Effect of different cooking method on vitamin E and K content and true retention of legumes and vegetables commonly consumed in Korea

Abstract

This study investigated the effect of cooking method on the content and true retention of vitamin E and K in legumes and vegetables, which are commonly consumed in Korea. Among eight isomers of vitamin E, α- and γ-tocopherol were detected at 0.44–1.03 and 2.05–2.11 mg/100 g, respectively, in legumes including chick pea, kidney bean, lentil, pea, and sword bean and they were decreased after boiling. Phylloquinone (vitamin K₁) was present at a range of 31.33 to 91.34 μg/100 g in legumes and true retention was reduced after boiling. In 21 kinds of vegetables, α-tocopherol and phylloquinone were present at 0.14–1.85 mg/100 g and 34.55–510.83 μg/100 g, respectively. α-Tocopherol and phylloquinone increased in most vegetables after cooking via blanching, boiling, steaming, and grilling. This study revealed that cooking changed vitamin E and K contents of legumes and vegetables and the changes were dependent on the type of food and cooking method.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10068-022-01206-9.

Introduction

Vitamins are organic compounds that are highly essential to the human body (Ravisankar et al., 2015) and are usually present at very low levels in foods accompanied by other compounds (Viñas et al., 2013). Their physical characteristics and contents are changed by cooking methods, such as boiling, microwaving, and grinding (Zhang and Hamauzu, 2004). In particular, temperature-sensitive vitamins are easily degraded, and elevated temperatures and long cooking times cause severe vitamin loss (Xu et al., 2014). For example, boiling and microwave cooking cause vitamin C loss in broccoli florets and stems (Zhang and Hamauzu, 2004). In contrast, heat treatment contributed to the increase in vitamin E in broccoli, which was attributed to the increase in vitamin extractability because cell walls were disrupted by softening tissues and inactivating oxidase enzymes (Lee et al., 2018). However, plant tissue damage releases enzymes, resulting in the loss of vitamins in raw fruits and vegetables (Knecht et al., 2015). For the consumption of foods, many foods are mostly cooked by heating, such as steaming, blanching, boiling, and microwaving (Lee et al., 2018; Sim et al., 2018). Several cooking processes would certainly bring about a number of changes in the physical characteristics and chemical composition of foods (Zhang and Hamauzu, 2004). Cooking leads to weight reduction owing to the loss of water and the influence of lipid oxidation (Sim et al., 2018). Additionally, cooking or heat treatments can have a significant impact on the vitamin content and lead to an inaccurate estimation of nutrient intake (Lee et al., 2018). Therefore, it is necessary to establish accurate nutritional information on the content and true retention of vitamins in various foods using different cooking methods.

Legumes and vegetables are excellent sources of vitamins, including vitamin C, E, and K (Prodanov et al., 2004; Zhang and Hamauzu, 2004; Boschin and Arnoldi, 2011). Several epidemiological studies have suggested that a diet rich in legumes and vegetables is associated with reduced risks of chronic diseases (Hu, 2002). Additionally, vegetables contain the highest amount of phylloquinone and provide approximately 60% of the total phylloquinone intake (Booth 2012). Legumes and vegetables are essential raw materials for the modern food industry for the production of protein, fiber, starch, and functional food ingredients (Prodanov et al., 2004; Lee et al., 2018). In general, legumes and vegetables are cooked via different cooking methods (Fabbri and Crosby, 2016). Previous studies have reported that cooking could result in the loss of nutrients from vegetables and legumes (Fabbri and Crosby, 2016). Some reports have suggested oxidizing enzymes being involved in the loss of vitamin during cooking (Lessin et al., 1997; Murillo et al., 1976). However, Knecht et al. (2015) reported that vitamin levels in baked vegetables were higher than those in raw vegetables. In addition, higher tocopherol content has been reported in heat-treated spinach and broccoli than in their respective raw forms (Chun et al., 2006). Due to the conflicting data on vitamin in some raw and cooked vegetables and legumes, the aim of the present study was to quantitate vitamin E and K in raw and cooked vegetables and legumes and to validate analytical method suitable for vitamin E and K. Therefore, changes in the nutritional value of various vegetables and legumes can be expected after different cooking treatments. In this study, the content and retention of vitamin E and K in legumes and vegetables commonly consumed in Korea were determined according to different cooking methods to establish the Korean Standard Food Composition Database.

Materials and methods

Chemicals

Eight different types of vitamin E (α-, β-, γ-, and δ-tocopherols and tocotrienols), sodium acetate, acetic acid, and zinc powder (particle size, < 63 μm) were purchased from Merck (Darmstadt, Germany). Vitamin K₁ (phylloquinone) was purchased from Wako (Tokyo, Japan). Vitamin K₂ (menaquinone), butylated hydroxytoluene (BHT), lipase (from Candida rugosa 1000 units/mg, type VII), monobasic potassium phosphate, and magnesium sulfate were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Potassium hydroxide, pyrogallol, and sodium chloride were obtained from Daejung Chemical Co. (Siheung, Korea). n-Hexane, isopropanol, ethanol, and ethyl acetate from J. T. Baker (Philipsburg, NJ, USA), as well as methanol, ethanol, dichloromethane, and water from Burdick & Jackson Co. (Muskegon, MI, USA), were all of analytical or high-performance liquid chromatography (HPLC) grade.

Sample preparation

All legumes and vegetables were purchased from a local retail market (Jeonju, Korea). For boiling, dried pea, chick pea, kidney bean, and lentil were added to distilled water (DW) that had just reached boiling in a glass pot (sample-to-DW ratio of 1:5, w/v). Ten grams of sword bean was added to 3 L of DW and then boiled. For boiling and blanching, the vegetables were added to boiled DW (1:10, w/v). Young pumpkins were divided into cubes of 0.5–1 cm per side. The eggplant used for blanching and streaming had a length of 10 cm and was thus cut into 1.5 cm per side. Sweet pumpkin was cut into pieces of 2 cm per side for boiling or divided into octuplicate for steaming. All blanched and steamed samples were cooked on a highlight cooktop using a strong flame. The cooking conditions are listed in Tables 1 and 2. The cooked samples were cut into small pieces, homogenized in a blender, and stored in a freezer at – 20 °C. Before pre-processing, the samples were thawed in a refrigerator at 4℃ overnight.

Table 1.

Cooking methods of legumes and vegetables commonly consumed in Korea

Common name	Scientific name		Description	Cooking method
Chick pea (Byeongalikong)	Cicer arietinum		Dried, boiled	Boiling with distilled water (1:5, w/v) for 1 h followed by cooling for 30 min
Kidney bean (Ggangnangkong)	Phaseolus vulgaris		Dried, boiled
Lentil (Lentilkong)	Brown	Lens culinaris Medik	Dried, boiled	Boiling with distilled water (1:5, w/v) for 15 min followed by cooling for 30 min
	Red	Lens culinaris Medik
Pea (Wandukong)	Pisum sativum L		Dried, boiled	Boiling with distilled water (1:5, w/v) for 1 h followed by cooling for 30 min
Sword bean (Jagdu)	Canavalia ensiformis DC		Dried, boiled	Boiling sword bean (10 g) in 3 L distilled water for 10 min followed by cooling for 30 min

Table 2.

Cooking methods of vegetables commonly consumed in Korea

Common name	Scientific name	Description	Cooking method
Aralia continentalis Kitag (Ttangdureub)	Aralia cordata	Raw, blanched	Blanching Ttangdureub (300 g) in 10 L boiled water for 1 min
Aralia elata var. elata (Chamdureub)	Aralia elata	Raw, blanched	Blanching Chamdureub (300 g) in 10 L boiled water for 1 min
Balsam pear (Yeoju)	Momordica charantia L	Raw, blanched	Blanching balsam pear with boiled water (1:5, w/v) for 1.5 min
Brussels sprout (Banguldadagi Yangbaechu)	Brassica oleracea var. gemmifera	Raw, blanched	Blanching Brussels sprouts with boiled water (1:5, w/v) for 1.5 min
		Raw, steamed	Steaming Brussels sprouts (700 g) in a steamer for 20 min followed by cooling
Butterbur (Meowi)
Petiole	Petasites hybridus	Raw, blanched	Blanching butterbur petiole (500 g) with 5 L boiled water for 1 min
Stem	Petasites hybridus	Raw, blanched	Blanching butterbur (the stem) with boiled water (1:10, w/v) for 10 min
Cabbage	Brassica oleracea var. capitata	Raw, boiled	Blanching cabbage with boiled water (1:5, w/v) for 5 min
		Raw, steamed	Steaming cabbage in a steamer for 20 min followed by cooling
Chinese chive (Hobuchu)	Allium tuberosum	Raw, blanched	Blanching Chinese chive with boiled water (1:10, w/v) for 20 s
		Raw, steamed	Steaming Chinese chive in a steamer for 20 min followed by cooling
Crown daisy (Ssukgot)	Glebionis coronaria	Raw, blanched	Blanching crown daisy with boiled water for 1 min
Curled mallow (Ahuk)	Malva verticillata	Raw, blanched	Blanching curled mallow with boiled water (1:10, w/v) for 1 min Soaking in cool water and removing moisture
Eggplant (Gaji)	Solanum melongena	Raw, blanched	Blanching eggplant with boiled water (1:10, w/v) for 2 min Removing moisture at 25 °C and cooling
		Raw, steamed	Steaming eggplant in a steamer for 2–3 min
		Raw, grilled	Grilled eggplant on a pan for 10 min
Gourd (Bak)	Lagenaria siceraria	Raw, blanched	Blanching crown daisy with boiled water for 1 min
Pumpkin
Sweet (Danhobak)	Kabocha squash	Raw, boiled	Boiling sweet pumpkin with distilled water (8 L) for 15 min
		Raw, steamed	Removing inedible parts, steaming in a steamer for 20 min
Old	Cucurbita moschata Duchesne	Raw, boiled	Boiling with distilled water for 15 min
		Raw, steamed	Steaming in steamer for 20 min
Young (Aehobak)	Cucucrbita moschata Duchesne	Raw, boiled	Boiling young pumpkin with boiled water (1:10) for 5 min
		Raw, steamed	Steaming young pumpkin in a steamer for 7–10 min
		Raw, grilled	Grilled young pumpkin on a pan on 10 min
Zucchini (Zucchini)	Cucucrbita pepo L	Raw, boiled	Boiling zucchini pumpkin with boiled water (1:5) for 10 min
Radish
Root	Raphanus sativus	Raw, blanched	Blanching with boiled water (1:10) for 0.5–1 min
Young	Raphanus sativus L	Raw, blanched	Blanching with boiled water (1:10) for 0.5–1 min
Water dropwort (Dolminali)	Oenanthe javanica	Raw, blanched	Blanching water dropwort with boiled water (1:10) for 0.5–1 min
Water spinach (Gongsimchae)	Ipomoea aquatica	Raw, blanched	Removing inedible parts and blanching water spinach with boiled water (1:10) for 30 min

Analysis of vitamin E

Vitamin E content was determined after saponification and extraction of food samples (Chun et al., 2006). For saponification, 20 mL of ethanol containing 6% pyrogallol was added to each sample (2–4 g) in a tube. After sonication for 5 min, 8 mL of 60% potassium hydroxide was added. The tube was flushed with nitrogen gas for 1 min and connected to an air condenser. The samples were digested at 75 °C for 50 min in a shaking water bath (JSSB-30 T, JS Research Inc., Gongju, Korea). The digested samples were cooled in an ice bath, and 30 mL of 2% sodium chloride was added. The saponified samples were extracted three times with 20 mL of extraction solvent (n-hexane: ethyl acetate = 90:10, v/v) containing 0.01% butylated hydroxytoluene and filtered with magnesium sulfate. The n-hexane extracts were transferred into a 50-mL volumetric flask, and the volume was made up of an extraction solvent. A 2-mL aliquot of the extract was evaporated under nitrogen gas and then mixed with n-hexane (1 mL). After being filtered with a 0.45-μm PTFE syringe filter (Whatman International Ltd., Piscataway, NJ, USA), vitamin E was analyzed by HPLC.

For the vitamin E analysis, an HPLC system (1260 Infinity, Agilent Technologies, Santa Clara, CA, USA) coupled with a fluorescence detector (FLD) was used and controlled by the Agilent ChemStation software (Agilent Technologies). Optimized chromatographic separation was achieved with a normal-phase column (Lichrosphere® Diol 100; 250 × 4 mm, i.d. 5 μm; Merck Millipore, Nottingham, UK) with a column temperature of 24℃. Vitamin E levels were estimated using an FLD set at λ_ex = 290 nm and λ_em = 320 nm. The mobile phase was n-hexane containing 1.3% isopropanol and isocratic elution. The flow rate and injection volume were 1 mL/min and 20 μm, respectively. The peaks were identified by comparing their retention times with the corresponding data obtained by analyzing the standard compounds. Quantification was performed using the external standard method. Vitamin E concentrations in the samples were calculated as the average peak area after duplicate injections and expressed in mg/100 g.

Calculation of α-tocopherol equivalents

Vitamin E concentrations were converted into activity equivalents (Park et al., 2016). Vitamin E was expressed as α-tocopherol equivalent (TE) as follows:

$$\begin{array}{cc}\hfill \alpha -\text{TE}=& \left(\alpha -\text{T}\text{content}\times 1\text{.0}\right)+\left(\beta -\text{T}\text{content}\times 0\text{.5}\right)\hfill \\ \hfill & +\left(\gamma -\text{T}\text{content}\times 0\text{.1}\right)+\left(\delta -\text{T}\text{content}\times 0\text{.01}\right)\hfill \\ \hfill & +\left(\alpha -\text{T3}\text{content}\times 0\text{.01}\right)+\left(\beta -\text{T3}\text{content}\times 0\text{.5}\right)\hfill \\ \hfill \end{array}$$

T: tocopherol. T3: tocotrienol.

Analysis of vitamin K

Vitamin K content was determined using the solvent extraction method. Lyophilized samples of raw and cooked legumes and vegetables (1 g) were ground and mixed with 30 mL of a dichloromethane-methanol (2:1, v/v) solvent. After homogenization, the extract was made up to 50 mL with methanol, and 2-mL aliquots of the extract were transferred to glass tubes. All solvents were evaporated under nitrogen gas and redissolved in 2 mL of n-hexane. Next, 8 mL of methanol–water (9:1, v/v) was added, and the sample was centrifuged for 5 min at 676×g (Gyrozen Co., Ltd., Daejeon, Korea). The supernatant (1 mL) was evaporated using nitrogen gas and redissolved in 1 mL of methanol. It was then filtered through a 0.45-μm PTFE syringe filter (Whatman International Ltd.) and analyzed using HPLC.

For vitamin K analysis, an HPLC system (1260 Infinity, Agilent Technologies) coupled with an FLD was used. The columns used were a Zorbax Eclipse XDB-C18 column (150 mm × 4.6 mm, 5 μm, Agilent Technologies) and a zinc-filled post column (2.0 mm × 50 mm, YNC Co., Wilmington, NC, USA). The mobile phase was methanol-dichloromethane (9:1, v/v). The flow rate was 1.0 mL/min, and the injection volume was 50 μL. Vitamin K content was estimated using an FLD set at λ_ex = 243 nm and λ_em = 430 nm at 25 °C. Quantification was performed using external calibration against phylloquinone and menaquinon. The vitamin K concentration in the samples was calculated using the average peak area after duplicate injections and expressed in μg/100 g.

Determination of true retention (TR) of vitamin E and K after cooking

The TR values were calculated for vitamin E and K content using the following formula:

$$\%\text{TR}=(\text{Nc}\times \text{Gc})/\left(\text{Nr}\times \text{Gr}\right)\times 100$$

Nc: nutrient amount per gram in a cooked food. Gc: weight of a cooked food in gram. Nr: nutrient amount per gram of a raw food. Gr: weight of food before cooking in gram.

Method validation

Method validation for vitamin E and K analyses was performed using the Association of Official Analytical Chemists (AOAC) guidelines and the ICH harmonized tripartite guidelines (ICH 2005; AOAC 2002). All certified reference materials were used as powder. The accuracy (recovery) of the method was evaluated on certified reference materials (SRM 3280, multivitamin; SRM 3235, soymilk), and the analytical values were compared with the corresponding certified values (a minimum of seven data points were required for validation). The recovery was calculated using the following equation:

$$\text{Recovery}\left(\%\right)=\left[\left(\text{Cs}-\text{Cp}\right)/\text{Ca}\right]\times 100$$

Recovery (%): recovery of added standard. Cs: vitamin content in a spiked sample. Cp: vitamin content in a sample. Ca: tocopherol standard added.

The precision of the vitamin E assay was evaluated from its repeatability and reproducibility, which were determined by analyzing commercial pine nuts and mixtures of broccoli and shiitake mushrooms (quality control sample) five times on the same day and once on five different days, respectively. The coefficient of variation (CV, %) was calculated for repeatability and reproducibility and then compared to the acceptance level of the AOAC guidelines (AOAC, 2002). Individual stock standard solution (50 mg/mL) of vitamin E (α-,β-,γ- and δ- tocopherol and tocotrienol) and vitamin K (phylloquinone and menaquinone) were prepared in hexane and methanol, respectively. The linearity of the vitamin E and K analysis was tested at six different concentrations of vitamin E (6.04 μg/mL, α-T; 1.25 μg/mL β-T; 12.40 μg/mL, γ-T; 3.96 μg/mL, δ-T; 2.38 μg/mL, α-T3; 1.94 μg/mL β-T3; 1.33 μg/mL, γ-T3; 1.06 μg/mL, δ-T3) and vitamin K standard solutions. All standard solutions were serially diluted. All analyses were performed in triplicate. The limit of detection (LOD) was calculated based on the detector signal-to-noise (S/N) ratio by multiplying the standard deviation of the S/N ratio by 3.3 and adding this to the average of the S/N ratio. The limit of quantitation (LOQ) was obtained by using 10 as the multiplying factor. The LOD and LOQ values are presented as μg/injection volume.

Quality control (QC)

The QC of vitamin E analysis was performed according to the AOAC guidelines (AOAC, 2002). The QC chart was plotted, and the analysis performance was controlled during the entire study. The QC sample, commercial pine nut, and a mixture of broccoli and shiitake mushrooms were analyzed more than 10 times to plot a QC chart with the mean and the control and action lines for vitamin E analysis. The control and action lines were calculated as follows:

$$\text{Upper and lower control line}=\text{mean}\pm \text{2 standard deviation}\left(\text{SD}\right)$$

$$\text{Upper and lower action line}=\text{mean}\pm \text{3 SD}$$

They were used to monitor the vitamin E assay for the samples during the entire study.

Statistical analysis

Statistical analyses were performed using SPSS Statistics (IBM Co., Chicago, IL, USA). Data are expressed as mean ± standard deviation (SD). Statistical comparisons were performed using one-way analysis of variance followed by Duncan’s multiple range test or Student’s t-test. The p < 0.05 was considered as statistically significant.

Results and discussion

Content and true retention of vitamin E

The compositions and true retentions of tocopherol and tocotrienol in legumes and their cooked products are shown in Table 3. And the chromatogram of vitamin E in representative sample presented in Supplementary Fig. 1. In legumes, α- and γ-tocopherol, among the eight isomers of vitamin E, were detected at 0.44–1.03 and 2.05–2.11 mg/100 g, respectively. Both α- and γ-tocopherol were not detected in dried kidney bean, boiled red lentil, and dried and boiled sword bean, among the six different types of legumes. Only dried and boiled chick pea and dried red lentil contained γ-tocopherol. The tocopherol content was then converted to α-TE for vitamin E activity equivalent. Dried chick pea showed the highest α-TE content (1.24 mg α-TE). After boiling, the α-tocopherol content of dried chick pea, brown lentil, and sword bean was reduced, with true retentions of 100.00% to 83.03–92.06%; moreover, α-tocopherol was not detected in red lentil. In some cases, blanching or boiling can increase vitamin E concentration through large losses of water-soluble components (Chun et al., 2006); however, most boiled legumes contain lower levels of vitamin E than dried legumes because of the loss of vitamin E through draining after soaking in the liquid (Prodanov et al., 2004). In legumes, hydration is a method of mass transfer from the seeds to the liquid phase, which results in a significant loss of vitamins (Prodanov et al., 2004). In the current study, chick pea, lentil, and sword bean were cooled for 30 min after boiling, which might cause the loss of vitamin E. For the dietary reference intake for Koreans, the recommended value of vitamin E intake is 12 mg/α-TE/day (Ministry of Health and Welfare and The Korean Nutrition Society, 2021). Daily vitamin E intakes in Korean males and females were reported to be 7.5 and 6.4 mg/α-TE/day, respectively (Ahn et al., 2017). However, the α-TE of raw and cooked legumes in the current study ranged to 0.47–1.24 mg α-TE; thus, vitamin E intake from legumes alone is not sufficient for daily use.

Table 3.

Content and true retention (TR) of vitamin E in legumes and vegetables according to the cooking method

Species	Common name			Description	Vitamin E (mg/100 g)			α-TE2)	TR (%)
					α-T1)	γ-T
Legumes	Chick pea			Dried	1.03 ± 0.10a3)		2.05 ± 0.07b	1.24 ± 0.09a	100.00
				Boiled	0.86 ± 0.02b		2.62 ± 0.30a	1.14 ± 0.05a	92.06
	Kidney bean			Dried	-4)		–	–	–
				Boiled	–		–	–	–
	Lentil	Brown		Dried	0.56 ± 0.06a		–	0.56 ± 0.06a	100.00
				Boiled	0.47 ± 0.02a		–	0.47 ± 0.02a	84.32
		Red		Dried	0.44 ± 0.01		2.11 ± 0.18	0.66 ± 0.01	–
				Boiled	–		–	–	–
	Pea			Dried	–		–	–	–
				Boiled	–		–	–	–
	Sword bean			Dried	0.70 ± 0.11a		–	0.70 ± 0.11a	100.00
				Boiled	0.58 ± 0.08a		–	0.58 ± 0.08a	83.03
Vegetables	Aralia continentalis Kitag			Raw	0.57 ± 0.02b		–	0.57 ± 0.02b	100.00
				Blanched	0.67 ± 0.05a		–	0.67 ± 0.05a	117.44
	Aralia elata var. elata			Raw	0.95 ± 0.13a		–	0.95 ± 0.13a	100.00
				Blanched	0.89 ± 0.00a		–	0.89 ± 0.00a	94.88
	Balsam pear			Raw	0.41 ± 0.02b		–	0.41 ± 0.02b	–
				Blanched	0.61 ± 0.03a		–	0.61 ± 0.03a	–
				Dried	3.69 ± 0.42a		1.90 ± 0.20	3.88 ± 0.44a	100.00
				Dried, boiled	0.74 ± 0.03b		–	0.74 ± 0.03b	19.18
	Brussels sprout			Raw	–		–	–	–
				Blanched	0.41 ± 0.00b		–	0.41 ± 0.00b	–
				Steamed	0.68 ± 0.05a		–	0.68 ± 0.05a	–
	Butterbur		Petiole	Raw	0.61 ± 0.01b		–	0.61 ± 0.01b	100.00
				Boiled	0.74 ± 0.03a		–	0.74 ± 0.03a	121.28
			Stem	Raw	–		–	–	–
				Blanched	0.46 ± 0.02a		–	0.46 ± 0.02a	–
	Cabbage			Raw	–		–	–	–
				Boiling	–		–	–	–
				Steamed	–		–	–	–
	Chinese chive			Raw	0.88 ± 0.00a		–	0.88 ± 0.00a	100.00
				Blanched	0.68 ± 0.05a		–	0.68 ± 0.05a	77.27
				Steamed	0.80 ± 0.12a		–	0.80 ± 0.12a	90.91
	Crown daisy			Raw	–		–	–	–
				Blanched	0.48 ± 0.01		–	0.48 ± 0.01	–
	Curled mallow			Raw	0.50 ± 0.03b		–	0.50 ± 0.03b	100.00
				Blanched	0.61 ± 0.02a		–	0.61 ± 0.02a	122.13
	Eggplant			Raw	–		–	–	–
				Blanched	–		–	–	–
				Steamed	–		–	–	–
				Grilled	–		–	–	–
	Gourd			Raw	–		–	–	–
				Blanched	–		–	–	–
	Pumpkin	Kabocha squash		Raw	1.85 ± 0.17b		1.60 ± 0.12b	2.01 ± 0.18ab	100.00
				Boiled	1.67 ± 0.01b		2.00 ± 0.09a	1.87 ± 0.02b	93.49
				Steamed	2.59 ± 0.27a		2.04 ± 0.26a	2.79 ± 0.30a	138.87
		Old		Raw	0.41 ± 0.01c		–	0.41 ± 0.01c	100.00
				Boiled	0.52 ± 0.02a		–	0.52 ± 0.02a	126.80
				Steamed	0.49 ± 0.05b		–	0.49 ± 0.05b	119.36
		Young		Raw	0.47 ± 0.01c		–	0.47 ± 0.01c	100.00
				Boiled	0.58 ± 0.03a		–	0.58 ± 0.03a	123.35
				Steamed	0.51 ± 0.01b		–	0.51 ± 0.01b	108.51
				Grilled	0.58 ± 0.01a		–	0.58 ± 0.01a	123.41
		Zucchini		Raw	0.39 ± 0.04a		–	0.39 ± 0.04a	100.00
				Boiled	–		–	–	–
	Radish	Korean (Root)		Raw	–		–	–	–
				Blanched	–		–	–	–
		Young		Raw	0.54 ± 0.04		–	0.54 ± 0.04a	100.00
				Blanched	0.61 ± 0.03		–	0.61 ± 0.03a	113.10
	Water dropwort			Raw	0.14 ± 0.02b		–	0.14 ± 0.02b	100.00
				Blanched	0.45 ± 0.06a		–	0.45 ± 0.06a	321.73
	Water spinach			Raw	0.91 ± 0.02a		–	0.91 ± 0.02a	100.00
				Blanched	–		–	–	–

¹⁾ T corresponds to tocopherol

²⁾ α-TE corresponds to α-tocopherol equivalent

³⁾ Each value is mean ± standard deviation. Means with different letters in a column are significantly different at p < 0.05

⁴⁾ – not detected

In raw 15 types of vegetables, α-tocopherol was the only detected vitamin E isomer, with content ranging from 0.14 to 1.85 mg/100 g (Table 3). Among these vegetables, only kobocha squash (Danhobak) contained γ-tocopherol (1.60 mg/100 g), with the highest amount of α-tocopherol (1.85 mg/100 g). Additionally, cabbage, eggplant, gourd, and Korean radish root did not contain vitamin E, despite the cooking treatments. Furthermore, dried balsam pear and steamed kobocah squash had the highest value of α-TE (3.88 and 2.79 mg α-TE). After cooking, including via blanching, boiling, steaming, and grilling, α-tocopherol content increased in most raw vegetables, but the true retention decreased in A. elata var. elata, dried balsam pear, Chinese chive, kobacha squash, and water spinach. This loss might be caused by a change in the water content after heat processing (Kim et al., 2021). Heat processing can increase vitamin E content through large losses of water-soluble components, and cooking with heat treatment contributes to the softening of tissues by cell wall disruption (Lee et al., 2018). Thus, cooking treatments including boiling, blanching, and steaming may change the cell matrix of raw vegetables (Lee et al. 2018; Ogawa et al., 2018). Although the α-TE values of vegetables (3.88–0.39 mg α-TE) were higher than those of legumes (0.47–1.24 mg α-TE), the differences were not large. In Korea, males and females obtain 23.1% and 22.4% of their α-tocopherol intake from vegetables, respectively (Ahn et al., 2017).

Content and true retention of vitamin K

The content and true retention of vitamin K in legumes and vegetables according to the cooking method are shown in Table 4. And the chromatogram of vitamin K in representative sample presented in Supplementary Fig. 2. Only vitamin K₁ (phylloquinone) was detected in most legumes and vegetables. In legumes, dried chick pea, dried brown and red lentils, and dried sword bean contained 31.33, 58.54, 33.61, and 91.34 μg/100 g phylloquinone. Furthermore, phylloquinone was not found in either dried or boiled kidney bean or pea as well as in boiled brown lentil and sword bean. Boiling reduced the true retention of phylloquinone in dried brown lentil and sword bean to 78.52% and 69.92%, respectively. A previous study reported that soaking and cooking treatments are highly efficient in reducing several antinutritional factors, such as α-galactosides, trypsin and chymotrypsin inhibitors, phytates, and lectin, which can decrease the availability of nutrients in legumes (Geraldo et al., 2022; Alsalman and Ramaswamy, 2020). Reducing these factors is essential for improving the nutritional quality of legumes (Prodanov et al., 2004). Heat treatment also causes the release of vitamin K from its location (Damon et al., 2005). Vitamin K is located in the chloroplast in plants, and the cooking processes break down plant cell walls to facilitate the release of vitamin K (Lee et al., 2018). Moreover, vitamin K is relatively heat-stable and is mostly retained during cooking (Dufossé and Galaup, 2010; McKeown et al., 2002). However, the vitamin K content of legumes decreased owing to boiling, similar to the vitamin E content (Table 3) because the water content of boiled legumes was higher than that of dried legumes. In a study by Prodanov et al. (2004), cooking with water caused considerable losses in some essential nutrients, although the antinutritional factor was reduced. Therefore, the water content of boiled legumes and the cooling process might have affected the reduction in vitamin K content.

Table 4.

Content and true retention (TR) of vitamin K in legumes and vegetables according to the cooking method

Species	Common name			Description	Vitamin K (µg/100 g)
					K1	TR (%)
Legumes	Chick pea			Dried	31.13 ± 1.921)	–2)
				Boiled	–	–
	Kidney bean			Dried	–	–
				Boiled	–	–
	Lentil	Brown		Dried	58.54 ± 1.09a	100.00
				Boiled	45.97 ± 1.32b	78.52
		Red		Dried	33.61 ± 2.60	–
				Boiled	–	–
	Pea			Dried	–	–
				Boiled	–	–
	Sword bean			Dried	91.34 ± 5.09a	100.00
				Boiled	63.87 ± 3.63b	69.92
Vegetables	Aralia continentalis Kitag			Raw	90.36 ± 3.70b3)	100.00
				Blanched	109.18 ± 6.63a	120.76
	Aralia elata var. elata			Raw	208.74 ± 11.52b	100.00
				Blanched	334.36 ± 5.82a	160.40
	Balsam pear			Raw	108.88 ± 2.06a	100.00
				Blanch	98.25 ± 0.17b	90.26
	Balsam pear (dried)			Raw	417.19 ± 21.36a	100.00
				Boiled	112.68 ± 3.65b	27.03
	Brussels sprout			Raw	331.25 ± 4.46b	100.00
				Blanched	340.71 ± 6.40ab	102.85
				Steamed	348.06 ± 9.41a	105.06
	Butterbur	Petiole		Raw	461.89 ± 0.18b	100.00
				Boiled	516.69 ± 25.88a	111.86
		Stem		Raw	34.55 ± 0.02b	100.00
				Blanched	42.70 ± 3.11a	123.59
	Cabbage			Raw	57.40 ± 0.97c	100.00
				Boiled	63.76 ± 2.76b	111.05
				Steamed	71.97 ± 0.11a	125.40
	Chinese chive			Raw	190.03 ± 1.42b	100.00
				Blanched	201.49 ± 6.98a	106.02
				Steamed	209.98 ± 1.93a	110.50
	Crown daisy			Raw	265.98 ± 8.03b	100.00
				Blanched	312.57 ± 3.82 a	117.52
	Curled mallow			Raw	510.83 ± 11.45b	100.00
				Blanched	567.53 ± 19.93a	111.10
	Eggplant			Raw	35.96 ± 1.13c	100.00
				Blanched	36.23 ± 0.37c	100.80
				Steamed	37.33 ± 0.35b	103.86
				Grilled	40.97 ± 0.12a	114.00
	Gourd			Raw	–	–
				Blanch	–	–
	Pumpkin		Kabocha squash	Raw	119.68 ± 1.44a	100.00
				Boiled	120.57 ± 5.85a	100.71
				Steamed	108.01 ± 4.23b	90.23
			Old	Raw	–	–
				Boiling	–	–
				Stream	–	–
			Young	Raw	42.28 ± 0.31b	100.00
				Boiled	54.46 ± 3.05a	128.78
				Steamed	58.31 ± 4.32a	137.87
				Grilled	57.80 ± 0.34a	136.71
			Zucchini	Raw	14.39 ± 0.78a	100.00
				Boiled	10.98 ± 0.45b	76.34
	Radish		Root	Raw	–	–
				Blanched	–	–
			Young	Raw	196.10 ± 11.37b	100.00
				Blanch	227.33 ± 13.73a	115.91
	Water dropwort			Raw	42.67 ± 0.64b	100.00
				Blanched	140.51 ± 5.24a	329.22
	Water spinach			Raw	123.02 ± 1.03b	100.00
				Blanched	347.53 ± 19.94a	282.42

¹⁾ –, not detected

²⁾ Each value is mean ± standard deviation. Means with different letters in a column are significantly different at p < 0.05

The content and true retention of vitamin K in raw and cooked vegetables are shown in Table 4. Similar to the findings in legumes, only vitamin K₁ (phylloquinone) was detected. The phylloquinone content of raw vegetables was in the range of 34.55–510.83 μg/100 g. Among these vegetables, curled mallow (Auk) had the highest phylloquinone content (510.83 μg/100 g), whereas gourd, old pumpkin, and Korean radish did not contain phylloquinone. After cooking via blanching, boiling, steaming, and grilling, the phylloquinone content of vegetables increased, except in blanched balsam pear, boiled dried balsam pear, steamed kabocha squash, and boiled zucchini. In particular, the true retention (TR) of vitamin K in blanched water dropwort and water spinach increased to 329.22% and 282.42%, respectively. In contrast, kabocha squash and zucchini lost phylloquinone content after steaming and boiling, with TR values of 90.23% and 76.34%, respectively. In the current study, most vegetables showed significant changes in the phylloquinone content after cooking, as shown by the high concentration of phylloquinone in cooked vegetables. Similarly, previous studies have reported that vitamin K increases in cooked vegetables such as broccoli, onion, potatoes, and carrots (Lee et al., 2018). Heat treatment helps release vitamin K (Damon et al., 2005) because the cooking processes may break down plant cell walls (Lee et al. 2018; Booth, 2012). Generally, heat treatment causes a significant loss of vitamins (Fanali et al., 2017); however, because vitamin K is relatively heat-stable, it is retained after cooking (Dufossé and Galaup 2010; McKeown et al., 2002). Therefore, cooking increased the vitamin K content of cooked vegetables in the present study.

Method validation

The analytical methods were validated in terms of accuracy and precision for all tested vitamins. The accuracies of the vitamin E and K analyses, measured using the standard reference materials SRM 3280 (soy milk) and SRM 3235 (multivitamin), are shown in Supplementary Table 1. The vitamin E content of SRM 3280 measured by HPLC-FLD was 237.76 mg/100 g, and the vitamin K content of SRM 3235 was 4.71 µg/ 100 g, with recovery of 110.38% and 98.47%, respectively. Precision was assessed based on repeatability (% coefficient of variation, %CV) and reproducibility (%CV), as shown in Table 5. All repeatability and reproducibility values were less than 5%. These results were below the acceptable precision level (15% for µg analyte/100 g sample) according to AOAC guidelines (AOAC, 2002). Therefore, the accuracy and precision of the HPLC-FLD method for vitamin E and K analyses in the current study were sufficient to provide reliable analytical data.

Table 5.

Precision of vitamin E and K analyses by HPLC-FLD

Sample	Compound	Parameter	Repeatability1)	Reproducibility2)
Pine nut	α-Tocopherol (mg/100 g)	Mean ± SD (CV, %)3)	5.89 ± 0.12 (3.96)	5.93 ± 0.28 (4.72)
	γ-Tocopherol (mg/100 g)	Mean ± SD (CV, %)	8.52 ± 0.36 (4.20)	8.39 ± 0.31 (3.72)
Mixture of broccoli and shiitake mushroom	Phylloquinone (µg/ 100 g)	Mean ± SD (CV, %)	838.66 ± 34.52 (4.12)	852.27 ± 31.21 (3.66)

¹⁾ Repeatability refers to the results of five independent determinations

²⁾ Reproducibility refers to the results of five independent determinations for a sample by analyzing five replicates at each of different assay days

³⁾ CV corresponds to coefficient of variation

The retention time of the vitamin E and K compound peaks of all samples analyzed by HPLC was the same as that of the standard compound. The linearity of α-T, γ-T, and phylloquinone showed excellent correlation coefficients (R²) of R² = 0.9999, R² = 0.9997, and R² = 0.9995 (Supplementary Table 2). The LOD and LOQ of HPLC analysis for vitamin E were 0.65 and 1.96 ng/injection volume for α-T and 0.95 and 2.87 ng/injection volume for γ-T, respectively. For vitamin K, the LOD and LOQ of HPLC analysis were 0.64 and 1.92 ng/injection volume for phylloquinone. These results were within the range of or lower than the published limits for all analytes determined by HPLC-FLD, highlighting the beyond-average sensitivity of the present method.

Analytical quality control (QC)

QC of the vitamin E and K assays was performed by constructing a QC chart throughout the study. The QC chart with control and action lines was set up according to the AOAC guideline (AOAC, 2002). The analytical vitamin E and K values of a QC chart were plotted whenever the samples were analyzed (Fig. 1). All analytical values were close to the mean and were included in the UCL and LCL.

Fig. 1.

Quality control (QC) chart for vitamin E and K analyses. (A)α-tocopherol, (B) γ-tocopherol, and (C)vitamin K₁. Upper and lower control lines, UCL and LCL, respectively; upper and lower action lines, UAL and LAL, respectively

In conclusion, the vitamin E and K content of legumes and vegetables was determined according to different cooking methods, including boiling, blanching, steaming, and grilling. The vitamin E and K content of legumes was decreased by cooking, as vitamin content is influenced by soaking in water after cooking. In contrast, vitamin E and K content increased in most vegetables after cooking. These results indicated that the cooking process caused changes in the vitamin E and K content, and these changes depended on the cooking method. Therefore, further research is needed to optimize the cooking procedures to enhance the retention of vitamin E and K.

Supplementary Information

Below is the link to the electronic supplementary material.

Funding

Funding was supported by Korea Rural Development Administration (Grant No. PJ014537).

Declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.