Effect of photoperiod and growth media on yield and antioxidant properties of wheatgrass juice of Indian wheat varieties

Abstract

The effect of photoperiod durations (16 h light:8 h dark vs 22 h light:2 h dark) and different doses (0.5x and 1x) of Murashige and Skoog medium on the yield and antioxidant characteristics of wheatgrass from hard, medium-hard and soft wheat varieties were analyzed. The average wheatgrass height and wheatgrass yield increased in MS media both under normal photoperiod as well as in water under prolonged photoperiod. An increase in total phenolic content (TPC) and ferric reducing antioxidant power (FRAP) of wheatgrass in different strengths of MS media under normal photoperiod was observed. Whereas, increase in protein content, chlorophyll (Chl) a, Chl b, total Chl, average TPC, DPPH inhibition and FRAP values were observed for wheatgrass of different varieties grown in water under prolonged photoperiod. The accumulation of polypeptides (PPs) of 92 kDa, 33 kDa, 23 kDa, 14 kDa, 12 kDa, and 10 kDa for wheatgrass shoot powder of different varieties was affected by strength of MS media and duration of photoperiod. On the contrary, wheatgrass juice powder showed major changes in the accumulation of PPs 33 kDa and 23 kDa PPs under varied strength of MS media and prolonged photoperiod.

Electronic supplementary material

The online version of this article (10.1007/s13197-020-04805-8) contains supplementary material, which is available to authorized users.

Introduction

Wheat (Triticum aestivum) is an important staple food of India and a wider segment of the population of the Asian sub-continent consumes processed wheat in various forms. Many studies have put forward that the consumption of whole wheat or whole grain shows a protective effect against chronic diseases (Aydos et al. 2011). While its germination results in the biosynthesis of vitamins, phenolics, and antioxidants (Kulkarni et al. 2006). Chlorophyll is the key active component in wheatgrass, which is claimed to have a role in the inhibition of the metabolic activity of carcinogens (Aydos et al. 2011). Chlorophyll comprises about 70% of the total chemical constituents of the wheatgrass juice. A derivative of chlorophyll that is chlorophyllin was shown to have a protective effect on mitochondria against oxidative damage (Kamat et al. 2000). Wheatgrass juice powder showed higher levels of glutamic acid, histidine, threonine, citrulline, arginine, gamma-aminobutyric acid (GABA) and leucine than that of pulse juice powder (Ghumman et al. 2017). Wheatgrass juice is also rich in antioxidants, vitamins, such as A, C, and E, etc., and minerals like iron (Fe), calcium (Ca), magnesium (Mg), and benzo(a)pyrene in a bioavailable form (Aydos et al. 2011). Ghumman et al. (2017) demonstrated that wheatgrass powder (WP) contained a higher proportion of K and Mg, as compared to pulse juice powder. Wheatgrass juice also possesses the superoxide scavenging and ferric reducing abilities (Kulkarni et al. 2006; Peryt et al. 1992). The presence of superoxide dismutase (SOD) in wheatgrass juice helps in lowering the effect of radiations and thus digests the toxin (Bar-Sela et al. 2007; Cao et al. 1996). The pH of wheatgrass juice is similar to blood i.e., 7.4. Because of this wheatgrass juice gets quickly absorbed into the blood and helps in detoxification of the body, digestion, improves blood flow, etc., (Bar-Sela et al. 2007). Wheatgrass juice has shown to have antioxidant and anti-inflammatory effects. Therefore, it can reduce the incidence of cancer (Calzuola et al. 2004). Indole compounds in wheatgrass increase the activity of the xenobiotic metabolic enzyme in the liver and intestinal mucosa, which might be responsible for the deactivation of carcinogens (Bonnesen et al. 2001).Wheatgrass juice can decrease the dose of medication during chemotherapy needed for blood and bone marrow building as seen in breast cancer patients (Bar-Sela et al. 2007)

Wheat being a C3 crop converts CO₂ into glucose in the presence of light, which is required for different metabolic pathways. Studies have shown that an increase in photoperiod from 16 to 22 h and a decrease in the dark period from 8 to 2 h improved the performance of crop and shorten the flowering and seed maturation time in wheat. The phenomenon of enhanced photoperiod and a decrease in dark period is new and popularly known as “speed breeding”. Speed-breeding can be also done following different methods like embryo culture, vernalization of grains, increasing exposure to light, etc. Speed breeding greatly helped plant breeders rapid selection of desirable traits and development of new varieties in a short time duration (Watson et al. 2018). However the effect of such rapid cultivation methods on the yield and antioxidant values of wheatgrass juice was not evaluated for Indian wheat varieties. The effect of prolong photoperiod and supplementation of the nutrient medium on the wheatgrass shoot and juice powder of hard (HW), medium-hard (MHW) and soft wheat (SW) varieties was studied. Therefore, the objective of the present study was to enhance the yield and antioxidant potential of wheatgrass juice by changing photoperiod conditions or by supply of nutrient media.

Materials and methods

Chemicals and reagents

Chemicals such as 2,2-diphenyl-1-picrylhydrazyl (DPPH), ( ±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), 2,3,5-triphenyltetrazolium chloride (TPTZ) coomassie brilliant blue G 250, 3,4,5-trihydroxybenzoic acid (Gallic acid), iron(III) chloride (ferric chloride), sodium carbonate, sodium acetate, Folin & Ciocalteu’s phenol reagent (FC reagent), acrylamide, bis-acrylamide, sodium dodecyl sulfate, ammonium persulfate, glycine,

N,N,N′,N′-tetramethylethylenediamine (TEMED), tris(hydroxymethyl)aminomethane (Tris Base) were of molecular biology/electrophoresis grade and purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Murashige & Skoog macroelements (10x) and microelements (100x) solutions and vitamins (100x) stocks were purchased from Himedia (Himedia Laboratories, Mumbai—400 086, India).

Plant material

Three wheat varieties viz. K307 (hard wheat), DBW39 (medium-hard wheat) and QBP12-11 (soft wheat) were procured from Indian Agricultural Research Institute (IARI), Pusa, New Delhi for this study.

Sample preparation

Wheat grains (50 g) soaked in autoclaved distilled water (ADW) for 30 min. Soaked grains were washed twice with ADW and spread on moist disc of filter papers previously placed in sterilized glass trays. Glass trays with wheat grains kept in plant growth chamber (Narang Scientific Works Pvt Ltd^®, New Delhi—110 064, India) under dark at 17 °C for first 2 days for germination. The lights were switched on third day post-germination of grains and glass trays were supplied with 0.5× and 1× Murashige and Skoog media (MS) media while controls were supplied with same volume of water. Plant growth chamber was equipped with fluorescent lights and wheatgrass were grown under normal photoperiod (16 h light:8 h dark) and prolong photoperiod (22 light: 2 h dark). Control and MS media supplied glass trays were kept moist with ADW for next 8 days. After 10 days, seedlings were harvested, weighed and a small proportion of wheatgrass was snap frozen in liquid N₂ gas and stored in −20 °C deep freezer for further analysis. While a large proportion was crushed using a Kalsi^® brand (Bhajan Singh & Sons, Ludhiana-141008) hand operated screw juicer to obtain juice. The juice thus obtained was freeze dried and also kept in −20 °C for further biochemical analysis. Intact wheatgrass plants with roots (triplicates) were carefully rooted up from Petri plates and height was measured using a stainless steel scale.

DPPH assay

It was performed using a modified procedure for smaller volumes performed by Nicklisch and Waite (2014). DPPH (2 mM) working solution was prepared in pure methanol and kept in dark and cool place. A standard curve for DPPH solution was prepared with different concentrations of Trolox solution (0, 2, 4, 6, 8 and 10 µg/µl) which was prepared in 80% methanol. A reaction mixture of 1 ml was prepared, of which 50 µl was sample extract, 50 µl was DPPH solution and rest 950 µl was 80% methanol. Reaction blank prepared by mixing 950 µl 80% methanol and 50 µl pure methanol. The reading for this assay was taken at 0 min and after 30 min. The experiment was carried out in duplicate. Thus absorbance values obtained at 515 nm were used in the following formula and %DPPH remaining was calculated (Nicklisch and Waite 2014; Brand-Williams and Berset 1995).

$$\%\mathrm{DPPH}\mathrm{remaining}=\frac{{\mathrm{Abs}}_{30\min }-{\mathrm{Abs}}_{\mathrm{sample}}}{{\mathrm{Abs}}_{30\min }-{\mathrm{Abs}}_{\mathrm{control}}}\times 100$$

FRAP assay

Ferric reducing antioxidant power (FRAP) was performed by following Benzie and Strain (1996) method. Trolox was used to make standard curve of different concentrations (Benzie and Strain 1996).

Chlorophyll estimation

Chlorophyll was estimated according to the method described by Hiscox and Israelstam’s method (1979). Briefly, 100 mg of WSP was poured into 15 ml falcon tube and mixed in 1 ml of DMSO solution. The absorbance of the solution was recorded at 645 nm and 663 nm. The absorbance values thus obtained were put in the following formula to calculate chlorophyll a and b (Hiscox and Israelstam 1979) content.

$$\text{Chl a}\left(\text{mg}/\text{g}\right)=\left[\left(12.7\ast \text{abs}@663\right)-\left(2.6\ast \text{abs}@645\right)\right]\ast \text{ml of DMSO per mg sample}$$

$$\text{Chl b}\left(\text{mg}/\text{g}\right)=\left[\left(22.9\ast \text{abs}@645\right)-\left(4.68\ast \text{abs}@663\right)\right]\ast \text{ml of DMSO per mg sample}$$

Total phenolic content

Total phenolic content (TPC) was carried out according to the method of Ainsworth and Gillespie (2007). TPC was estimated using the standard curve of different concentrations of gallic acid (Ainsworth and Gillespie 2007).

Protein estimation

Protein content was done according to Bradford’s method (1976). A standard curve of BSA (bovine serum albumin) in presence of Bradford solution was drawn for concentration of protein (BSA) ranging from 0 to 10 µg at 595 nm wavelength (Bradford 1976).

Protein profiling

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of wheatgrass shoot powder and wheatgrass juice powder was carried out according to the modified method of Laemmli (1970). Briefly, 100 mg of fine leaf powder or freeze dried wheatgrass juice powder (WJP) was weighed in pre-sterile eppendorff tubes (ET) and 1.0 mL of extraction buffer [50 mM Tris buffer (pH 6.8), 0.1% sodium dodecyl sulfate, 4% β-mercaptoethanol, 20% glycerol, and 15 µl/mL protease inhibitor cocktail (Sigma-Aldrich, USA)] was added. Soluble proteins were estimated by using Bradford’s method (1976) and 40 µg of protein solution was transferred on to fresh ETs and equal volume of 2 × Laemmli buffer [100 mM Tris buffer (pH 6.8); 4% SDS; 2% β-mercaptoethanol; 20% glycerol; 0.04% bromophenol blue] was mixed together and loaded on to the wells. The electrophoresis was carried out at 35 mA constant current and gels were stained with coomassie brilliant blue R250 (CBB-R250) dye (50% methanol; 10% glacial acetic acid; 0.2% w/v CBB-R250). Stained gels were destained by using destaining solution (20% methanol and 12% glacial acetic acid) and scanned with HP Scanjet 4010 scanner at 600 dots per inch resolution. AlphaEaseFC® v6.0.0 was used for the determination of molecular weight of different polypeptide bands (Laemmli 1970; Kawakatsu et al. 2008).

Statistical analysis

All analysis was performed at least in triplicate. Data obtained were analyzed using statistical package IBM^® SPSS^® v23.0 (SPSS Inc., Chicago, IL, USA, 2015) for Windows 7.

Results and discussions

Wheatgrass height and yield

The average wheatgrass height (WH) of 11.3 cm, 15.5 cm, and 17.0 cm, respectively in water, 0.5x, and 1xMS media under normal photoperiod (16 h light: 8 h dark) against 15. 0 cm, 15.3 cm, and 14.7 cm, respectively under prolong photoperiod (22 light: 2 h dark) was observed (Table 1). Therefore, the height of WG grown in different strengths of MS media under normal light conditions increased significantly. The prolonged photoperiod increased WH in water and decreased in 0.5 × and 1 × MS media. Though, WH from prolonged photoperiod in water and MS media was higher than from the normal photoperiod. The statistical analysis revealed a significant effect of varieties, MS media and photoperiod on WH. The effect of varieties on WH was the highest followed by MS media (Table 3). The significant interaction effect of variety with photoperiod and MS media, photoperiod x MS media and variety x photoperiod x MS media on WH was also observed. However, the interaction effect of variety with photoperiod was the highest on WH. WH of 11.5 cm, 9.8 cm, and 12.5 cm, respectively for wheatgrass of K307, DBW39 and QBP12-11in water under normal photoperiod against an increase up to 13 cm, 16 cm and 15.8 cm, respectively for these in water under prolong photoperiod was observed. These findings thus revealed that a prolong photoperiod increased WH and its effect on DBW-39 was much higher (Table 3). An increase in plant height under prolonged light conditions was also observed for the chrysanthemum. Height of 42.70 cm and 46.25 cm, internodal length of 2.63 cm and 2.70 cm, and leaf number of 29.75 and 32.75, respectively for Chrysanthemum cv. Zembla plants grown under normal and prolong photoperiod was reported (Kumar and Singh 2017). Therefore, prolong photoperiod increased WH in water more effectively, whereas, the effect of MS media on WH under normal photoperiod was more pronounced.

Table 1.

Effect of growth media and photoperiod on various physiological parameters of wheatgrass of different varieties

		Photoperiod
Parameters	Treatment	16 h light:8 h dark			22 h light:2 h dark
Plant height(cm)	Variety	Water	0.5xMS	1 × MS	Water	0.5xMS	1 × MS
	K307	11.5	13.5	16	13	12.8	14
	DBW39	9.8	16	16.5	16	17.8	15.6
	QBP12-11	12.5	17	18	15.8	15.3	14.5
	Average	11.3	15.5	17.0	15.0	15.3	14.7
Wheatgrass yield(G)	K307	6.77	7.42	9.67	7.46	8.84	10.08
	DBW39	8.68	9.40	10.94	9.22	10.48	11.26
	QBP12-11	7.41	7.70	10.59	7.97	10.34	10.30
	Average	7.6	8.2	10.4	8.2	9.9	10.6

MS media: Murashige and Skoog basal medium

Table 3.

F value of data shown in Table 1 and 2 for wheatgrass powder of hard, medium-hard and soft wheat varieties

Source	WGY	WH	PC	Chl a	Chl b	Total Chl	DPPH	TPC	FRAP
Var	23,467**	891.71**	7327**	37.31**	36.59**	138.67**	15,569**	87,119**	62,631**
PhP	17,568**	198.75**	86,545**	20.91**	80.67**	188.16**	284,273**	302,177**	8,997,969**
MS	57,958**	693.22**	11,560**	0.99NS	0.19NS	0.75NS	70,383**	22,479**	1,335,023**
Var *PhP	267.57**	1558.11**	41,954**	25.63**	16.75**	71.36**	8780**	11,442**	17,913**
Var *MS	489.97**	181.36**	2843**	3.9*	11.27**	23.63**	34,346**	50,956*	135,985**
PhP *MS	5640**	63.54**	22,572**	5.2*	6.59**	9.92**	16,236**	37,755**	360,394**
Var *PhP *MS	1100**	216.58**	16,062**	2.3NS	7.35**	11.20**	24,846**	44,619**	106,902**

NS: non-significant; Var = variety; PhP: photoperiod; MS: Murashige and Skoog basal media. WGY: wheatgrass yield; WH: wheatgrass height; PC: protein content; Chl a: chlorophyll a; Chl b: chlorophyll b; Total Chl: total chlorophyll; DPPH: 2, 2-diphenyl-1-picrylhydrazyl; TPC: total phenolic content; FRAP: ferric reducing antioxidant power assay

^*p ≤ 0.05; ** p ≤ 0.005

Average wheatgrass yield (WGY) of 7.6 g, 8.2 g, and 10.4 g, respectively for water, 0.5 × and 1 × MS media under normal photoperiod against 8.2 g, 9.9 g, and 10.6 g, respectively under extended photoperiod was observed (Table 1). These results thus revealed that wheatgrass exposed to long photoperiod led to an increase in WGY both in water and media. Statistical analysis also revealed the significant effect of varieties, MS media and photoperiod on WGY; however, the effect of MS media on WGY was higher as compared to variety and photoperiod. The interaction effect of variety with photoperiod, and MS media, as well as photoperiod x MS media on WGY, was also significant. However, the interaction effect of photoperiod x MS media on WGY was higher as compared to others. WGY of 0.5 × and 1 × MS media was higher both under normal and long photoperiods. However, the increase in WGY was higher in response to long photoperiodic conditions. WGY showed a positive correlation with WH (r² = 0.557, p ≤ 0.005), therefore, the height of wheatgrass also linked to yield.

Physico-chemical properties of wheatgrass

Protein content

Protein content (PC) of wheatgrass shoot powder (WSP) varied in response to MS media and photoperiod. WSP from different varieties showed average PC 12.2, 11.3 and 15.1 mg/G FW, respectively grown in water, 0.5 × and 1 × MS under normal light conditions, against 15.7, 15.6 and 15.1 mg/G FW, respectively under prolong light conditions (Table 2). Statistical analysis revealed a significant effect of varieties, photoperiod, and MS media on the PC of WSP (Table 3). However, the effect of photoperiod on the PC of different wheatgrass varieties was highly significant. Furthermore, the interaction effect of variety, MS media and photoperiod on the PC of WSP were significant (p =  < 0.005). However, the interaction effect of varieties x photoperiod on the PC was the highest followed by photoperiod x MS media (Table 3). These results thus demonstrated that PC was largely affected by varieties and photoperiod. Major changes in PC of DBW-39 and QBP12-11 were observed. PC of wheatgrass DBW39 and QBP12-11 was 9.96 and 11.0 mg/G FW, respectively for water under normal photoperiod was observed against 15.50 mg/G FW for K307 (Table 2). PC increased in response to extended photoperiod. PC of 15.40 and 18.20 mg/G FW for DBW39 and QBP12-11 grown in water under normal photoperiod against PC of 13.63 mg/G FW for K307 under prolonged photoperiod was observed (Table 2). Yunze and Shuangsheng (2014) also reported that extension in light duration before and after flowering resulted in an increase in starch content, whereas the accumulation of protein in wheat seeds was decreased along with grain yield. These findings indicated no adverse effect of prolonged photoperiod on PC of different Indian wheatgrass varieties.

Table 2.

Effect of growth media and photoperiod on various physico-chemical parameters of wheatgrass of hard, medium-hard and soft wheat varieties

Source		Photoperiod
		16 h light:8 h dark			22 h light:2 h dark
Protein content(mg/G FW)	Variety	Water	0.5 × MS	1 × MS	Water	0.5 × MS	1 × MS
	K307	15.50	13.50	15.28	13.63	14.73	16.25
	DBW39	9.96	11.40	16.70	15.40	15.75	11.70
	QBP12-11	11.00	8.96	13.33	18.20	16.45	17.30
	Average	12.2	11.3	15.1	15.7	15.6	15.1
Chl a (average)	K307	0.34	0.31	0.35	0.43	0.44	0.42
	DBW39	0.33	0.35	0.35	0.35	0.36	0.37
	QBP12-11	0.34	0.30	0.32	0.33	0.33	0.24
	Average	0.33	0.32	0.34	0.37	0.38	0.34
Chl b (average)	K307	0.37	0.43	0.45	0.50	0.56	0.54
	DBW39	0.47	0.47	0.43	0.46	0.47	0.53
	QBP12-11	0.47	0.35	0.36	0.43	0.44	0.42
	Average	0.44	0.42	0.41	0.46	0.49	0.50
Chl a + b (Avg)	K307	0.72	0.74	0.80	0.92	1.00	0.96
	DBW39	0.80	0.81	0.78	0.82	0.83	0.90
	QBP12-11	0.81	0.65	0.68	0.77	0.77	0.66
	Average	0.78	0.73	0.75	0.84	0.87	0.84
Total TPC(µg GA equivalent TPC/G FW)	K307	1003.01	1243.05	1142.97	1241.85	1244.77	1194.24
	DBW39	1064.90	1056.52	1149.55	1186.41	1160.56	1162.80
	QBP12-11	1107.40	1031.31	1122.28	1117.51	1130.62	1155.48
	Average	1058	1110	1138	1182	1179	1171
Average % DPPH inhibition	K307	44.34	41.06	36.39	44.85	45.81	42.59
	DBW39	40.88	39.86	38.78	46.96	39.53	40.35
	QBP12-11	39.43	39.50	38.80	44.37	41.17	46.06
	Average	42	40	38	45	42	43
FRAP(µM Trolox equivalent FRAP/G FW)	K307	885.90	1062.44	1107.94	1192.30	1242.00	1276.01
	DBW39	927.01	988.27	1163.27	1262.86	1204.09	1233.59
	QBP12-11	1011.61	1016.53	1154.27	1214.63	1182.88	1345.79
	Average	942	1022	1142	1223	1210	1285

MS media: Murashige and Skoog basal medium

Chlorophyll a, chlorophyll b and total chlorophyll content

Chlorophyll (Chl) is a key active component in wheatgrass that actively involved in the inhibition of the metabolic activation of carcinogens (Aydos et al. 2011). Therefore, chlorophyll content in WSP cultivated under normal and prolonged light conditions was also estimated. Average Chl a content between 0.32 to 0.34 mg/G FW for WSP of different varieties grown in water and MS media under normal light conditions against 0.34 to 0.38 mg/G FW under prolong photoperiod was observed. While Chlorophyll b (Chl b) content between 0.41 to 0.44 in water and MS media for WSP of different varieties under normal light conditions against 0.46 to 0.50 mg/G FW under prolong light conditions was observed (Table 2). Statistical analysis revealed a significant effect of varieties and photoperiod on Chl a and Chl b content (Table 3). Statistical analysis further revealed significant interaction effect of varieties with photoperiod and MS media, as well as photoperiod x MS media on Chl a and Chl b content of wheatgrass of different varieties. However, the interaction effect of varieties x photoperiod was significantly higher as compared to varieties x MS media. WSP of K307 showed greater changes in chlorophyll a and chlorophyll b content in response to prolonged photoperiod as compared to WSP of DBW-39 and QBP12-11 (Table 3). Chl a content ranged between 0.31 and 0.35 mg/G FW for WSP of K307 grown under normal light condition, whereas, it was observed between 0.42 to 0.44 mg/G FW for WSP of K307 grown under long photoperiod (Table 2). Similar observations for chlorophyll b content in WSP of K307 were also observed. Chl b content ranged between 0.37 and 0.45 mg/G FW and 0.50–0.56 mg/G FW, respectively for WSP of K307 grown under normal and long photoperiod conditions. These observations thus demonstrated the greater effect of varieties and photoperiod on Chl a and Chl b content.

The average total chlorophyll content between 0.73 and 0.78 mg/G FW and 0.84–0.87 mg/G FW, respectively for WSP grown under normal light and prolong light conditions was observed. (Table 2). Statistical analysis also revealed a greater effect of varieties and photoperiod on total chlorophyll content, however, the effect of later was higher. Statistical analysis also revealed highly significant (p ≤ 0.005) interaction effect of varieties x photoperiod on total chlorophyll content as compared to photoperiod x MS media, and variety x photoperiod x MS media (Table 3). Total chlorophyll content of WSP from K307 was also greatly influenced by photoperiod and total chlorophyll content ranged between 0.72 to 0.80 mg/G FW under normal photoperiod against between 0.92 and 1.00 mg/G FW for long photoperiod. These findings thus revealed that total chlorophyll content of WSP from K307 was highly influenced by photoperiod and MS media as compared to WSP of DBW-39 and QBP12-11. Earlier studies have shown negative correlation of chlorophyll content with length of photoperiod for tomato and pepper plants (Dorais et al. 1996). Furthermore, photoperiod significantly affected Chl a and Chl b and total chlorophyll content in basil plants (Avgoustaki 2019). Total chlorophyll content of 3.45 and 3.65 mg/G FW, respectively for control and long photoperiod grew Chrysanthemum cv. Zembla plants were reported by Kumar and Singh (2017). Higher chlorophyll content indicates higher photosynthesis rates which thus led to higher ash/essential mineral content, which are essentially important co-factors for several metabolic and pigment biosynthesis pathways (Ghumman et al. 2017).

Total phenolic content (TPC)

The average TPC value of 1058, 1110, and 1138 µg GA equivalent TPC/G FW, respectively in water, 0.5 × , and 1 × MS media for WSP of different varieties under normal light conditions against 1182, 1179, and 1171 µg GA equivalent TPC/G FW, respectively under prolong light conditions was observed (Table 2). Statistical analysis revealed a significant effect of varieties, MS media and photoperiod on average TPC values, however, the effect of photoperiod was higher as compared to varieties and MS media (Table 3). The interaction effect of varieties, MS media and photoperiod was also significant; however, the interaction effect of variety with MS media as well as photoperiod x MS media on TPC was significantly higher as compared to variety x photoperiod, etc. (Table 3). Our findings demonstrated an increase in TPC values for different wheatgrass in water under prolonged photoperiod. TPC of 1003, 1064 and 1107 µg GA equivalent TPC/G FW, respectively for WSP of K307, DBW39 and QBP12-11 under normal photoperiod against 1242, 1186, 1118 µg GA equivalent TPC/G FW, respectively under prolong photoperiod was observed (Table 2). These findings thus revealed that changes in TPC value for WSP of K307 was higher in response to photoperiod as compared to other varieties. These observations thus revealed that, under normal photoperiod, MS media boosted the biosynthesis of different phenolics. These results also exhibited an increase in TPC for wheatgrass grown in MS media, which was also increased for wheatgrass grown in water or MS media under prolong photoperiod. Varietal changes in TPC for Polish wheat were also reported by Kowalska et al. (2019). The highest TPC of 1377.96 μg/g in the flag leaf of Trappe wheat than that TPC of 1004.93 μg/g for Kandela was exhibited. Variations in TPC content for the flag leaf of different spring and winter wheat varieties was also reported (Kowalska et al. 2019). Gallic acid content of 1.76 mg/G for wheatgrass shoot powder of Raj3765 against 0.82 mg/G for PBW343 was observed. Whereas, the ferulic acid content of 0.13 mg/G for wheatgrass shoot powder of Raj3765 against 0.16 mg/G for PBW343 was observed (Ghumman et al. 2017). These findings thus revealed inter-varietal differences in TPC for wheat varieties.

The average TPC values of 8.5, 9.4 and 10.3 µg gallic acid equivalent AOA/mg dry weight of WJP (µg GAE AOA/mg DW WJP), respectively in water, 0.5 × and 1 × MS media under normal photoperiod against 8.8, 10.5, 9.8 µg GAE AOA/mg DW WJP under prolong photoperiod were observed (Table 4). Therefore, MS media and photoperiod highly influenced the phenolic content in different wheatgrasses. Statistical analysis also revealed that varieties, MS media and photoperiod significantly (p ≤ 0.005) affected TPC and the effect of MS media was the highest aside from varieties and photoperiod. The interaction effect of variety x photoperiod; variety x MS media; MS media x photoperiod; and variety x MS media x photoperiod was also significant, however, the interaction effect of MS media x photoperiod and variety x MS media x photoperiod was more pronounced (Table 5). On the other hand, Özköse et al. (2016) also demonstrated TPC of 293 mg GAE/L for the juice of the first cut of wheatgrass supplemented with fertilizers against 324 mg GAE/L for the juice of water grown wheatgrass. These values were 289 and 342 mg GAE/L, respectively for juice of fertilizer supplemented and water grown wheatgrass of the second cut. It was, therefore, likely that TPC in wheat was affected by growing conditions.

Table 4.

Effect of growth medium and photoperiod on the physico-chemical properties of wheatgrass juice powder obtained from hard, medium-hard and soft wheat varieties

Source		Photoperiod
Parameters	Treatment	16 h light:8 h dark			22 h light:2 h dark
	variety	Water	0.5 × MS	1X MS	Water	0.5 × MS	1 × MS
Avg DPPH (µg Trolox equivalent AOA/mg dw of juice)	K307	4.83	5.96	5.60	5.07	4.29	3.75
	DBW39	5.78	5.94	3.73	3.12	3.29	3.9
	QBP12-11	7.63	6.54	3.16	4.12	5.52	5.07
	Average	6.1	6.2	4.2	4.1	4.2	4.2
Total TPC (µg GA equivalent TPC/G FW)	K307	7.17	8.21	7.56	8.21	24.17	31.45
	DBW39	6.68	7.81	6.96	6.65	34.09	6.78
	QBP12-11	8.75	35.7	9.12	23.77	11.06	30.95
	Average	8.5	9.4	10.3	8.8	10.5	9.8
FRAP (µM Trolox equivalent FRAP/G FW)	K307	0.17	0.19	0.18	0.19	0.29	0.13
	DBW39	0.19	0.18	0.17	0.15	0.15	0.13
	QBP12-11	0.22	0.44	0.18	0.27	0.17	0.37
	Average	0.19	0.27	0.18	0.21	0.20	0.21

MS media: Murashige and Skoog basal medium

Table 5.

F value of data shown in Table 1 and 2 for wheatgrass juice powder of hard, medium-hard and soft wheat varieties

Source	DPPH	TPC	FRAP
Var	887**	58,021**	11.04**
PhP	1031**	79,578**	1.79**
MS	3611**	185,715**	0.15**
Var *PhP	211**	22,550**	0.66**
Var *MS	152**	18,286**	0.72**
PhP *MS	1027**	24,503**	2.19**
Var *PhP *MS	1001**	120,609**	7.07**

NS: non-significant; Var = variety; PhP: photoperiod; MS: Murashige and Skoog basal media. WGY: wheatgrass yield; WH: wheatgrass height; PC: protein content; Chl a: chlorophyll a; Chl b: chlorophyll b; Total Chl: total chlorophyll; DPPH: 2, 2-diphenyl-1-picrylhydrazyl; TPC: total phenolic content; FRAP: ferric reducing antioxidant power assay

^* = p ≤ 0.05; ** = p ≤ 0.005

Ghumman et al. (2017) reported that wheatgrass powder accumulated the highest levels of sinapic acid followed by gallic acid as compared to other polyphenols, present in free- or bound form. While wheatgrass juice powder showed the higher levels of the free-form of sinapic-, ferulic- and p-coumaric acid, as compared to gallic-, chlorogenic- and vanillic acid. Ghumman et al. (2017) demonstrated that sinapic acid and ferulic acid appeared to be the main polyphenols, which play a key role in determining the antioxidant potential of wheatgrass powder.

Antioxidant activity

DPPH assay

Growth media and photoperiod affected DPPH inhibition assay in a variety-dependent manner. Average DPPH percentage inhibition (DPI) for WSP of different varieties was decreased with an increase in the strength of MS media. The average DPPH inhibitory values of WSP of different varieties between 38 to 42% under normal light condition were observed against 42% to 45% under prolong light conditions (Table 2). Significant effect of varieties, MS media and photoperiod on DPPH inhibitory value was revealed by statistical analysis. However, the effect of MS media on DPI was much larger than photoperiod and varieties (Table 3). The interaction effect of variety x photoperiod, MS media, photoperiod x MS media, and variety x photoperiod x MS media on DPI was also significant, but the interaction effect of variety x MS media was higher than that of other factors (Table 3). DPI decreased for different wheatgrass under normal light conditions. However, the effect of long photoperiod on DPI was differential and variety-dependent. DPI between 42.59% and 45.81% for WSP of K307 was observed against 39.53–46.96% for WSP of DBW39 under prolong photoperiod (Table 2). However, DPI of 44.37%, 41.17%, and 46.06%, respectively, for WSP of QBP12-11 grown under water, 0.5 × and 1 × MS media and prolonged light conditions were observed (Table 2).

The average DPI of 6.08, 6.15 and 4.16 µg Trolox equivalent AOA/mg dry weight of WJP (µg TE AOA/mg DW WJP) in water, 0.5 × and 1 × MS media under normal photoperiod was observed against 4.10, 4.37 and 4.24 µg TE AOA/mg DW WJP under prolong photoperiod (Table 4). Therefore, a decline in DPI in response to MS media and prolonged photoperiod was observed. Statistical analysis revealed that varieties, MS media and photoperiod significantly affected the antioxidant potential of wheatgrasses from different varieties (Table 5). However, the effect of MS media on DPI was much higher besides of varieties and photoperiod. The interaction effect of the MS media x photoperiod was greater than varieties with photoperiod and MS media (Table 5). The differential response of different wheatgrass varieties for MS media and photoperiod may be responsible for a decline in average DPI values. An increase in DPI value of 4.83 and 5.96 µg TE AOA/mg DW for WJP of water, 0.5 × and 1 × MS grew K307 was observed against 5.78 and 5.94 µg TE AOA/mg DW WJP, respectively for WJP of DBW39 in water and 0.5 × MS media under normal light conditions. A decline in DPPH values of 5.6 and 3.73 µg TE AOA/mg DW WJP for K307 and DBW39, respectively, in 1 × MS media under normal photoperiod was observed. Similar results were also observed for these wheatgrass varieties under prolonged photoperiod (Table 4). These findings thus exhibited that photoperiod differentially affected the antioxidant potential of different wheat varieties; therefore, screening of wheatgrass varieties with high antioxidant potential is highly desired for speed breeding-mediated enhancement in nutraceutical potential of different wheatgrass juice. Furthermore, under normal photoperiod, MS media decreased the biosynthesis of nutraceutical compounds, which may be attributed to a higher synthesis of proteins and starch molecules, as depicted from higher WGY and PC content in WSP of different varieties.

Akbas et al. (2017) reported antioxidant activity between 0.30 and 0.06 mg DPPH/g powder of WJP encapsulated in different ratios of whey protein isolates (W) and maltodextrin (MD). The antioxidant activity of WJP influenced with different planting conditions, harvesting period, and varieties and the presence of phenolics compounds and flavonoids widely determines the antioxidant activity of wheatgrass (Aydos et al. 2011; Kulkarni et al. 2006). Phenolics are responsible for hydrogen donating ability in different plant samples which was related to their DPPH radical scavenging activity (Benincasa et al. 2015; Brand-Williams et al. 1995). While flavonoids containing samples also possesses strong antioxidant activity due to the presence of double bonds in the C-ring. The structure and hydroxyl group arrangement of flavonoids are responsible for its radical scavenging activity. Therefore, higher chlorophyll content may attribute to higher DPPH inhibition activity in wheatgrass under prolonged photoperiod as well as under MS media. Higher radical scavenging capacity linked to the pronounced anticancer activity, and also to reduced oxidative damage to deoxyribose nucleic acid (DNA) and lower lipid peroxidation (Singh et al. 2012). Therefore, higher chlorophyll content and TPC in WJP of wheatgrass under a long photoperiod may attribute to increased DPPH inhibitory activity.

FRAP assay

The average FRAP values between 942 and 1142 µM Trolox equivalent TPC/G FW for WSP of different varieties grown in water and MS media under normal light conditions against 1210–1285 µM GA equivalent TPC/G FW under prolong light conditions were observed (Table 2). Statistical analysis revealed a significant effect of photoperiod and MS media on average FRAP values, however, the effect of photoperiod was higher as compared to varieties and MS media. The interaction effect of varieties, MS media and photoperiod was also significant; however, the interaction effect of photoperiod x MS media on TPC was more pronounced as compared to variety x photoperiod and varieties x MS media (Table 3). Therefore, changes in photoperiod greatly influenced the FRAP of wheatgrass from different varieties. TPC of 886, 927 and 1012 µM Trolox equivalent TPC/G FW for K307, DBW39 and QBP12-11 in water under normal photoperiod was observed against 1192, 1263 and 1215 µM Trolox equivalent TPC/G FW under prolong photoperiod (Table 2). On the contrary, an increase in FRAP value in response to MS media was not significantly influenced under prolonged photoperiod, but was increased in response to MS media under normal photoperiod. FRAP values showed positive correlation to Chl b (r² = 0.376, p ≤ 0.005), DPPH (r² = 0.396, p ≤ 0.005) and TPC (r² = 0.695, p ≤ 0.005). Therefore, photoperiod more prominently affected FRAP potential of different varieties as compared to MS media.

Wheatgrass juice powder (WJP) showed average FRAP values between 0.18 and 0.27 µg Trolox equivalent AOA/mg dry weight of WJP (µg TE AOA/mg DW WJP) in water and MS media under normal photoperiod against 0.20 to 0.21 µg TE AOA/mg DW WJP under long photoperiod (Table 4). The strength of MS media and light conditions largely influenced the FRAP potential of wheatgrass juice. Average FRAP values for WJP were increased in response to 0.5 × MS media under normal photoperiod and showed a similar increase under prolonged photoperiod also. Statistical analysis also revealed pronounced effect of varieties on FRAP values of WJP, and the interaction effect of variety x MS media x photoperiod was also highly significant (Table 4). FRAP values of 0.17, 0.19 and 0.22 µg TE AOA/mg DW WJP of K307, DBW39 and QBP12-11, respectively in water under normal photoperiod against 0.19, 0.15 and 0.27 µg TE AOA/mg DW WJP were observed. These findings showed higher diversity in FRAP values of WJP of different varieties (Table 4). The higher increase in FRAP values for QBP12-11 in response to prolonged photoperiod was observed as compared to other wheat varieties. Furthermore, 1 × MS media showed a negative effect on FRAP values both under normal and prolonged photoperiod, which implies that wheatgrass juice produced from water grown seedling under prolong photoperiod possessed better nutritional attributes. These findings also demonstrated that prolong photoperiod did not adversely affect the nutraceutical profile of wheatgrass juice of different wheat varieties.

SDS-PAGE analysis of WSP and WJP

SDS-PAGE revealed the presence of polypeptide (PPs) bands between 150 and 9 kDa (± 2 kDa) in WSP of different varieties (Fig. 1a). Greater effect of MS media and prolong photoperiod on the accumulation of major PPs of 150 kDa, 61 kDa, and 9 kDa in WSP of different varieties was observed. Higher accumulation of 150 kDa, 61 kDa PPs and 9 kDa in WSP of DBW39 in 0.5 × MS media under normal photoperiod was observed. While, decrease in the accumulation of major PPs in WSP of different varieties in 0.5 × and 1 × MS under prolonged photoperiod was observed (Fig. 1a). The effect of prolong photoperiod on wheatgrass of different varieties was much higher as compared to wheatgrass grown in MS media under normal light conditions. Previous studies established the identity of 61 kDa and 9 kDa PPs as subunits of Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which are the major PPs in the leaves of green plants. Therefore, increase in the levels of rubisco proteins may be positively associated with prolong photoperiod. Higher levels of protein accumulation in wheatgrass under the prolong photoperiod may also be associated to higher accumulation of these PPs. Therefore, an improvement in the physico-chemical properties of wheatgrass of prolong photoperiod may also be linked to light-induced increase in protein and starch metabolism in wheatgrass of different varieties.

Figure 1.

a SDS-PAGE analysis of total proteins isolated from wheatgrass shoot powder of different Indian bread wheat varieties. 18/6: 18 h light and 6 h dark; 22/2: 22 light and 2 h dark period; kDa: kilo Dalton; MS: Murashige and Skoog medium. b SDS-PAGE analysis of total proteins of wheatgrass juice obtained from different Indian bread wheat varieties. 18/6: 18 h light and 6 h dark; 22/2: 22 light and 2 h dark period; kDa: kilo Dalton; MS: Murashige and Skoog medium.

The SDS-PAGE analysis of total proteins in WJP of different varieties revealed the presence of polypeptides (PPs) between 92 and 10 kDa (± 2 kDa). The major effect of MS media and prolong photoperiod on the accumulation of major PPs of 92 kDa, 33 kDa, 23 kDa, 14 kDa, 12 kDa, and 10 kDa of different wheat varieties was observed (Fig. 1b). Major changes in 33 kDa PP was observed, therefore, integrated densitometry analysis of 33 kDa PP of WJP of different varieties was carried out (Fig. 1S). An increase in the storage of 33 kDa PP for WJP of K307 and QBP12-11 in different doses of MS media under normal photoperiod was observed. However, the levels of 33 kDa PP of DBW39 in 0.5 × MS media decreased followed by a rise in 1 × MS media (Fig. 1S). Therefore, growing wheatgrass in the presence of MS media significantly enhanced the accumulation of 33 kDa PP in WJP of different wheat varieties, which was adversely affected for DBW39 and QBP12-11 under prolonged photoperiod (Fig. 1S). A positive effect of prolong photoperiod and growth media on the accumulation of 33 kDa PP in WJP of different varieties. Previous studies have also reported that 26 SOD genes of SOD1 and SOD2 are encoded by wheat genome with molecular weight between 14.2 and 43.4 kDa. Among these 26 wheat SODs, 32.3 kDa, 22.2 kDa, 25.3 kDa, 24.6 kDa were also encoded in wheat genome and the cytosolic localization of these SODs was also predicted in silico (Jiang et al. 2018). Studies have shown that wheatgrass are rich source of peroxidases, superoxide dismutases, and cytochromes, etc. (Parit et al. 2018). It is therefore, likely that 23 kDa and 33 kDa PP of WJP may be superoxide dismutase (SOD) enzyme, which strongly acts as a key antioxidant enzyme under different abiotic stress conditions (Fig. 1b). The identification of these major polypeptides by using peptide-fingerprinting technology (MALDI-ToF MS) is underway.

Conclusion

The present study deciphered that photoperiod and the different doses of growth media influenced the nutraceutical and protein characteristics of WSP and WJP of different varieties. Under normal photoperiod, the average plant height, yield, protein content, TPC and FRAP values increased in response to 0.5 × and 1 × MS media. While, the average WGY, Chl-b, FRAP increased and PC, Chl-a, total chlorophyll, DPPH, and TPC decreased in response to different doses of MS media and prolong photoperiod. Still, these values remained higher from wheatgrasses grown in water and MS media under normal light conditions. WJP was also analyzed for different physico-chemical properties. WJP showed an increase in average DPPH, FRAP and TPC in 0.5 × MS media under normal photoperiod, while an increase in values for these parameters in 0.5 × MS media under prolong photoperiod was also observed. These results revealed that the yield and nutraceutical value of wheatgrass may have increased by either supply of different doses of MS media or by increasing duration of the light period from 16 h light and 8 h dark to 22 h light and 2 h dark. Further studies on antioxidant profiling of polyphenols by using high-performance liquid chromatography are underway.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

ASV and NS conceived the idea while ASV and KKB performed experiments, recorded and analyzed data. ASV and NS jointly wrote MS. Laboratory facilities provided by NS and AK to perform experiments.