Effect of different cooking methods on the content of vitamins and true retention in selected vegetables

Abstract

This study evaluated the effect of different cooking methods including blanching, boiling, microwaving and steaming on the content of vitamins in vegetables. True retention was estimated using the yield expressed as a ratio of the weight of the cooked sample to the weight of the raw sample. The retention of vitamin C ranged from 0.0 to 91.1% for all cooked samples. Generally, higher retention of vitamin C was observed after microwaving with the lowest retention recorded after boiling. Cooked vegetables were occasionally higher contents of fat-soluble vitamins, including α-tocopherol and β-carotene, than that of their fresh counterparts, but it depends on the type of vegetables. Microwave cooking caused the greatest loss of vitamin K in crown daisy and mallow; in contrast, it caused the least loss of vitamin K in spinach and chard. Cooking may cause changes to the contents of vitamins, but it depends on vegetables and cooking processes.

Introduction

Vitamins are organic compounds and vital nutrients that cannot be synthesized and thus must be obtained through the diet. Although vitamins are usually needed in minute amounts for normal physiological functions such as maintenance, growth, and development, insufficient intake of vitamins gives rise to specific deficiency syndromes [1]. Many reports have also found various health benefits of vitamins. In detail, ascorbic acid and α-tocopherol have anti-aging and redox state regulation effects as powerful antioxidants [2, 3], α-tocopherol and β-carotene could be synergistic antioxidants and may prevent several cancers [4–6], and vitamin K intake is associated with reduced cardiovascular disease and vascular calcification [7].

Vegetables are excellent sources of vitamins including β-carotene (provitamin A), ascorbic acid, and vitamins E and K [8]. Several epidemiological studies have suggested that diets rich in vegetables are associated with reduced risks of chronic diseases [9]. Therefore, consumption of vegetables has rapidly increased in recent years due to their health benefits [10]. Prior to consumption, vegetables are usually cooked using heat treatments such as steaming, blanching, boiling, and microwaving. It is well known that cooking alters the nutritional value of fresh vegetables. However, whilst several studies have focused on how cooking changes the nutritional components and phytochemical content of vegetables, few have investigated the true retention of vitamins following exposure to different cooking methods [11–13]. Additionally, cooking or heat treatments can have a significant impact on the content of vitamins, and lead to an inaccurate estimation of nutrient intake. Therefore, it is necessary to establish nutritional information on the retention of vitamins from vegetables by the different processing methods. The true retention is an important component defining the ultimate importance of vitamins in the consumed vegetables.

The objectives of this study were to investigate the effect of different cooking methods including blanching, boiling, microwaving, and steaming, on the content and true retention of vitamins (i.e. β-carotene, ascorbic acid and vitamins E and K) in vegetables. In addition, analytical method validation parameters such as accuracy and precision were determined to ensure the validity of vitamin analysis.

Materials and methods

Materials

Ten vegetables including broccoli, chard, mallow, potato, sweet potato, carrot, crown daisy, pellia leaf, spinach, and zucchini were purchased from a local retail market (Chungbuk, Korea). Vitamin K and β-carotene were obtained from Waco Pure Chemical Industries (Osaka, Japan). Sodium acetate, acetic acid, zinc powder (particle size < 60 μm), α-tocopherol and γ-tocopherol were purchased from Merck (Darmstadt, Germany). Ascorbic acid, monobasic potassium phosphate, metaphosphoric acid, potassium hydroxide, and potassium carbonate were purchased from Sigma-Aldrich (St. Louis, MO, USA). All reagents and solvents were analytical and high performance liquid chromatography (HPLC) grade.

Cooking methods

The fresh vegetables were cleaned, washed, and cut into pieces before cooking treatment. All vegetables have been well done but firm to the bite. After removing the main part of the stem, the broccoli was cut into floweret pieces. The width, height, and length of the pieces were 4, 4, and 8, respectively. The carrot, potato and sweet potato were divided into quarters and cut into many cubes 2 cm on a side. The zucchini cut into rectangular pieces which has a width of 4 cm, a height of 2 cm, and a length of 4 cm. The detailed cooking conditions were listed in Table 1. Four different cooking methods were tested: boiling, blanching, steaming, and microwaving. For boiling, vegetables were added to distilled water that had just reached the boil in a glass pot (1:5, food/water). Cooking time was 5 min for broccoli, chard, mallow, carrots, sweet potato, crown daisy, perilla leaf, and spinach, and 20 min for potato and zucchini. For blanching, vegetables were added to boiled distilled water in a glass pot (1:5, food/water) and cooked on a moderate flame. Cooking time was 1 min for broccoli, chard, mallow, zucchini, crown daisy, sweet potato, perilla leaf, and spinach, 3 min for carrots, and 5 min for potato. After boiling and blanching treatments, samples were drained, stored at − 80 °C and lyophilized. For steaming, vegetables were steamed in a closed stainless steel pot using a stainless-steel steam basket above boiling distilled water for 5 ~ 20 min. Microwaving was carried out in a domestic microwave oven (Samsung, Suwon, Republic of Korea) without water. Sample were placed in a glass dish on the rotating plate of the oven and exposed to full power (700 W; 2452 MHz) for 2–5 min. After steaming and microwaving treatments, samples were stored at − 80 °C and lyophilized.

Table 1.

Cooking condition of vegetables

Common name	Scientific name	Boiling time (min)a	Blanching time (min)b	Steaming time (min)c	Microwaving time (min)d
Broccoli	Brassica oleracea	5	1	10	2
Chard	Beta vulgaris	5	1	10	3
Mallow	Amaranthus tricolor	5	1	10	2
Potato	Solanum tuberosum	20	5	20	3
Sweet potato	Ipomoea batatas	20	5	20	3
Carrot	Daucus carota	12	3	15	4
Crown daisy	Chrysanthemum coronarium	5	1	10	1
Perilla leaf	Perilla frutescens	5	1	10	1
Spinach	Spinacia oleracea	5	1	10	3
Zucchini	Cucurbita pepo	12	3	15	4

^aBoiled with 500 mL boiling distilled water and put it in the colander to drain for 2 min

^bBlanched with 500 mL boiling distilled water and put it in the colander to drain for 2 min

^cAfter adding distilled water in a steamer and boiled, steamed over medium heat

^dPlaced in microwave and cooked on both side

Analysis of vitamins C

Lyophilized samples of raw and cooked vegetables (0.2 g) were ground and added to 30 mL of 3% metaphosphoric acid solution and homogenized at 11,000 rpm for 2 min using a T25 basic ULTRA-TURRAX^® homogenizer (IKA-Werke GmbH & Co. KG, Staufen, Germany). The volume was made up to 50 mL with 3% metaphosphoric acid solution. The extract (2 mL) was centrifuged at 12,000 rpm for 3 min, and the supernatant filtered through a 0.45 μm polyvinylidene difluoride (PVDF) membrane filter (Whatman International Ltd., Maidstone, UK). All samples were immediately analyzed using an HPLC system, equipped with a PU-2089 pump, an AS-2057 auto injector, and a MD-2010 UV–vis variable wavelength detector (JASCO Corp., Tokyo, Japan). Separation was carried out in a CrestPak C18S column (150 × 4.6 mm, i.d., 5 μm, JASCO Corp.), and the isocratic elution was carried out with 0.1% trifluoroacetic acid in distilled water as a mobile phase for 15 min (flow rate 0.8 mL/min). The peak was read at 254 nm using an UV detector and quantification was determined via external calibration against ascorbic acid.

Analysis of vitamins E

Vitamin E content was determined using saponification extraction as outlined by Lee et al. [14], but with some modifications. Lyophilized samples (1.0 g) were ground and mixed with 20 mL of ethanol containing pyrogallol (6% wt/vol) and 8 mL of potassium hydroxide solution (60% wt/vol) and heated at reflux (70 °C, 50 min) with shaking in a water bath (NTS-1300, Eyela, Japan). The mixture was cooled in an ice bath and 20 mL of 2% sodium chloride solution and 20 mL of n-hexane:ethyl acetate (85:15, v/v) containing 0.1% butylated hydroxytoluene (BHT) were added as the extracting solvent. After vortexing for 2 min, the phases were allowed to separate, and extracted three times with 20 mL portions of the extracting solvent. The organic phases were collected and made up to 50 mL and the resulting solution, containing vitamin E, was evaporated under nitrogen gas. The residues were redissolved in n-hexane, filtered through a 0.45 μm polytetrafluoroethylene (PTFE) membrane filter (Whatman) and analyzed using an HPLC system equipped with a PU-2089 pump, an AS-2057 auto injector, and an FP-2020 fluorescence detector (JASCO Corp.). Analysis of vitamin E was performed on a LiChrosphere^® Diol 100 column (250 × 4 mm, i.d., 5 μm, Merck, Berlin, Germany) using a mobile phase of hexane/isopropanol (98.7:1.3, v/v) at a flow rate of 1.0 mlL/min. Peaks were detected by fluorescence using an excitation wavelength of 290 nm and an emission wavelength of 330 nm. Quantification was determined via an external calibration against α- and γ-tocopherols.

Analysis of vitamins K

Vitamin K was determined using the solvent extraction method outlined by Jakob and Elmadfa [15], but with some modifications. Lyophilized samples of raw and cooked vegetables (1 g) were ground and added to 30 mL of dichloromethane: methanol (2:1, v/v) and homogenized at 11,000 rpm for 2 min using a T25 basic ULTRA-TURRAX^® homogenizer. The extract was made up to 50 mL with methanol and 2 mL aliquot of the extract was transferred to glass tubes and evaporated under nitrogen gas. The residue was redissolved with n-hexane, and 8 mL of methanol:water (9:1, v/v) was added prior to centrifuging for 5 min at 2,000 rpm. The hexane layer was collected and evaporated to dryness. The residue was redissolved with methanol, filtered through a 0.45 μm PTFE membrane, and analyzed using an HPLC system equipped with a PU-2089 pump, an AS-2057 auto injector, and an FP-2020 fluorescence detector (JASCO Corp.). Analysis of vitamin K was performed on a Zorbax Eclipse XDB-C18 column (150 × 4.6 mm, i.d., 5 μm, Agilent, Palo Alto, USA) using a mobile phase of methanol:dichloromethane (9:1, v/v) and a flow rate of 1.0 mL/min. Peaks were detected by fluorescence using an excitation wavelength of 243 nm and an emission wavelength of 430 nm. Quantification was determined via an external calibration against phylloquinone.

Analysis of β-carotene

Lyophilized samples (1.0 g) were ground and carried out saponification for extraction of β-carotene from vegetables, as previously described by Lee et al. [14], but with some modifications. All samples were analyzed using an HPLC system equipped with a PU-2089 pump, an AS-2057 auto injector, and an MD-2010 UV–Vis variable wavelength detector (JASCO Corp.). Separation was carried out in a CrestPak C18S column (150 × 4.6 mm, i.d., 5 μm, JASCO Corp.). Analysis of β-carotene was performed on an Ascentis RP-Amide column (150 × 4.6 mm, i.d., 5 μm, Supelco, Bellefonte, USA) using a mobile phase of acetonitrile:methanol (7:3, v/v) and a flow rate of 1.0 mL/min. Peaks were measured at 473 nm using a UV detector and quantification was determined by external calibration against β-carotene.

Determination of true retention during cooking

True retention (TR) values for all vitamins were calculated using the following formula [16]:

$$\text{TR}(\%)=\frac{\text{Nutrient}\text{content}\text{per}\text{g}\text{of}\text{cooked}\text{food}\times \text{g}\text{of}\text{food}\text{after}\text{cooking}}{\text{Nutrient}\text{content}\text{per}\text{g}\text{of}\text{raw}\text{food}\times \text{g}\text{of}\text{food}\text{before}\text{cooking}}\times 100$$

Method validation

All analysis methods were validated by determining the precision (repeatability and reproducibility) and accuracy (recovery) [14]. The precision of the assay was defined as the coefficient of variation (CV); there were at least five repetitions. The recovery was calculated by the following equation: R% = [(Cs − Cp)/Ca] × 100, where R (%) is the recovery of added standard; Cs the vitamin content in spiked sample; Cp the vitamin content in sample; Ca the vitamin added.

Statistical analysis

One-way analysis of variance (ANOVA) was performed using SAS version 9.4 (SAS Institute, Cary, USA). Results were compared using Duncan’s test with a significance level of p < 0.05.

Results and discussion

The effect of cooking methods on vitamin C content in vegetables

The nutritional importance of vitamin C (l-ascorbic acid; 2,3-endiol-l-gulonic acid-γ-lactone) as an essential water-soluble vitamin is well established. Vitamin C is a cofactor in numerous physiological reactions, including collagen gene expression, peptide hormone activation, and carnitine synthesis; it is also an effective antioxidant. Therefore, adequate intake of vitamin C from food is vital for normal functioning of the human body [17]. In this study, Table 2 shows the content of vitamin C in raw and cooked vegetables after exposure to different cooking methods. Vitamin C content varied widely between the raw vegetables tested; it was not detected in mallow, crown daisy, and perilla leaf with the lowest content found in carrots (39.92 mg/kg of fresh weight), and the highest in broccoli (668.04 mg/kg of fresh weight). These findings are consistent with the results of previous investigations by Bureau et al. [18], reporting that raw broccoli was a higher in vitamin C content than most other raw vegetables such as spinach and carrot, with levels of 583, 237, 107, and 19 mg/kg of fresh weight reported for broccoli, spinach, zucchini, and carrot, respectively. Concentration of vitamin C in raw vegetables in the present study were higher than those reported by Bureau et al. [18], but these differences might be due to the fact that Bureau et al. [18] used frozen vegetables. Also, it is well known that vitamin content is varied with cultivar and growing conditions. Vitamin C is a water-soluble and temperature-sensitive vitamin, so is easily degraded during cooking, and elevated temperatures and long cooking times have been found to cause particularly severe losses of vitamin C [12]. In this study, boiling destroyed vitamin C in almost all the samples, with nutrient retention ranging from 0 to 73.86%; the greatest loss was found in boiled chard. Blanching also destroyed vitamin C in the samples, indicated by the retention that ranged from 57.85 to 88.86%, with the greatest loss found in blanched spinach. Steaming treatment significantly reduced the retention of vitamin C in all vegetables except broccoli; retention ranged from 0 to 89.24%. Microwaving had less of an impact on vitamin C content, with high retention (> 90%) observed for spinach, carrots, sweet potato, and broccoli. Steaming and microwaving retained higher concentrations of vitamin C than boiling because of the reduced contact with water at relatively low temperatures. Using minimal cooking water and cooking for shorter time periods should result in higher vitamin C retention.

Table 2.

The contents and true retention of vitamin C and vitamin K in fresh and cooked vegetables

Sample	Cooking treatment	Vitamin C		Vitamin K
		CWa (mg/kg)	TRb (%)	CWa (mg/kg)	TRb (%)
Broccoli	Raw	668.04c	100.00b	1.54a	100.00a
	Boiled	370.04d	52.85d	1.59a	98.89a
	Blanched	615.42c	88.86c	1.69a	108.81a
	Steamed	761.48b	111.21a	1.90a	123.08a
	Microwaved	836.15a	112.76a	1.71a	101.76a
Chard	Raw	163.04a	100.00a	1.59c	100.00b
	Boiled	0.00c	0.00c	2.95a	116.23a
	Blanched	124.58b	59.69b	2.38b	116.73a
	Steamed	0.00c	0.00c	2.24b	105.60ab
	Microwaved	173.43a	67.64b	2.82a	112.62ab
Mallow	Raw	NDc	ND	3.41a	100.00ab
	Boiled	ND	ND	3.00a	81.75b
	Blanched	ND	ND	2.17b	56.71 cd
	Steamed	ND	ND	2.66ab	69.33bc
	Microwaved	ND	ND	2.03b	44.28d
Potato	Raw	58.30a	100.00a	0.02b	100.00b
	Boiled	30.57c	49.79c	0.03a	141.16a
	Blanched	44.73b	73.20b	0.02b	94.71b
	Steamed	51.08b	83.65b	0.03a	75.98c
	Microwaved	62.98a	76.86b	0.03ab	95.32b
Sweet potato	Raw	131.35b	100.00a	ND	ND
	Boiled	95.20c	73.86b	ND	ND
	Blanched	88.92c	67.70bc	ND	ND
	Steamed	81.32c	59.44c	ND	ND
	Microwaved	183.73a	100.21a	ND	ND
Carrot	Raw	39.92b	100.00a	0.04b	100.00a
	Boiled	56.62d	55.33c	0.04b	85.02ab
	Blanched	32.15 cd	72.71b	0.03b	71.86b
	Steamed	33.49bc	70.51b	0.04b	69.87b
	Microwaved	63.00a	92.02a	0.06a	85.35ab
Crown daisy	Raw	ND	ND	1.19c	100.00a
	Boiled	ND	ND	1.70a	100.11a
	Blanched	ND	ND	1.31bc	89.67a
	Steamed	ND	ND	1.54ab	89.42a
	Microwaved	ND	ND	0.73d	49.80b
Perilla leaf	Raw	ND	ND	3.18c	100.00b
	Boiled	ND	ND	4.74b	116.66b
	Blanched	ND	ND	6.42ab	171.67a
	Steamed	ND	ND	7.80a	215.65a
	Microwaved	ND	ND	6.21ab	169.88a
Spinach	Raw	337.48b	100.00a	2.34c	100.00ab
	Boiled	220.14d	40.12c	3.69ab	94.93ab
	Blanched	283.96c	57.85b	3.35bc	97.27ab
	Steamed	262.78 cd	44.75c	3.61ab	87.70b
	Microwaved	499.26a	91.10a	4.67a	121.21a
Zucchini	Raw	151.30bc	100.00a	0.19c	100.00a
	Boiled	131.82c	63.71b	0.23b	87.09b
	Blanched	163.86b	87.01a	0.23b	93.78ab
	Steamed	199.53a	89.24a	0.25b	90.22ab
	Microwaved	205.80a	92.73a	0.29a	101.95a

All values are means of duplicate cooking trial and duplicate analysis. Mean values in a row followed by different superscript letters are significantly (p < 0.05) different (Duncan’s multiple range test)

^a CW cooked weight

^bTR(%) = (Nc*Gc)/(Nr*Gr)*100, Nc = nutrient contents per gram of sample after cooking, Gc = gram of sample after cooking, Nr = nutrient content per gram of sample before cooking, Gr = gram of sample before cooking

^c ND not determined

The effect of cooking methods on vitamin K content in vegetables

Vitamin K, a fat-soluble vitamin, is well known for its beneficial role in blood coagulation and bone metabolism. Although phylloquinone (vitamin K1) and menaquinone (vitamin K2) are the two naturally occurring forms of vitamin K, phylloquinone levels in vegetables and fruits range from extremely low to quite high [19]. In this study, the content and true retention of vitamin K in raw vegetables and their changes after cooking are shown in Table 2. The highest amounts of vitamin K were found in green leafy vegetables (chard, mallow, crown daisy, perilla leaf and spinach; 1.59, 3.41, 1.19, 3.18, and 2.34 mg/kg of fresh weight, respectively) and green flowers (broccoli; 1.54 mg/kg of fresh weight). However, root vegetables including potato and sweet potato had low amounts of vitamin K. This is expected, as it is well known that phylloquinone is found at highest concentrations in green leafy vegetables [20]. The retention of vitamin K in cooked vegetables ranged from 44.28 to 216.65%, and our results show that microwaving caused the highest loss of vitamin K in crown daisy and mallow but the lowest loss in spinach and chard. Cooking fresh chard and perilla leaf lead to a significant change in vitamin K content, with a trend towards higher concentrations of vitamin K in cooked vegetables than in the corresponding raw samples, although this was not consistent across all comparisons. Similar results were described by Damon et al. [21] for several vegetables such as broccoli, onion, potatoes and carrots. The effect of cooking on vitamin K has not yet been fully investigated, but increases may be because heat treatment causes vitamin K to be released. Vitamin K is located in the chloroplast in plants and the cooking process may breakdown the plant cell wall, thereby releasing vitamin K and making it available for detection by HPLC. Moreover, vitamin K is relatively heat stable and is thus retained after the cooking process [22]. In contrast, there was no apparent effect of cooking on the vitamin K content of mallow and carrot.

The effect of cooking methods on vitamin E

Vitamin E consists of four tocopherols (α-, β-, γ-, and δ-) and the corresponding tocotrienols (α-, β-, γ-, and δ-) which contain unsaturated side chains. Among the tocochromanol family, α-tocopherol is believed to present the most biological antioxidant activity, mainly attributed to inhibition of membrane lipid peroxidation [23]. In addition, the synergy effect between vitamin C and vitamin E in protecting against lipid peroxidation in liposomes has been reported [24]. The content and true retention of α-tocopherol, γ-tocopherol, and total tocopherol in raw and cooked vegetables are presented in Table 3. In raw vegetables, α-tocopherol was the major tocochromanol with amounts ranging from 0.39 to 92.07 mg/kg of fresh weight. In contrast, a low level of γ-tocopherol was determined in raw broccoli, mallow, crown daisy, perilla leaf, and zucchini, with levels ranging from 0.00 to 5.17 mg/kg of fresh weight. Levels are in agreement with other studies that show green leafy vegetables have higher vitamin E, occurring mainly as α-tocopherol and situated inside chloroplasts [25]. Cooking fresh broccoli, chard, mallow, crown daisy, perilla leaf, spinach, and zucchini lead to a significant increase in α-tocopherol, while cooking potato, sweet potato, and carrot lead to a significant decrease in α-tocopherol. Green leafy or flower vegetables have a higher retention of α-tocopherol than root vegetables, which may be attributed to the increased extractability of α-tocopherol following denaturation of proteins and a complete breakdown of the cell wall in plants which occur as a result of cooking. Moreover, there was a trend toward higher retention of γ-tocopherol in cooked rather than raw vegetables. This high content of vitamin E in cooked samples could be attributed to two reasons: (1) The effect of heat treatment encountered during domestic cooking may cause softening of the tissue by cell disruption in plants and consequently result in the release of vitamin E from the lipids and then become more available for extraction and (2) the heat treatment may also abolish the activity of tocopherol oxidase, which was found in all parts of plant like roots, stems, leaves, flowers and fruits [26]. It has been suggested that oxidizing enzymes maybe involved in the loss of vitamin E during food processing [27]. Plant tissue damage, caused by cutting or mixing could activate oxidizing enzymes involved in the loss of vitamin E due to the collapse of cell compartments [28], but heat treatment could deactivate endogenous oxidative enzymes [29].

Table 3.

The contents and true retention of vitamin E and β-carotene in fresh and cooked vegetable

Sample	Cooking treatment	Vitamin E						β-Carotene
		α-Tocopherol		γ-Tocopherol		Total-tocopherol		CWa (mg/kg)	TRb (%)
		CWa (mg/kg)	TRb (%)	CWa (mg/kg)	TRb (%)	CWa (mg/kg)	TRb (%)
Broccoli	Raw	14.87b	100.00b	2.98c	100.00c	17.85b	100.00b	2.56ab	100.00ab
	Boiled	25.39a	166.96a	4.18b	133.36b	29.57a	161.16a	2.33bc	89.40ab
	Blanched	25.84a	170.19a	4.23b	137.07b	30.07a	164.28a	2.01c	78.23b
	Steamed	24.20a	161.62a	5.17a	169.20a	29.37a	162.34a	2.71ab	105.93a
	Microwaved	23.73a	144.59a	4.82ab	145.36b	28.55a	144.86a	3.02a	109.71a
Chard	Raw	10.74d	100.00c	NDc	ND	10.74d	100.00c	15.22d	100.00b
	Boiled	23.50a	136.71a	ND	ND	23.50a	136.71a	30.06a	124.22a
	Blanched	15.73c	114.50b	ND	ND	15.73c	114.50b	21.28c	109.36ab
	Steamed	15.00c	104.31bc	ND	ND	15.00c	104.31bc	25.40b	125.37a
	Microwaved	17.40b	102.80c	ND	ND	17.40b	102.80c	26.78b	112.06a
Mallow	Raw	6.43b	100.00b	2.67c	100.00b	9.11b	100.00b	34.99b	100.00ab
	Boiled	10.33a	149.86a	4.10b	149.36a	14.43a	147.78a	41.39ab	109.92ab
	Blanched	9.81a	139.53a	4.24b	146.05a	14.05a	141.33a	46.42a	119.16a
	Steamed	10.49a	145.34a	4.45b	148.88a	14.94a	146.25a	33.89b	85.61b
	Microwaved	10.33a	122.94ab	4.89a	140.50a	15.23a	128.12ab	46.56a	101.94ab
Potato	Raw	0.56a	100.00a	ND	ND	0.56a	100.00a	ND	ND
	Boiled	0.43 cd	71.38c	ND	ND	0.43 cd	71.38c	ND	ND
	Blanched	0.50ab	84.11b	ND	ND	0.50ab	84.11b	ND	ND
	Steamed	0.39d	66.15 cd	ND	ND	0.39d	66.15 cd	ND	ND
	Microwaved	0.47bc	59.96d	ND	ND	0.47bc	59.96d	ND	ND
Sweet potato	Raw	5.68bc	100.00a	ND	ND	5.68bc	100.00a	ND	ND
	Boiled	5.44c	97.18ab	ND	ND	5.44c	97.18ab	ND	ND
	Blanched	5.56c	98.24ab	ND	ND	5.56c	98.24ab	ND	ND
	Steamed	6.28b	107.59a	ND	ND	6.28b	107.59a	ND	ND
	Microwaved	7.06a	88.72b	ND	ND	7.06a	88.72b	ND	ND
Carrot	Raw	3.74b	100.00a	ND	ND	3.74b	100.00a	29.38a	100.00a
	Boiled	3.04c	69.46b	ND	ND	3.04c	69.46b	16.23 cd	47.36c
	Blanched	3.49bc	84.26ab	ND	ND	3.49bc	84.26ab	19.02bc	58.29b
	Steamed	3.98b	89.79a	ND	ND	3.98b	89.79a	14.06d	40.02d
	Microwaved	5.55a	86.62ab	ND	ND	5.55a	86.62ab	20.66b	40.80d
Crown daisy	Raw	3.72d	100.00c	0.21c	100.00c	3.93d	100.00c	7.78b	100.00a
	Boiled	8.23ab	148.40ab	0.40ab	135.35bc	8.63ab	147.30ab	6.34b	57.38b
	Blanched	6.98bc	149.52ab	0.40ab	150.33ab	7.38bc	149.54ab	8.06b	86.10ab
	Steamed	9.01a	159.11a	0.53a	165.77a	9.54a	159.62a	10.67a	96.83a
	Microwaved	6.17c	130.74b	0.28bc	108.26c	6.46c	129.60b	7.70b	83.34ab
Perilla leaf	Raw	42.01c	100.00b	0.89b	100.00c	42.90c	100.00b	42.63a	100.00a
	Boiled	78.29b	151.61a	1.48a	138.00a	79.77b	156.22a	34.26b	65.72c
	Blanched	70.26b	146.89a	1.37a	134.53ab	71.42b	146.61a	42.74a	84.85b
	Steamed	92.07a	192.44a	1.34a	130.47abc	93.40a	191.14a	36.23ab	72.75bc
	Microwaved	74.98b	163.86a	1.03b	104.65bc	76.02b	162.62a	39.03ab	81.25b
Spinach	Raw	20.03d	100.00b	ND	ND	20.03d	100.00b	27.56d	100.00ab
	Boiled	52.00b	158.37a	ND	ND	52.00b	158.37a	46.54ab	102.43ab
	Blanched	44.82c	151.64a	ND	ND	44.82c	151.64a	35.50 cd	86.83b
	Steamed	57.56a	165.42a	ND	ND	57.56a	165.42a	41.18bc	84.67b
	Microwaved	56.68a	174.45a	ND	ND	56.68a	174.45a	51.58a	114.67a
Zucchini	Raw	2.99b	100.00 cd	1.70b	100.00a	4.69d	100.00ab	0.72ab	100.00a
	Boiled	4.74a	116.24a	2.42ab	103.96a	7.16ab	111.83a	0.41b	42.13b
	Blanched	3.56b	96.25d	1.89ab	90.56a	5.46c	93.74b	0.66ab	74.33a
	Steamed	4.60a	105.01bc	2.26ab	91.73a	6.86b	99.89ab	0.87a	82.47a
	Microwaved	4.93a	112.17ab	2.71a	105.88a	7.64a	110.98a	0.82a	77.21a

All values are means of duplicate cooking trial and duplicate analysis. Mean values in a row followed by different superscript letters are significantly (p < 0.05) different (Duncan’s multiple range test)

^a CW cooked weight

^bTR(%) = (Nc*Gc)/(Nr*Gr)*100, Nc = nutrient contents per gram of sample after cooking, Gc = gram of sample after cooking, Nr = nutrient content per gram of sample before cooking, Gr = gram of sample before cooking

^c ND not determined

The effect of cooking methods on β-carotene

Carotenoids are the precursors of vitamin A and those commonly occurring in nature include α-, β-, and γ-carotene, lycopene, and cryptoxanthin, which are converted to vitamin A in the human body. Among these precursors, β-carotene accounts for more than 90% of total carotenoids in vegetables and plays a crucial role in a major proportion of vitamin A activity [30]. Vitamin A is vital for maintaining the integrity of epithelial tissue growth, proper functioning of the retina and immune system [31]. The content and true retention of β-carotene in raw vegetables and its changes after cooking are shown in Table 3. The β-carotene content of raw vegetables ranged from 0.72 to 42.63 mg/kg of fresh weight, but it was not detected in potato or sweet potato. The β-carotene retention of cooked vegetables was in the range of 40.02–125.37%. It is known that β-carotene extractability may be influenced by cooking, but it can be enhanced or not [18]. The present study showed that β-carotene retention of cooked broccoli, chard, mallow, and spinach was higher than in the corresponding raw samples except for several cooking processes. Carotenoids including β-carotene are found in the chloroplasts of all green plant tissues, where they occur in the photosynthetic pigment-protein complexes, which have an inhibitory effect on extractability of β-carotene from the vegetable matrix. Cooking of foods could increase the extraction of carotenoids by softeningplant walls and disrupting carotenoid-protein complexes [32, 33]. It is assumed that an increased extractability may be associated with improved bioavailability. In contrast, β-carotene retention of cooked carrot, crown daisy, perilla leaf, and zucchini was lower than the raw samples. Our results agree with those of a previous study, which reported to a decrease in β-carotene in carrots after boiling [34]. This low thermal stability of β-carotene observed for carrot could be due to the different intracellular location of β-carotene. Carotene in carrots was found to be located in crystalline chromoplasts with membranes rich in polar lipids. Thermal treatment of carrots could cause the alteration of the physical state of carotenes. In particular, blanching allowed the solubilization of carotenes by cellular lipids [35]. Moreover, the difference in the β-carotene retention of cooked vegetables might be attributed to the extent of loss of carotene caused by dripping during the cooking process.

Method validation

The analytical methods used in this study were validated in terms of accuracy and precision for all tested vitamins. The precision and accuracy for vitamins in broccoli are shown in Table 4. Accuracy of the method was measured based on recovery (%). The recoveries for vitamin C, α-tocopherol, γ-tocopherol, vitamin K and β-carotene were 105.15 ± 3.81, 98.68 ± 1.80, 116.55 ± 4.74, 99.88 ± 3.00 and 102.27 ± 1.18, respectively. Precision of the method was assessed based on the repeatability (% coefficient of variation, %CV) and reproducibility (%CV). The repeatability and reproducibility was less than 5%, except for γ-tocopherol and β-carotene. The higher %CV for γ-tocopherol and β-carotene might have been caused by the lower content found in broccoli. The proposed analytical method was validated in terms of specificity for vitamin E and vitamin K. In general, the results from the method validation are satisfactory.

Table 4.

Precision and accuracy for vitamins in broccoli

Type of vitamin	Parameter	Precision		Accuracya
		Repeatabilityb	Reproducibilityc	Recovery (%)
Vitamin C	Meand	682.66	665.05	105.15
	SD	15.82	19.35	3.81
	CV (%)	2.32	2.91	3.63
α-Tocopherol	Mean	4.26	5.24	98.68
	SD	0.12	0.19	1.80
	CV (%)	2.78	3.56	1.82
γ-Tocopherol	Mean	0.95	1.10	116.55
	SD	0.04	0.07	4.74
	CV (%)	4.20	6.07	4.07
Vitamin K	Mean	1.42	1.46	99.88
	SD	0.04	0.07	3.00
	CV (%)	3.07	4.69	3.01
β-Carotene	Mean	0.52	0.54	102.27
	SD	0.01	0.03	1.18
	CV (%)	2.14	5.48	1.15

^aAccuracy is a measure of the closeness of the analytical result to the true value determined by analyzing a spiked sample

^bRepeatability was evaluated using five independent analyses of replicate sample performed on a given day

^cReproducibility was evaluated using five independent analyses of replicate sample performed on a different day

^dConcentration of vitamins expressed as mg/100 g as dry weight basis

In conclusion, the present study examined the effect of different cooking methods including blanching, boiling, microwaving, and steaming, on the content and true retention of β-carotene, ascorbic acid, vitamins E and vitamin K in vegetables. Cooking may cause changes to the contents of vitamins and it depends on the type of vegetables and the method of cooking methods. Therefore, further research is needed to optimize cooking procedures to enhance retention of vitamins.