits, vegetables, legumes, grains, and nuts, which had attracted increasing attention due to their potential health benefits 37. In addition to antioxidant properties, procyanidins had been reported to exhibit anticancer 38, anti-infectious, anti-inflammatory, cardioprotective, antimicrobial, antiviral, antimutagenic, wounding healing, antihyperglycemic as well as anti-allergic activities 39. Moreover, polyphenol compounds like quercetin were reported to have neuroprotective properties attributed to their inhibiting activity against enzyme acetylcholinesterase 40. Other polyphenols, such as genistein, daidzein, and glycitein were main phytoestrogens in the form of isoflavones. Phytoestrogens can also suppress the clinical symptoms of menopause caused by a decrease in the production of endogenous estrogen. Several studies had proven the protective effects of phytoestrogens on cardiovascular disease which could decrease total cholesterol and improve heart function 41. Linalool was present at rather high concentration levels in pod extract. This kind of phytochemical was acyclic monoterpene which was an important odorous constituent in a series of plant aromas. Linalool and linalool-rich essential oils were also known to exhibit various biological activities such as antimicrobial, anti-inflammatory, anticancer, anti-oxidant properties. In fact, several in vivo studies have confirmed various effects of linalool on the central nervous system 42. Fortification of green soybean milk with GSP extracts enhanced both the antioxidant activity and the phytochemical content and variety of the products, thus its increasing nutritional values.