Efficient thermal treatment of radish (Raphanus sativus) for enhancing its bioactive compounds

Abstract

Old preserved radish (OPR), a traditional pickled-food of Asia, contains the healthy bioactive compounds, such as phenols and flavonoids. To preserve the phenols levels in radish by thermal treatment, which are decreased due to the polyphenol oxidase activity during long storage. Range of thermal processing evaluated to retain the maximum phenols level in the radish while processed at temperatures of 70 °C, 80 °C and 90 °C for 30 days. In this study, the bioactive compounds and antioxidant activity of thermal processing radish (TPR) were evaluated and compared with commercial products of OPR. Results showed the best condition of thermal processing, 80°C for 30 days, could increase the values of phenols, flavonoids and antioxidant activity that were 2.27, 2.74 and 2.89 times, respectively. When comparing the thermally processed radish or TPR with OPR, TPR has a higher content of phenols and flavonoids, indicating that the thermal processing was effective to increase the content of functional compounds in radish and significantly improved its nutritional values.

Introduction

The radish, Raphanus sativus, is the Brassicaceae family and a popular root vegetable throughout East Asia. In the previous studies, the radish was found to have bioactive compounds, such as vitamins C, lignin, phenols, flavonoids, and isothiocyanates (Banihani 2017). Besides, sulforaphane is a compound of radish used in the Chinese herbal medicines which inhibit cell proliferation of human cancer cells (Wu et al. 2020). Moreover, other studies also address numerous bioactivities of radish such as antioxidant, anti-inflammatory and anti-diabetic activities (Banihani 2017; Manivannan et al. 2019).

The root of radish has been consumed worldwide in the form of pickles, salads and vegetables due to the high nutritional values (Manivannan et al. 2019). Among these processing foods, a special radish pickled food, OPR was made from the fresh white radish dehydrated with salt, pressed, and pickled in a ceramic container without adding other ingredients and being preserved for years (Wei 2022; Li, et al. 2021). The OPR has a long shelf life, and can be kept at room temperature up to 20 ~ 50 years (Wang, et al. 2010). During the preserved period, the amino acids and reducing sugars lead to a Maillard reaction, turning its black appearance (Liu et al. 2016) and increasing its functional components level. Recent studies show that OPR contains bioactive compounds like phenols, flavonoids among others (Li, et al. 2021; Hsieh 2021). Previous studies reported that phenolic compounds of OPR elicit certain health benefits, such as antioxidant activity, antibacterial activity, decreased serum lipid levels and lipid-lowering activity (Li, et al. 2021). The OPR requires long preparation time for storage. The longer the OPR is stored, the higher its value would become. The price of OPR can range from 100  to 10,000 US dollars/kg, reflecting the high value of OPR. Nevertheless, the OPR processing needs to spend time on storage for many years. In addition, the phenol content of OPR often decreases due to the activity of polyphenol oxidase in OPR during prolonged storage (Liu et al. 2016). Therefore, the process needs to ameliorate to prevent the loss of phenol content and preserve its antioxidant properties.

Thermal treatment is a widely used processing on food (Ando et al. 2017). After thermal processing, the foods have various chemical reactions such as the Maillard reaction, caramelisation and lipid oxidation such the non-enzymatic browning reaction to make the appearance change (Zhang et al. 2016; Bai 2021). Besides the colour change, such processing can change the ingredient and property in food, also increase bioactive compounds and change the constituents (Zhang et al. 2016; Choi et al. 2014). Take ginseng for instance, when ginseng is steamed at 100 °C, the ginseng turns to red whereas the ginseng turn in black when it is steamed at 120 °C (Kim et al. 2013). The report also shows that the concentration of less-polar ginsenoside which has anticancer activity increases significantly during ginseng processing at 120 °C (Kim et al. 2013). Moreover, similar thermal processing has also applied to the bitter melon and garlic foods (Hsieh 2021; Lee 2020; Sun et al. 2019). For example, the thermal processing of garlic at 70 °C for 30 days, it increases the phenol content in processed black garlic products (Zhang et al. 2016). Thermal processing of bitter melon at 70 °C for 12 days increases 5.12 times phenols level and increases the inhibitory effect of α-glucosidase up to 96.65% (Hsieh 2021). In addition, Maillard reaction products, which are responsible for the development of colour, are also regarded as a source of compounds related to enhanced antioxidation activity during the heat treatment of foods (Çelik et al. 2018). The results clearly showed that thermal processing has significantly improved the function properties of processed food than their unprocessed forms.

According to a report, the texture of radish changes and their cell structures are significantly broken during the thermal treatment above 70 °C (Ando et al. 2017). However, there is no reports so far addressing the effects of thermal processing on the radish and describing their functional components such as phenols, flavonoids and antioxidant activity. Therefore, the aim of this study is to evaluate the effect of different temperatures (70 °C, 80 °C and 90 °C) on the TPR processing from fresh radish and investigate the bioactive compounds and their antioxidant activity. The content of phenols and flavonoids change were analysed during thermal processing and compared with commercially available OPR.

Materials and methods

Materials

The radish used in this study was the Gan Zi variety, cultivated in the Yongkang District and Rende District, Tainan, Taiwan. The different OPR was purchased from Tainan market, Taiwan. All the chemicals used in this study were high performance liquid chromatography grade and obtained commercially.

Sample processing

TPR was manufactured at three different heating temperatures (70 °C, 80 °C and 90 °C) with 90% relative humidity. All treatments lasted for 30 days and the contents were analysed every 3 days.

All radish samples were lyophilised and ground to obtain powder form for later use. Each sample weighed 1 ± 0.01 g and was homogenised with 40 mL of distilled water, extracted with an ultrasonic equipment at room temperature for 30 min and filtered with filter paper. The obtained radish extract solution was stored at 4°C.

Colour change

The colour attributes of radish samples were measured with a colorimeter (TC-8600A, Tokyo Denshoku, Japan). Before testing, the colorimeter was calibrated using a Minolta standard white reflector plate. The data were presented as L (lightness), a (redness) and b (yellowness) values of the Hunter colour system (Choi et al. 2014). All measurements were done in triplicates. The colour attributes obtained were used to calculate total colour differences (ΔE) in Eqs. (1) as follows:

$$\mathrm{\Delta }E=\sqrt{{\left(L-L_0\right)}^2+{\left(a-a_0\right)}^2+{\left(b-b_0\right)}^2}$$ 1

Phenol content

The phenol content of the radish extract solution was determined by the Folin–Ciocalteu method. Firstly, a 50 μL of the radish extract solution was added, followed by 50 μL of Folin–Ciocalteu reagent and 0.2 mL of 20% Na₂CO₃ solution prior to vortexing the final mix. After 15 min of incubation at room temperature, 1 mL of ultra pure water was added into the mix. Finally, the absorbance was measured in a spectrophotometer (Thermo-1510, Thermo scientific, USA) at 725 nm and sample results were determined against the gallic acid equivalents (GAE) calibration curve. Results are presented in mg GAE/g of dry weight (d.w.) (Goyeneche et al. 2015).

Flavonoids content

The flavonoids content of the radish extract solution was determined using the Aluminium(III) chloride method. 100 μL of the radish extract solution were mixed with 100 μL of 2% methanolic AlCl₃ 6H₂O. The mixture was incubated for 10 min at room temperature and the absorbance of the mixture was measured in a spectrophotometer at 430 nm and compared with a quercetin equivalents (QE) calibration curve. Results are presented in mg QE/gdw (Lou et al. 2014).

1,1-diphenyl-2-picrylhydrazyl (DPPH) Radical Scavenging Activity Assay

Firstly, 50 μL of the radish extract solution was added to a 1.0 mL solution of 0.1 mmol/L DPPH dissolved in ethanol and mixed well via vortexing. After 60 min of incubation at room temperature in the dark, the absorbance was measured in a spectrophotometer at 517 nm (Lou et al. 2014). The DPPH radical scavenging activity is presented in Eq. (2) as percent inhibition:

$$\frac{A_{\text{control}}-A_{\text{sample}}}{A_{\text{control}}}\times 100\%$$ 2

A_control: Blank (distilled water + DPPH).

A_sample: Sample (extract solution + DPPH).

2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging activity assay

The ABTS radical scavenging activity of the radish extract solution was determined by Trolox equivalent antioxidant capacity (TEAC) methods. The 7 mmol/L ABTS solution was mixed well with 2.45 mmol/L K₂S₂O₈ (potassium perdisulfate) in a 1:1 ratio and incubated for 12 to 16 h at 4°C in the dark. 10 μL of the radish extract solution were added to each well, followed by 200 μL of ABTS work solutions and incubated at room temperature in the dark for 6 min. Absorbance was measured in a spectrophotometer at 734 nm and compared to a TEAC equivalents calibration curve (Sun and Wang 2018). Results are presented in mmol/L Trolox/gdw.

Ferric reducing antioxidant power (FRAP) activity assay

The FRAP of the radish extract solution was determined using the method with slight modification (Choi et al. 2014). The FRAP solutions included 300 mmol/L acetate buffer (3.1 g CH₃COONa· 3H₂O mixed with 16 mL CH₃COOH to 1L, pH 3.6), 10 mmol/L TPTZ (2,4,6-tripyridyl-s-triazine) solution dissolved in 40 mmol/L HCl and 20 mmol/L FeCl₃·6H₂O solution. The working solution was prepared by mixing acetate buffer, TPTZ solution and a 20 mmol/L FeCl₃·6H₂O solution in a 10:1:1 ratio. Then 12.5 μL of the radish extract solution was added with 187.5 μL of the FRAP solution and incubated the mix at room temperature in the dark for 30 min. Absorbance was measured by spectrophotometer at 593 nm and results of samples determined against the TEAC equivalents calibration curve. Results are presented in mmol/L Trolox/gdw.

Reducing power activity assay

The reducing power activity of the radish extract solution was determined using a method with slight modification. At first, 150 μL of the radish extract solution was mixed with 375 μL 200 mmol/L sodium phosphate buffer (pH 6.6,) and 375 μL 1% potassium ferrocyanide. The mixture was then incubated at 50 °C in a water bath for 20 min and cooled down. Later, 300 μL of 0.62 mol/L trichloroacetic acid solution was added to terminate the reaction and centrifuged at 1006.2 × g for 10 min. 800 μL of the supernatant was then mixed with 200 μL of deionised water and 500 μL of 6.17 mmol/L ferric chloride solution. The absorbance of the resulting solution was measured at 700 nm against deionised water as a blank. The absorbance value of the radish extract solution was higher when stronger reducing power was present (Choi et al. 2014).

Statistical Analysis

All values are expressed with means ± standard deviations. Statistical data analysis was performed with one-way Analysis of variance (ANOVA) and implemented by means of dispersion analysis using SPSS 20 software (2011; IBM Institute, Inc., Chicago, IL, USA). Significant differences were determined using Duncan's multiple range test at p < 0.05.

Results and discussion

The colour changes in TPR

The variability in colour change and overall appearance of radish depends on the different temperatures used which are summarised in Table 1. It was observed that the ∆E increases as per thermal processing time increases. Thermal processing for 30 days at 70°C, 80°C and 90°C, yielded ∆E values of 57.21, 57.79 and 52.90, respectively. These ∆E values increased continuously as processing temperatures increased from 70 and 80°C, possibly due to the Maillard reaction (Zhang et al. 2016). The reaction contains a reactive reducing sugar, reacting with an amino acid to produce N-substituted glycosylamine. The glycosylamine is unstable and therefore quickly undergoes to yield the Amadori rearrangement product, which forms aldehydes, ketones, or enolization. The products then undergo a series of reactions, generating volatile products. In the last stage, the products polymerised into Melanoidins, yielding a brown coloration(Çelik et al. 2018).

Table 1.

The value of ∆E changes in TPR at 70 °C, 80 °C and 90 °C during different thermal processing

Processing time (days)	Temperature (ºC)
	70 °C	80 °C	90 °C
0	0 ± 0Ae	0 ± 0Ae	0 ± 0Ab
3	43.23 ± 1.23Bd	48.41 ± 1.26Ad	50.98 ± 1.63Aa
6	49.90 ± 1.43Ac	53.16 ± 0.39Ac	51.55 ± 2.64Aa
9	53.20 ± 1.28Ab	53.47 ± 0.60Abc	52.30 ± 1.24Aa
12	52.77 ± 0.93Ab	53.72 ± 1.52Abc	51.93 ± 1.06Aa
15	54.97 ± 2.40ABab	55.10 ± 1.62Abc	53.68 ± 1.34Ba
18	56.29 ± 0.80Aa	55.63 ± 0.50Aab	52.47 ± 2.75Ba
21	56.18 ± 0.80Aa	56.27 ± 3.01Aab	52.68 ± 1.55Ba
24	56.74 ± 1.56Aa	56.53 ± 1.23Aab	51.81 ± 0.93Ba
27	57.57 ± 1.52Aa	57.56 ± 1.64Aa	51.80 ± 2.34Ba
30	57.21 ± 1.50Aa	57.79 ± 0.72Aa	52.90 ± 2.70Ba

Results are expressed as means ± standard deviation (n = 3). Different upper-case letter superscripts in the same row indicate significant difference at p < 0.05. Different lower-case letter superscripts in the same column indicate significant difference at p < 0.05

The result summarised in Table 1 showed that the colour change did not increase when processing temperatures reached 90°C after 6 days. In fact, ∆E was 52.47, at 90°C after 18 days, which is significantly lower than the values that obtained at 70°C and 80°C equal to 56.29 and 55.63, respectively. The result shown in Fig. 1A describe that the radish processed at 90°C exhibiting no significant colour change after 3 days, moreover, it has yielded a hard-bone-dry texture appearance and a burning smell. In addition, Fig. 1B showed that the high temperature causes rapid loss of surface moisture of the radish and facilitates quick drying prior to caramelisation. Therefore, the radish processing at 90ºC was not desirable. The previous study showed that high temperatures in thermal processing have a negative effect, such as black garlic presented with a hard texture and an obvious burning smell when processed at 90 °C (Zhang et al. 2016), these facts explain why higher settings are not used when processing the radish for storage food preparation.

Fig. 1.

The appearance (A) and the cross (B) section of TPR at 70 °C, 80 °C and 90 °C during different thermal processing

The content of phenols and flavonoids in TPR

The content of phenols and flavonoids changes in radish at different temperatures is shown in Fig. 2. The phenol content decreased from 3.37 mg/gdw to 1.88 and 2.92 mg/gdw during the first 3 days at 70°C and 80°C, respectively. Increase in content after the 3^rd day could probably be the reason that the heat sensitive phenols degraded at high temperature. Also, the thermal processing would let the phenol compounds migrate to the permeate solution and lead to the phenols loss due to the increased diffusion flow rate, and interfere with the selectivity of cell membranes (Moreno et al. 2016). After the 3^rd day, the phenol content gradually increased and reached to 6.05 and 7.64 mg/gdw at 70°C and 80°C on the 30th day, respectively. In contrast, the flavonoids content of radish at 70°C and 80°C increased continuously from 0.76 mg/gdw to 1.82 and 2.08 mg/gdw respectively. It was also found that the content of phenols and flavonoids increased first at 90°C, attaining maximum on the day 9th, 6.93 and 2.04 mg/gdw, respectively and subsequently decreased in the later days.

Fig. 2.

The content changes in the phenols (A) and the flavonoids (B) of TPR at 70 °C, 80 °C and 90 °C during different thermal processing (n = 3)

A different study showed that the content of phenols and flavonoids increased after thermal processing. Light and medium roasting of coffee beans yield a higher content of phenols and flavonoids by 10 to 50% as compared to green beans and dark roasted beans (Hečimović et al. 2011). Phenols in black garlic exhibited a similar trend, increasing 2.5 times from initial value 2 mg/gdw to 5 mg/gdw after thermal processing for 12 days at 75°C (Sun and Wang 2018). Studies have pointed out that thermal treatment could increase the free phenol content by decreasing the esterification form, glycoside form, breaking the ester bonding, and releasing the phenols from phenolic-containing macromolecules (Molaveisi 2019); or induced cell wall rupture and lead to the softening of fruits and vegetables to affect the extraction of phenolic substances. The study pointed out that the phenols are released from cells by thermal treatment and increase the absorbance efficiency (Sreeramulu and Raghunath 2010). The study has shown that both of the free and bound phenols could absorb, especially the free form could be freely available and more readily absorbed and exert beneficial bioactivities up on consumption (Molaveisi 2019). Then, phenols absorbance efficiency increases when they are in free form (Sreeramulu and Raghunath 2010). The report also showed that the flavonoids content in mushrooms increases and occurs in the free form after thermal treatment (Sun and Wang 2018), thus exerting more prominent bioactivity. These results show that the appropriate thermal processing conditions could increase the content of phenols and flavonoids, however excess temperature causes their losses.

Antioxidant activity of TPR

The changes in antioxidant activity of radish at different temperatures is shown in Fig. 3. The antioxidant activity of TPR approximately conformed to the content of phenols and flavonoids due to the similar trends during the thermal processing. When processed at 70°C and 80°C, the DPPH scavenging rate of radish increased from 36.92% to 64.53 and 70.53% in 30 days, respectively (Fig. 3A). The ABTS scavenging rate increased from 21.63 mmol/L Trolox /gdw to 29.68 and 38.57 mmol/L Trolox /gdw in 30 days, when treated at 70°C and 80°C, respectively (Fig. 3B). And the FRAP value increased from 13.54 mmol/L Trolox /gdw to 31.40 and 38.81 mmol/L Trolox /gdw in 30 days, for the same respective temperatures (Fig. 3C). The reducing power increased from 1.38 absorbance value to 2.22 and 2.67 in 30 days, at 70°C and 80°C respectively (Fig. 3D). The antioxidant activity would continuously increase due to the increase in phenols, also showed that the antioxidant activity possibly significantly exerted by the phenol content (Sreeramulu and Raghunath 2010). The study also shows that, when treated at 90ºC the antioxidant activity of radish exhibited the highest values, 67.37%, ABTS scavenging rate 38.53 mmol/L Trolox /gdw, FRAP 36.03 mmol/L Trolox /gdw and reducing power 2.67 in 9 days and decreased thereafter. Compared with the content of phenols and flavonoids, the antioxidant activity of TPR approximately conformed to the content of phenols and flavonoids due to the similar trends during the thermal processing.

Fig. 3.

The value of TPR changes in the anti-oxidant activity, including (A) DPPH scavenging rate; (B) ABTS scavenging rate; (C) FRAP; (D) Reducing power (n = 3), at 70 °C, 80 °C and 90 °C during different thermal processing

The previous study also shows that antioxidant activity increases after thermal processing. When roasting coffee, light roasts have 50% more antioxidant activity as compared to green beans (Hečimović et al. 2011). Antioxidant activity in black garlic also increased 6 times after 12 days of thermal processing at 75°C (Sun and Wang 2018). With honey over 10 days of thermal processing, the antioxidant activity of DPPH scavenging increased from 40 to 60, 70 and 90% at 45, 55 and 65ºC, respectively, suggesting that the higher thermal processing temperature remarkably increase the antioxidant which was according to increasing content of phenols and flavonoids (Molaveisi 2019). In addition to the content of phenols and flavonoids, the Maillard reaction products also contribute in antioxidant activity (Çelik et al. 2018). The previous findings also support the increasing trend of antioxidant activity of thermal processing which has been observed during the TPR processing in this study.

Comparison of the antioxidant properties and activity of TPR and commercially available OPR

The content of phenols and flavonoids change in TPR and commercially available OPR is shown in Table 2 and, confirmed that thermal processing increases the phenol content and it was maximum 7.64 mg/gdw at 80°C. Compared to commercially available OPR (2.36 to 4.29 mg/gdw), phenol content in thermal processing was about 1.78 times higher. Flavonoids content exhibited a similar increasing trend (2.08 mg/gdw) while comparing with commercially available OPR (0.25 to 0.72 mg/gdw) and was 2.89 times higher. The difference in antioxidant activity of TPR and commercially available OPR is also depicted in Table 2. The antioxidant activity also showed the similar increasing trend which was in line with the increased content of phenols and flavonoids. Overall, the radish after the thermal processing could offer about 1.79 to 3.86 times higher phenols and flavonoids and thus claim better health effects possibility than commercially available OPR.

Table 2.

Phenols content, flavonoids content and antioxidant activity of TPR and commercially available OPR

Sample Number	Processing temperature and time	Phenols content (mg/g)	flavonoids content (mg/g)	DPPH scavenging rate (%)	ABTS scavenging rate (mmol Trolox/g)	FRAP (mmol Trolox /g)	Reducing power
OPR A	Room temp. for 10 years	4.29 ± 0.05c	0.72 ± 0.05c	23.60 ± 0.30c	16.59 ± 1.04c	10.06 ± 0.60c	0.91 ± 0.03c
OPR B	Room temp. for 10 years	2.35 ± 0.07e	0.25 ± 0.01e	16.18 ± 0.29e	8.56 ± 0.11e	4.36 ± 0.43e	0.51 ± 0.02e
OPR C	Room temp. for 20 years	3.02 ± 0.04d	0.62 ± 0.20d	17.31 ± 0.24d	13.68 ± 1.25d	8.71 ± 0.50d	0.62 ± 0.02d
TPR 80 °C	80 °C for 30 days	7.64 ± 0.07a	2.08 ± 0.13a	70.53 ± 0.28a	38.57 ± 0.61a	38.81 ± 0.22a	2.67 ± 0.06a

Results are expressed as means ± standard deviation (n = 3). Different superscripts in the same column indicate significant difference at p < 0.05

OPR A: Meinong Mount Yueguang, Bai Yu old preserved radish; OPR B: Di’s old preserved radish; OPR C: Gao’s old preserved radish; TPR: thermal processing radish

The report showed that increasing phenols levels changes the characteristic of the product and has higher antibacterial and antioxidant activity (Li et al. 2021). Nevertheless, the other previous study showed that thermal treatment inactivates the polyphenol oxidase, allowing for endogenous biotransformation of precursor or intermediates turning into flavonoids to increase the content of phenols and flavonoids (Molaveisi 2019). Because the thermal treatment inactivated polyphenol oxidase and turned bound phenols into free form, TPR had a higher content of phenols and flavonoids, as well as antioxidant activity than commercially available OPR.

Conclusions

In the present study, optimum thermal process was determined to improve the phenolic compounds in TPR. Selected thermal processing speed up the processing of TPR and effectively improved the bioactive compound concentration as well as antioxidant property of TPR as compared to unprocessed sample. Thermal processing of radish at 80ºC determined as best condition. Compared to commercially available OPR, TPR exhibited higher bioactive compound concentration and antioxidant activity. Further studies can provide better insights about other bioactive compounds and their bioactivities. Optimized thermal process can be adopted for commercial production of TPR to offer better nutritional values and health benefits to the consumers.

Abbreviations

OPR

Old preserved radish

TPR

Thermal processed radish

GAE

Gallic acid equivalents

QE

Quercetin equivalents

DPPH

1,1-Diphenyl-2-picrylhydrazyl

ABTS

2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid

TEAC

Trolox equivalent antioxidant capacity

FRAP

Ferric reducing antioxidant power

TPTZ

2,4,6-Tripyridyl-s-triazine

Authors' contribution

MY: Data curation (equal); Formal analysis (equal); Methodology (equal); Supervision (equal); Writing-original draft (equal); Writing-review & editing (equal). C-YH: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Investigation (equal); Validation (equal). Y-AC: Conceptualization (equal); Investigation (equal); Resources (equal); Writing-review & editing (equal). J-TW: Funding acquisition (equal); Resources (equal). AKP: Validation (equal); Writing-review & editing (equal). C-DD: Validation (equal); Writing-review & editing (equal). C-KC: Conceptualization (equal); Validation (equal); Visualization (equal); Writing-original draft (equal); Writing-review & editing (equal). M-CL: Investigation (equal); Writing-review & editing (equal). C-WH: Conceptualization (lead); Methodology (lead); Project administration (lead); Resources (lead); Validation (equal); Writing-review & editing (lead).

Not applicable.

Funding

This work was supported by the Tainan City Government, Taiwan, Republic of China (Grant No. 108SBIR-Biotechnology 12). This research was partially supported by the Ajins Biomedical Co., Ltd (AS-2019001-aa1).

Availability of data and material

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Code availability

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Declarations

Conflict of interest

The authors declare no conflict of interest.

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All authors have read and agreed to the published version of the manuscript