Lactuca sativa L. losses and wastes as a source of biobased ingredients

TL;DR

This paper explores how waste from different lettuce varieties contains valuable nutrients and bioactive compounds that could be used in food and pharmaceutical industries.

Contribution

The study identifies specific lettuce waste varieties rich in bioactive compounds, offering new opportunities for sustainable utilization.

Findings

Lollo Rossa lettuce waste contains high levels of beneficial compounds like organic acids, tocopherols, and phenolics.

Little Gems waste is notable for its high content of nonessential amino acids such as glutamine and asparagine.

Lollo Rossa extracts showed strong antioxidant activity, and some lettuce varieties displayed antimicrobial effects.

Abstract

The growing global population and increasing consumer focus on healthy eating challenge the agricultural sector to ensure both sustainable food production and safety. Lettuce (Lactuca sativa Mill.), the most cultivated leafy vegetable worldwide, can lose up to 40% of its weight during processing. This study investigates the biochemical composition of waste from six lettuce varieties, Curly‐leafed, Iceberg, Little Gems, Romaine, Frisée and Lollo Rossa, collected from losses during cultivation, transport, storage and processing.

Lollo Rossa showed the highest levels of beneficial compounds, including organic acids (189 g kg−1 dry weight (dw)), tocopherols (292 mg kg−1 dw), folates (2.7 mg kg−1 dw), carotenoids (1.1 mg g−1 dw) and essential minerals. Little Gems waste stood out for its content of nonessential amino acids, particularly glutamine (63 mg g−1 dw), asparagine (17.4 mg g−1 dw) and glutamic acid (6.5 mg g−1 dw). Lollo Rossa also exhibited high levels of phenolic compounds (29.77 mg g−1 extract), including anthocyanins such as cyanidin‐O‐hexoside and cyanidin‐3‐(6″‐malonyl)glucoside. Decoction extracts from Lollo Rossa showed the strongest antioxidant activity, while hydroethanolic extracts from four varieties displayed antimicrobial activity against Yersinia enterocolitica.

These findings highlight the potential of lettuce waste as a source of valuable bioactive compounds, supporting sustainable practices and value‐added applications in the food and pharmaceutical industries. © 2026 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Figures

Representative samples of lettuce waste and losses. (A) Curly‐leafed, (B) Iceberg, (C) Little Gems, (D) Romaine, (E) Frisée and (F) Lollo Rossa.

Chromatogram obtained using HPLC–FL for 5‐MTHF‐monoglutamate in the Curly‐leafed sample. The 5‐MTHF peak was identified at a retention time of 6.41 min.

Phenolic profile of hydroethanolic extract of Lollo Rossa variety, recorded at 330 nm (A) and 370 nm (B). The peaks are identified in Table 2.

Tables

Table 1. Content of organic acids, vitamins (tocopherols and folates), carotenoids and minerals in lettuce waste samples (mean ± SD, n = 3)

Variety type	Curly‐leafed	Iceberg	Little Gems	Romaine	Frisée	Lollo Rossa
Organic acids (g kg−1 dw)
Oxalic acid	52.8 ± 0.5e	83.6 ± 0.1c	106 ± 1a	58.9 ± 0.1d	31.5 ± 0.4f	85.9 ± 0.3b
Malic acid	22.1 ± 0.1c	16.0 ± 0.5e	35 ± 1b	19 ± 1d	15 ± 1f	50 ± 1a
Succinic acid	37 ± 1e	43 ± 1d	58 ± 1a	45 ± 1c	25 ± 1f	53 ± 1b
Fumaric acid	tr	tr	tr	tr	tr	tr
Total	112 ± 1e	142.5 ± 2c	198 ± 2a	123 ± 1d	72 ± 2f	189 ± 1b
Tocopherols (mg kg−1 dw)
α‐Tocopherol	120 ± 1c	46.9 ± 0.2f	60.2 ± 0.4e	114.3 ± 0.1d	154.5 ± 0.2b	180 ± 1a
γ‐Tocopherol	74.8 ± 0.4d	30.2 ± 0.1f	81 ± 1c	67 ± 1e	143 ± 1a	112 ± 1b
Total	195 ± 1c	77.1 ± 0.1f	141 ± 1e	181 ± 1d	298 ± 1a	292 ± 1b
Folates (mg kg−1 dw)
Total	3.5 ± 0.1b	3.52 ± 0.03a	1.67 ± 0.02e	2.75 ± 0.01d	3.12 ± 0.03c	2.7 ± 0.1d
Carotenoids (expressed as lutein, mg g−1 dw)
Total	0.5 ± 0.4b	0.09 ± 0.01d	0.12 ± 0.01d	0.19 ± 0.02c,d	0.38 ± 0.02b,c	1.1 ± 0.1a
Minerals (g kg−1 dw)
K	34.0 ± 0.6a,b	31.3 ± 0.3b	34.5 ± 0.4a	25.7 ± 0.9c	35.0 ± 0.8a	36.2 ± 0.8a
Na	2.1 ± 0.1b	0.304 ± 0.004c	1.6 ± 0.2b	1.7 ± 0.1b	3.7 ± 0.2a	0.53 ± 0.03c
Ca	6.4 ± 2.3b	3.5 ± 0.2c,d	4.0 ± 0.4c	3.1 ± 0.7d	4.3 ± 0.1c	9.2 ± 0.5a
Mg	2.2 ± 1.0a	1.2 ± 0.1c,d	1.8 ± 0.2a,b	1.2 ± 0.2d	1.6 ± 0.1b	1.6 ± 0.1b,c
Fe	0.14 ± 0.03b	0.037 ± 0.003d	0.06 ± 0.01c	0.06 ± 0.01c	0.07 ± 0.01c	0.506 ± 0.004a
Mn	0.05 ± 0.01a	0.022 ± 0.002c	0.0247 ± 0.0001c	0.041 ± 0.004b	0.0348 ± 0.0001b	0.054 ± 0.005a
Cu	0.01 ± 0.003b,c	0.003 ± 0.001e	0.009 ± 0.001c	0.011 ± 0.001b	0.0065 ± 0.0003d	0.049 ± 0.004a
Zn	0.11 ± 0.03a	0.02 ± 0.01f	0.06 ± 0.01d	0.09 ± 0.01b	0.032 ± 0.004e	0.068 ± 0.005c

Table 2. Molecular formula, exact mass [M + H], retention time (Rt) and free amino acid content in lettuce waste samples (mean ± SD, n = 3)

Compound	Molecular formula	Exact mass [M + H]+	Rt (min)	Quantification (mg g−1 dw)
				Curly‐leafed	Iceberg	Little Gems	Romaine	Frisée	Lollo Rossa
Essential amino acids
Histidine	C6H9O2N3	156.07654	6.53	1.6 ± 0.1b	1.61 ± 0.01b	1.85 ± 0.03a	1.9 ± 0.1a	0.76 ± 0.03d	1.16 ± 0.01c
Isoleucine	C6H13O2N	132.10164	2.21	1.23 ± 0.01e	1.698 ± 0.001c	3.2 ± 0.1a	3.0 ± 0.2b	0.38 ± 0.02f	1.55 ± 0.02d
Leucine	C6H13ON	132.10164	2.46	1.79 ± 0.02d	2.37 ± 0.03c	2.63 ± 0.01b	3.5 ± 0.1a	0.51 ± 0.03f	1.7 ± 0.1e
Lysine	C6H14O2N2	147.11256	6.78	0.5 ± 0.03e	0.67 ± 0.05c	0.72 ± 0.01b	0.84 ± 0.04a	0.282 ± 0.001f	0.541 ± 0.002d
Methionine	C5H11O2NS	150.05809	2.585	0.046 ± 0.001c	0.058 ± 0.003a	0.057 ± 0.001b	0.026 ± 0.002e	0.030 ± 0.001d	0.022 ± 0.001f
Phenylalanine	C9H11O2N	166.08598	2.09	1.28 ± 0.03d	1.29 ± 0.03d	2.2 ± 0.1a	1.91 ± 0.03b	1.09 ± 0.01e	1.7 ± 0.1c
Threonine	C4H9O3N	120.06526	4.72	2.6 ± 0.1d	3.6 ± 0.1c	4.5 ± 0.1a	4.05 ± 0.04b	1.6 ± 0.1e	1.6 ± 0.1e
Tryptophan	C11H12O2N2	205.09685	2.03	0.69 ± 0.03c	0.42 ± 0.01d	0.94 ± 0.01a	0.79 ± 0.03b	0.67 ± 0.03c	1.0 ± 0.1a
Valine	C5H11O2N	118.08604	3.02	2.26 ± 0.04d	2.81 ± 0.01c	3.1 ± 0.1a	2.9 ± 0.1b	1.1 ± 0.1f	1.4 ± 0.1e
Nonessential amino acids
Alanine	C3H7O2N	90.05476	4.54	2.1 ± 0.1d	3.08 ± 0.01a	2.54 ± 0.05b	2.28 ± 0.03c	1.7 ± 0.1e	1.38 ± 0.02f
Arginine	C6H14O2N4	175.11861	6.57	0.68 ± 0.01e	1.03 ± 0.01c	2.3 ± 0.1b	3.0 ± 0.2a	0.93 ± 0.01d	0.95 ± 0.01d
Asparagine	C4H8O3N2	133.06056	5.78	6.5 ± 0.1c	4.8 ± 0.1f	17.4 ± 0.1a	5.66 ± 0.01e	9.3 ± 0.1b	6.2 ± 0.3d
Aspartic acid	C4H7NO4	134.0448	9.59	1.5 ± 0.1d	1.40 ± 0.04e	1.92 ± 0.01a	1.48 ± 0.02c	0.70 ± 0.04f	1.69 ± 0.02b
Glutamic acid	C5H9O4N	148.0603	8.25	4.0 ± 0.1e	3.94 ± 0.01e	6.5 ± 0.1a	4.5 ± 0.1b	4.1 ± 0.1d	4.3 ± 0.2c
Glutamine	C5H10O3N2	147.07617	5.79	48 ± 2d	66 ± 2a	63 ± 1b	61 ± 1c	25 ± 1e	22 ± 1f
Glycine	C2H5NO2	76.03919	5.34	0.138 ± 0.004d	0.169 ± 0.001b	0.198 ± 0.003a	0.159 ± 0.001c	0.116 ± 0.004e	0.095 ± 0.004f
Proline	C5H9O2N	116.0703	3.00	0.79 ± 0.03e	1.25 ± 0.01c	2.07 ± 0.02a	1.91 ± 0.05b	0.58 ± 0.01f	0.88 ± 0.05d
Serine	C3H7O3N	106.04965	5.74	2.4 ± 0.1c	3.61 ± 0.01b	4.1 ± 0.1a	4.0 ± 0.1a	2.3 ± 0.1c	1.4 ± 0.1d
Tyrosine	C9H11O3N	182.08086	2.95	0.531 ± 0.002d	0.55 ± 0.01c	0.88 ± 0.01a	0.80 ± 0.01b	0.38 ± 0.01f	0.41 ± 0.03e

Table 3. Retention time (Rt), wavelengths of maximum absorption in the visible region (λ max), mass spectra data, tentative identification of phenolic compounds and anthocyanins in lettuce waste samples

Peak	Rt (min)	λ max (nm)	[M − H]−	MS2	MS3	MS4	Tentative identification
1	5.44		341	179	135		Caffeic acid hexoside
2	7.51	326	353	191			5‐Caffeoylquinic acid
3	7.73		725	681, 639, 573, 505	505, 577, 601	301	Quercetin‐3‐O‐(6″‐malonylglucoside)‐7‐O‐glucoronide
4	8.65	255, 349	711	667	505, 463, 625, 547, 489, 301	300, 3301, 271, 255, 153	Quercetin‐3‐O‐(6″‐malonylglucoside)‐7‐O‐glucoside
5	10.68	327	295	179, 133, 135			Caffeoylmalic acid
6	12.11		337	191, 163			5‐p‐Coumaroylquinic acid
7	13.64	280, 328, 514	449	287			Cyanidin‐O‐hexoside
8	15.29	328	473	311, 293	149, 179		Chicoric acid
9	15.31		677	515	353, 179, 341, 335, 192, 173	191, 179	Tricaffeoylquinic acid
10	15.31		651	447, 489, 285	284, 255, 227, 151, 327, 357		Kaempferol‐acetylhexoside‐hexoside
11	18.48	256, 351	477	301			Quercetin‐O‐glucoronide
12	18.92	254, 347	461	285			Luteolin‐O‐glucoronide
13	20.5		549	505	301, 300, 463, 271, 255, 179, 151		Quercetin‐3‐(6″‐malonyl)glucoside
14	22.32	266, 346	461	285			Kaempferol‐O‐glucoronide
15	23.15		505	301, 463, 445	179, 255, 228, 151		Quercetin acetyl hexoside
16	23.31	271, 343, 516	535	287, 449			Cyanidin‐3‐(6″‐malonyl)glucoside

Table 4. Quantification of phenolic compounds and anthocyanins found in hydroethanolic extract and decoction preparations of lettuce waste samples (n = 3, mean ± SD)

Compound	Quantification (mg g−1 extract)		Student t‐test
	Hydroethanolic extract	Decoction	P
	Curly‐leafed
5‐Caffeoylquinic acid	0.20 ± 0.01	0.41 ± 0.01	<0.001
5‐p‐Coumaroylquinic acid	tr	tr	‐
Chicoric acid	0.05 ± 0.01	0.23 ± 0.01	<0.001
Quercetin malonyl hexoside	0.05 ± 0.01	nd	‐
Total phenolic acids	0.25 ± 0.01	0.64 ± 0.01	<0.001
Total flavonoids	0.05 ± 0.01	nd	‐
Total phenolic compounds	0.30 ± 0.01	0.64 ± 0.01	<0.001
	Little Gems
Caffeic acid hexoside	0.02 ± 0.01	nd	‐
5‐Caffeoylquinic acid	0.11 ± 0.01	0.19 ± 0.01	0.036
Chicoric acid	0.03 ± 0.01	0.60 ± 0.01	<0.001
Total phenolic compounds	0.16 ± 0.01	0.79 ± 0.01	<0.001
	Romaine
5‐Caffeoylquinic acid	0.21 ± 0.02	0.17 ± 0.01	0.796
Chicoric acid	0.05 ± 0.01	0.05 ± 0.01	0.001
Total phenolic compounds	0.26 ± 0.01	0.22 ± 0.01	0.697
	Frisée
5‐Caffeoylquinic acid	0.63 ± 0.01	0.81 ± 0.04	<0.001
Tricaffeoylquinic acid	tr	nd	‐
Kaempferol‐acetylhexoside‐hexoside	0.46 ± 0.01	nd	‐
Chicoric acid	0.59 ± 0.02	2.76 ± 0.04	<0.001
Kaempferol‐O‐glucoronide	0.57 ± 0.01	0.60 ± 0.01	0.207
Total phenolic acids	1.22 ± 0.01	3.57 ± 0.04	<0.001
Total flavonoids	1.03 ± 0.01	0.60 ± 0.01	0.300
Total phenolic compounds	2.25 ± 0.01	4.16 ± 0.03	<0.001
	Lollo Rossa
5‐Caffeoylquinic acid	5.58 ± 0.01	5.1 ± 0.1	0.001
Cyanidin‐O‐hexoside	0.497 ± 0.002	‐	‐
Quercetin‐3‐O‐(6″‐malonylglucoside)‐7‐O‐glucoronide	nd	0.57 ± 0.01	‐
Quercetin‐3‐O‐(6″‐malonylglucoside)‐7‐O‐glucoside	0.81 ± 0.03	0.64 ± 0.01	<0.001
Caffeoylmalic acid	0.83 ± 0.04	0.62 ± 0.02	<0.001
5‐p‐Coumaroylquinic acid	tr	tr	‐
Chicoric acid	4.7 ± 0.3	3.8 ± 0.2	<0.001
Quercetin‐O‐glucoronide	1.61 ± 0.07	1.11 ± 0.04	0.137
Luteolin‐O‐glucoronide	1.96 ± 0.05	6.06 ± 0.06	<0.001
Quercetin malonyl hexoside	15.3 ± 0.1	7.5 ± 0.3	<0.001
Quercetin acetyl hexoside	0.50 ± 0.01	0.55 ± 0.01	0.005
Cyanidin‐3‐(6″‐malonyl)glucoside	0.706 ± 0.005	‐	‐
Total phenolic acids	11.1 ± 0.1	9.5 ± 0.1	0.008
Total flavonoids	20.17 ± 0.06	16.46 ± 0.09	<0.001
Total phenolic compounds	29.77 ± 0.09	25.99 ± 0.1	<0.001
Total anthocyanins	1.20 ± 0.01	‐	‐

Table 5. Antioxidant activity of hydroethanolic extract and decoction preparations of lettuce varieties waste samples (mean ± SD, n = 3)

		Curly‐leafed	Iceberg	Little Gems	Romaine	Frisée	Lollo Rossa	Positive control: trolox (mg mL−1)
Antioxidant activity (EC50, mg mL−1)
DPPH	Decoction	1.6 ± 0.2b	3.8 ± 0.1a	1.02 ± 0.03d	1.2 ± 0.1c	0.87 ± 0.03e	0.104 ± 0.004f	0.043 ± 0.002
	Hydroethanolic	6.3 ± 0.6b	6.6 ± 0.3a	4.13 ± 0.4c	3.4 ± 0.2d	2.1 ± 0.2e	0.27 ± 0.02f
Reducing power	Decoction	0.61 ± 0.01a	0.6 ± 0.1a,b	0.55 ± 0.03a,b	0.5 ± 0.1b	0.2 ± 0.1c	0.2 ± 0.1c	0.029 ± 0.003
	Hydroethanolic	0.5 ± 0.1b	0.27 ± 0.02c	0.7 ± 0.2a	0.5 ± 0.1b	0.68 ± 0.01a	0.2 ± 0.1c
TBARS	Decoction	0.8 ± 0.1b	3.1 ± 0.1a	0.87 ± 0.06b	1.0 ± 0.2b	0.5 ± 0.2c	0.08 ± 0.01d	0.0058 ± 0.0006
	Hydroethanolic	1.3 ± 0.4b	1.5 ± 0.8a,b	1.6 ± 0.1a,b	0.49 ± 0.03c	0.12 ± 0.02c	1.9 ± 0.1a

Table 6. Antimicrobial activity (mg mL−1) of hydroethanolic extract and decoction preparations of lettuce waste samples

			Curly‐leafed	Iceberg	Little Gems	Romaine	Frisée	Lollo Rossa		Streptomycin	Methicillin	Ampicillin
Gram‐negative bacteria
E. cloacae	Hydroethanolic	MIC	>10	>10	>10	>10	>10	>10	MIC	0.007	n.t.	0.15
		MBC	>10	>10	>10	>10	>10	>10
	Decoction	MIC	>10	>10	>10	>10	>10	>10	MBC	0.007	n.t.	0.15
		MBC	>10	>10	>10	>10	>10	>10
E. coli	Hydroethanolic	MIC	10	5	10	5	5	10	MIC	0.01	n.t.	0.15
		MBC	>10	>10	>10	>10	>10	>10
	Decoction	MIC	10	10	10	10	10	2.5	MBC	0.01	n.t.	0.15
		MBC	>10	>10	>10	>10	>10	>10
P. aeruginosa	Hydroethanolic	MIC	>10	>10	>10	>10	>10	>10	MIC	0.06	n.t.	0.63
		MBC	>10	>10	>10	>10	>10	>10
	Decoction	MIC	5	10	5	5	5	2.5	MBC	0.06	n.t.	0.63
		MBC	>10	>10	>10	>10	>10	>10
S. enterica	Hydroethanolic	MIC	5	5	5	5	5	5	MIC	0.007	n.t.	0.15
		MBC	>10	>10	>10	>10	>10	>10
	Decoction	MIC	10	10	5	5	5	1.25	MBC	0.007	n.t.	0.15
		MBC	>10	>10	>10	>10	>10	>10
Y. enterocolitica	Hydroethanolic	MIC	0.007	0.007	0.007	0.007	0.015	>10	MIC	0.007	n.t.	0.15
		MBC	>10	>10	>10	>10	>10	>10
	Decoction	MIC	5	1.25	1.25	1.25	5	5	MBC	0.007	n.t.	0.15
		MBC	>10	>10	>10	>10	>10	>10
Gram‐positive bacteria
B. cereus	Hydroethanolic	MIC	>10	10	>10	>10	>10	10	MIC	0.007	n.t.	n.t.
		MBC	>10	>10	>10	>10	>10	>10
	Decoction	MIC	10	>10	>10	>10	>10	10	MBC	0.007	n.t.	n.t.
		MBC	>10	>10	>10	>10	>10	>10
L. monocytogenes	Hydroethanolic	MIC	>10	2.5	>10	2.5	5	>10	MIC	0.007	n.t.	0.15
		MBC	>10	>10	>10	>10	>10	>10
	Decoction	MIC	10	10	10	10	10	5	MBC	0.007	n.t.	0.15
		MBC	>10	>10	>10	>10	>10	>10
S. aureus	Hydroethanolic	MIC	10	>10	>10	>10	>10	5	MIC	0.007	0.007	0.15
		MBC	>10	>10	>10	>10	>10	>10
	Decoction	MIC	10	5	5	10	5	10	MBC	0.007	0.007	0.15
		MBC	>10	>10	>10	>10	>10	>10
Antifungal activity										Ketoconazole
A. brasiliensis	Hydroethanolic	MIC	5	10	>10	10	10	2.5	MIC	0.06
		MFC	>10	>10	>10	>10	>10	>10
	Decoction	MIC	5	10	10	5	2.5	5	MFC	0.125
		MFC	>10	>10	>10	>10	>10	>10
A. fumigatus	Hydroethanolic	MIC	2.5	10	10	10	10	5	MIC	0.5
		MFC	>10	>10	>10	>10	>10	>10
	Decoction	MIC	2.5	10	10	10	10	2.5	MFC	1
		MFC	>10	>10	>10	>10	>10	>10

Full text

INTRODUCTION

The exponential population growth and the concern of consumers to maintain a balanced diet are currently among the biggest challenges for the agricultural sector, which must ensure a sustainable food supply and food security at the same time. However, in addition to food security, there is also concern about the demand for agricultural products with high nutritional value and with bioactive properties.1, 2, 3

Agriculture is one of the major activities practiced worldwide to produce food, and as scientific and technological advances continue to progress, along with population growth, balancing food demand and supply has become a significant concern.4, 5 The intensification of agriculture has resulted in the generation of large amounts of agri‐food surpluses, waste and losses (WL) generated along the entire food supply chain, with notable losses occurring in agricultural areas due to inadequate harvesting and handling practices, adverse weather conditions, as well as improper storage conditions, transport damage and inadequate packaging.6, 7 According to FAO,8 approximately 42% of the fruits and vegetables produced worldwide are lost or wasted even before reaching the consumer. These wastes have the potential to be reused for the development of new biodynamic‐based products since they are rich sources of nutrients and bioactive compounds.

Particularly, Lactuca sativa L., commonly known as lettuce, is a widely consumed leafy vegetable that holds great nutritional and economic value. As with any agricultural crop, the production and processing of lettuce generate significant amounts of WL.

Waste refers to lettuce that is discarded or deemed unfit for human consumption due to quality issues or damage during handling, transportation or storage. Lettuce waste can occur at any stage of the supply chain, from the farm to the retailer, and contributes to the overall food waste problem.9

Losses, specifically in the context of lettuce, encompass the quantity of lettuce that is lost or reduced in quality before it reaches the consumer. Losses can occur due to factors such as improper harvesting techniques, unfavorable weather conditions or inadequate postharvest handling and storage practices. These losses not only result in economic losses for producers but also represent a missed opportunity to provide nutritious food to consumers.10

This industrial WL presents a challenge in terms of proper disposal and can have negative environmental impacts if not managed efficiently. However, recent research has shown that these WL materials may possess untapped potential as a valuable source of bioactive compounds.11 Lettuce is cultivated on 1.22 million hectares, and its global production is approximately 27.66 million tons.12 Its consumption is mainly raw in salads, contributing significantly to the nutritional content of diets, as there is a higher retention of nutrients compared to other vegetables that are usually consumed cooked or processed.13, 14 It is a vegetable rich in vitamins (A, K and C), polyphenols, flavonoids, carotenoids and minerals although its nutritional value is underestimated due to its high water content (95%).14, 15 In this paper, we explore the biochemical methodologies employed in analyzing the WL materials and discuss the identified bioactive compounds. By shedding light on the underutilized potential of L. sativa WL, its biochemical characterization can provide valuable insights into WL composition, and potential utilization.

MATERIALS AND METHODS

Plant material

A local company, stockist and distributor, in the city of Bragança, northern Portugal, provided six samples of L. sativa. The samples consisted of lettuce residues and losses from different varieties, namely Lollo Rossa, Curly‐leafed and Frisée (var. crispa), Iceberg (var. capitata) and Little Gems and Romaine (var. longifolia) lettuce (Fig. 1). The samples were freeze‐dried (FreeZone 4.5, Labconco, Kansas City, MO, USA) and reduced to a fine powder that was stored in a sealed plastic bag.

Representative samples of lettuce waste and losses. (A) Curly‐leafed, (B) Iceberg, (C) Little Gems, (D) Romaine, (E) Frisée and (F) Lollo Rossa.

Extract preparations

Hydroethanolic extractions were performed according to a previously described procedure.16 Briefly, 3 g of each sample was twice suspended in 80 mL of ethanol–water (80:20 v/v) and stirred for 1 h at room temperature. After extraction, the suspensions were filtered with Whatman No. 4 paper and the ethanol was removed by reduced pressure using a rotary evaporator (Büchi R‐2010, Flawil, Switzerland) and the aqueous fractions were frozen and lyophilized.

For decoction, 3 g of plant material was used with the addition of 100 mL of boiling distilled water for 5 min. Then the samples were left to stand for 5 min and filtered through Whatman No. 4 paper.16 The extracts obtained were frozen and freeze‐dried.

Chemical compound characterization

The study was based on biological replicates, as lettuces were provided from 10–15 individual plants per species, and these were pooled to form a composite sample representative of each species at the sampling site. For each composite sample, all analyses were performed in triplicate (n = 3) as analytical replicates to ensure accuracy and reproducibility of the measurements.

Organic acids

Organic acids were assessed by following the procedure previously described and optimized by Pereira et al.17 The analysis was performed using a Shimadzu 20A series UFLC and detection was carried out with a photodiode array, using 215 nm as the preferred wavelength. The organic acids found were quantified by comparing the area of the peaks with calibration curves obtained from commercial standards of oxalic, malic and fumaric acids purchased from Sigma‐Aldrich (St Louis, MO, USA). For quantitative analysis, the calibration curves were determined from different standard compounds. The results were expressed in grams per kilogram of dried weight (dw).

Vitamins (tocopherols and folates)

Tocopherols were determined by following the procedure previously described by Spréa et al.16 The analysis was performed using a high‐performance liquid chromatography (HPLC) system and a fluorescence (FL) detector (FP‐2020; Jasco) programmed for excitation at 290 nm and emission at 330 nm. The compounds were identified by chromatographic comparisons with authentic standards. Quantification was based on the FL signal response of each standard using the IS (tocol) method and the calibration curves obtained from the commercial standards of α‐ and γ‐tocopherol purchased from Matreya (PA, USA). The results were expressed in milligrams per kilogram dw.

To analyze the different forms of natural folates with vitamin B_9_ activity, a method optimized by Morales et al.18 was used. The procedure consisted of converting the chemical forms into 5‐CH_3_‐H_4_folate and quantifying them by HPLC–FL. The sample was extracted with 100 mmol L^−1^ Na_2_HPO_4_·2H_2_O buffer at pH 7 and 80 °C for 15 min, followed by centrifugation at 6000 × g for 15 min. A 10 mL aliquot of the supernatant was incubated at 37 °C for 3 h with rat serum and chicken pancreas solution (1 mg mL^−1^ in 100 mmol L^−1^ phosphate buffer, pH 7) to release folates from the plant matrix. The folates were then reduced with NaBH_4_, passed through activated SPE/SAX cartridges and measured by HPLC–FL using a Lichrospher 100 column with RP 18 end cap. A gradient of acetonitrile and 100 mmol L^−1^ sodium phosphate buffer (pH 4.4) was used at a flow rate of 0.4 mL min^−1^. Chromatographic peaks were identified by comparing retention times of the commercial standard 5‐CH_3_‐H_4_folate. Quantification was based on the FL signal response, with the total folate content expressed in milligrams per kilogram dw.

Carotenoids

A mass of each sample (50 to 75 mg) was extracted with 5 mL of absolute ethanol in an ultrasonic bath (P Selecta, Ultrasounds H‐D, 25 kHz) for 5 min at a temperature of 25 °C.19 The mixture was then centrifuged (5 min, 4 °C, 14 000 × g) to obtain the extract (supernatant). The residue was submitted to further extractions, and the procedure repeated until the supernatant was colorless. The extracts were combined, concentrated (rotatory evaporator, Heidolph Hei‐VAP Silver 4, T ≤ 37 °C) and transferred to a volumetric flask, with the volume made up to an exact volume with ethanol for spectrophotometric monitoring (300–600 nm, ZUZI 4255/50 spectrophotometer). The total carotenoid content of each extract was calculated using the specific extinction coefficient of lutein in ethanol (E1cm1% = 2550). The results were expressed as milligrams per gram dw.

Mineral content

The mineral composition was determined according to an AOAC protocol.20 Briefly, a freeze‐dried sample (250 mg) was digested with 10 mL of 65% nitric acid in a microwave extraction system at 200 °C and 1600 W for 30 min and then made up to a final volume of 50 mL with distilled water. The mineral content in terms of potassium (K), sodium (Na), calcium (Ca), magnesium (Mg), iron (Fe), manganese (Mn), zinc (Zn) and copper (Co) was determined through atomic absorption spectrophotometry (Perkin Elmer 1100B, Waltham, MA, USA).21 The results were expressed in grams per kilogram dw.

Free amino acids

The determination of free amino acids was performed following the procedure described by Lima et al.,22 with adaptations appropriate to our samples. A 2.5 g portion of each sample was extracted with 40 mL of a water–acetonitrile (1:1 v/v) solution. The mixture was vortexed for 5 min and then sonicated for 10 min at room temperature. Finally, samples were centrifuged at 9000 × g for 10 min at 4 °C and the supernatant was collected and filtered through a 0.2 μm nylon membrane filter. Amino acid determination was performed using an ultra‐HPLC electrospray ionization (ESI) mass spectrometry (MS) system (Vanquish, Thermo Scientific, San Jose, CA, USA) coupled to an Orbitrap mass spectrometer (Exploris 120, Thermo Scientific). Chromatographic separation was achieved on a bioZen column (100 × 2.1 mm, 2.6 μm; Phenomenex, Torrance, CA, USA), maintained at 40 °C. The mobile phases were (A) 10 mmol L^−1^ ammonium formate (pH 3.1) and (B) acetonitrile containing 10 mmol L^−1^ ammonium formate at pH 3.1 (90:10 v/v). The elution gradient was as follows: 100% to 99% A (0–2 min), 99% to 97% A (2–3 min), 97% to 50% A (3–8 min), held at 50% A until 9 min, followed by re‐equilibration to initial conditions for 6 min. The flow rate was 0.5 mL min^−1^ and the injection volume was 5 μL. The Orbitrap was equipped with a heated ESI source operating in positive ion mode. Ionization conditions were: spray voltage, 4000 V; vaporizer temperature, 350 °C; sheath gas flow rate, 50 (arbitrary units); auxiliary gas flow rate, 10 (arbitrary units); ion transfer tube temperature, 325 °C. Selected ion monitoring mode at a resolution of 60 000 was used to collect data. Instrument parameters included RF lens voltage at 70%, standard AGC target, automatic maximum injection time and a scan width of 2 m/z. Peak identification was conducted by accurate mass and comparison of the retention time of the compounds in the samples with those of authentic standards. Calibration curves were established for each of the analyzed amino acids, and results were expressed in milligrams per gram dw.

UHPLC analysis of phenolic compounds and anthocyanins

The phenolic compounds were evaluated in the hydroethanolic extracts and decoction preparations prepared as described above and re‐dissolved in ethanol–water (80:20 v/v) and water, respectively, corresponding to a final concentration of 10 mg mL^−1^. The analysis was performed using a Dionex Ultimate 3000 UPLC (Thermo Scientific, San Jose, CA, USA), supplied with a diode array detector (DAD; 280 and 370 nm as the chosen wavelengths for phenolic compounds and 520 nm for anthocyanins) and combined with an ESI mass detector. Identification of each phenolic compound was focused on the chromatographic data obtained (retention time, UV–visible spectra and mass), and compared to existing standard compounds, or earlier defined data in the literature, using Xcalibur® software (ThermoFinnigan, San Jose, CA, USA). The quantitative assessment of the discovered compounds was achieved using seven‐level calibration curves built on the UV signal of available standard compounds. When precise standards were not available, the calibration curves of the most comparable standards were used. Working procedures were previously described in detail by the authors.23 The results were expressed in milligrams per gram of extract.

Bioactive properties

The evaluation of the bioactive properties of the obtained extracts (hydroethanolic and decoction) was performed in vitro.

Antioxidant activity

Antioxidant activity was evaluated in hydroethanolic extracts and decoction preparations using two chemical assays (DPPH radical scavenging effect and reducing power) and one cell‐based assay (thiobarbituric acid reactive substances (TBARS)).

The DPPH radical scavenging activity was evaluated using an ELX800 microplate reader (Bio‐Tek, Instruments Inc., Winooski, VT, USA). The activity was calculated based on decolorization using the following formula: ADPPH−AS/ADPPH]×100, where AS represents the absorbance of the sample‐containing solution at 515 nm and ADPPH is the absorbance of the DPPH solution.24 Additionally, the reducing power was determined by measuring the ability to convert Fe^3+^ into Fe^2+^. The absorbance reading was taken at 690 nm using the same microplate reader mentioned above.

For the TBARS assay, both extracts were re‐dissolved in water, and then diluted from 10 to 0.078125 mg mL^−1^. Lipid peroxidation inhibition in porcine (Sus scrofa) brain cell homogenates was estimated by the decline in TBARS formation and, consequently, in the color strength of malondialdehyde–thiobarbituric acid, measuring the absorbance at 532 nm. The inhibition extent was calculated using the formula: [(A − B)/A] × 100%, where A and B are related to the absorbance of the control and extract sample, respectively.16 Results were expressed in EC_50_ values (mg mL^−1^), which indicate the sample concentration required to achieve 50% antioxidant activity in the DPPH and TBARS assays or an absorbance of 0.5 in the reducing power assay. Trolox (Sigma‐Aldrich, St Louis, MO, USA) was used as positive control. The extraction solvents (ethanol–water (80:20 v/v) and water) were used as negative controls for the DPPH and reducing power assays, whereas Tris buffer and water were used as negative controls for the TBARS assay.

Antimicrobial activity

Gram‐positive (Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus) and Gram‐negative (Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica and Yersinia enterocolitica) bacteria were selected to test the antibacterial activity of the extracts prepared as described above.

For antifungal activity, Aspergillus brasilliensis and Aspergillus fumigatus were used. The results were presented as the concentrations that resulted in complete inhibition of bacterial growth (minimal inhibition concentration, MIC), through the microdilution method coupled to rapid colorimetric assay with p‐iodonitrotetrazolium chloride as described by Pires et al.,25 as well as minimal bactericidal concentration (MBC) and minimal fungicidal concentration (MFC) values. The positive controls were streptomycin, methicillin, ampicillin, ketoconazole and bifonazole (Sigma‐Aldrich, St Louis, MO, USA), whereas the negative control was 5% dimethyl sulfoxide.

Statistical analysis

The results are presented as mean ± standard deviation. A Student's t‐test was applied to assess significant difference in phenolic compound results, with a significance level of α = 0.05. For the other parameters, a one‐way analysis of variance was performed, followed by Tukey's HSD test (α = 0.05) to evaluate significant differences among samples. All analysis was conducted using SPSS Statistics software (IBM SPSS Statistics for Windows, Version 22.0, Armonk, NY).

RESULTS AND DISCUSSION

Organic acid, vitamin, carotenoid and mineral compositions

The results obtained for the composition of organic acids, tocopherol, total folates, total carotenoids and minerals in the different lettuce varieties studied are presented in Table 1.

Regarding organic acid composition, oxalic, malic, succinic and fumaric acids were identified in all the analyzed samples (Table 1). Among them, oxalic acid stood out due to its high content, ranging from 31.5 to 106 g kg^−1^ dw (Frisée and Little Gems varieties, respectively). In comparison, fumaric acid was only detected in trace amounts.

However, López et al.,26 in a study conducted with three types of lettuce of different sizes, namely regular (Romaine), intermediate (Mini‐Romaine) and small (Little Gems), detected a greater diversity of organic acids. In their study, malic, citric, fumaric, glutamic, tartaric, quinic, α‐ketoglutaric, succinic, shikimic and malonic acids were identified in all samples, with malic acid being present in higher concentrations (ranging from 1.92 to 2.53 g (100 g)^−1^ dw).

Among the tocopherol isoforms, both α and γ were identified in all six samples studied (Table 1), with α‐tocopherol being the predominant isoform, detected at higher concentrations ranging from 46.9 to 180 mg kg^−1^ in Iceberg and Lollo Rossa varieties, respectively. α‐Tocopherol, one of the main isomers of vitamin E, is a potent antioxidant agent. Due to its high concentrations in vegetables, it is believed to play a role in reducing the risk of certain diseases.27 Among the samples, Frisée had the highest content of tocopherols (298 mg kg^−1^ dw), while Iceberg had the lowest content (77.1 mg kg^−1^ dw). Our results are consistent with those reported by Byrdwell et al.,28 who also identified α‐tocopherol as the predominant isoform in lettuce samples, with concentrations ranging from 2.1 to 10.5 mg(100 g)^−1^ dw. Another study reported the identification of all four isoforms (α, β, δ, γ) in samples of Iceberg lettuce (capitata variety) and Swordleaf (longifolia variety), the values ranging from 0.05 to 6.5 mg (100 g)^−1^ dw and 0.125 to 27.5 mg (100 g)^−1^ dw, respectively.29 The results obtained in that study are consistent with our findings, in the case of Iceberg lettuce, they reported a higher concentration for the α isoform (6.5 mg (100 g)^−1^ dw).

The results obtained for total folates (vitamin B_9_) revealed significant variation among the analyzed lettuce samples. Values ranged from 1.67 mg kg^−1^ dw in Little Gems to 3.52 mg kg^−1^ dw in Iceberg lettuce. This variation may be attributed to several factors, including genetic differences among varieties, agronomic conditions and postharvest conditions that influence folate stability.30 The Iceberg (3.52 mg kg^−1^ dw), Curly‐leafed (3.5 mg kg^−1^ dw; Fig. 2) and Frisée (3.12 mg kg^−1^ dw) samples exhibited the highest levels of vitamin B_9_, suggesting that these varieties may have a greater capacity for folate retention throughout the production cycle. These results contrast with those reported by other authors, who found significantly higher folate levels in Iceberg lettuce, ranging from 600 to 860 μg (100 g)^−1^ dw.31 Regarding the Little Gems variety, our results revealed the lowest total folate content (1.67 mg kg^−1^ dw), in contrast to findings by other authors who identified this variety as having one of the highest concentrations of 5‐methyltetrahydrofolate (5‐MTHF) – the main biologically active form of folate – among the samples studied.26 On the other hand, varieties such as Frillice and Romaine have consistently been identified as good sources of folates,31 which partially aligns with our findings, as Curly‐leafed lettuce exhibited high levels of vitamin B_9_.

Chromatogram obtained using HPLC–FL for 5‐MTHF‐monoglutamate in the Curly‐leafed sample. The 5‐MTHF peak was identified at a retention time of 6.41 min.

Regarding the content of carotenoids (expressed as lutein) present in each sample (Table 1), there was a fluctuation between 0.09 and 1.1 mg g^−1^ dw, with Iceberg exhibiting the lowest content, whereas Lolo Rossa had the highest content. Studies have reported that carotenoids play crucial roles in human health, primarily due to their provitamin A activity and antioxidant capacity.32 In a study conducted on different varieties of green and red lettuce, it was concluded that the carotenoid content was also higher in red lettuce varieties compared to green lettuce varieties,33 which is in agreement with our study. In general, when comparing the results obtained in our work with the studies on lettuce varieties conducted previously by other authors, it is evident that food waste and food losses do not lose their chemical properties.

According to the obtained results, all samples contained macrominerals such as K, Na, Ca and Mg, along with microminerals including Fe, Mn, Cu and Zn. As indicated in Table 1, the samples were particularly rich in macrominerals, especially K, while micromineral contents were comparatively lower. All varieties exhibited similar results, with the Lollo Rossa variety standing out due to higher values in almost all minerals. Consistent with previous studies, K was identified as the predominant mineral compound in the analyzed samples.34, 35, 36 However, there were discrepancies in minor compounds, including Cu,36 Fe35 and even Cd.34

Free amino acid composition

Table 2 presents the data for the free amino acid profiles of the different lettuce varieties – identified based on the exact mass and retention time of the compounds. The presence of free amino acids in plant foods is of great importance, not only for their nutritional value, but also because they can influence sensory properties, improve nutrient bioavailability and provide functional properties, including antioxidant activity. In addition, free amino acids are readily absorbed by the body, which makes them relevant for diets that require rapid nutrient absorption or metabolic support.37, 38

In general, it was observed that the Little Gems variety presented the highest concentrations for most of the free amino acids analyzed, especially glutamine (63 mg g^−1^ dw), asparagine (17.4 mg g^−1^ dw) and glutamic acid (6.5 mg g^−1^ dw), which suggests a nutritional profile particularly rich in nonessential amino acids. These results are in agreement with those of a previous study that highlighted a higher concentration of nonessential amino acids and a predominance of glutamine in samples of lettuce cv. Frillice.39

Regarding nonessential amino acids, glutamine stood out as the most abundant compound in all varieties. This amino acid plays a fundamental metabolic role, as it functions as the main energy substrate for cells with a high proliferation rate and, in addition, it is a precursor in the biosynthesis of several compounds, which reinforces its physiological relevance.40

Regarding essential amino acids, Little Gems once again stood out with high levels of threonine (4.5 mg g^−1^ dw), isoleucine (3.2 mg g^−1^ dw) and valine (3.1 mg g^−1^ dw), reinforcing its potential as a complementary source of plant proteins of nutritional interest.

In contrast, the Lollo Rossa variety had the lowest levels for most of the free amino acids analyzed, which may be related to natural deterioration processes and storage conditions, which accelerate the degradation of these compounds for energy production and cell maintenance after harvest.41, 42

Phenolic compound and anthocyanin characterization

The tentative identification of the phenolic compounds presents in the Lactuca sativa L. extracts, as well as the retention times, maximum absorbance (λ max), molecular ion ([M − H]^−^) and main ion fragments of each phenolic compound are described in Table 3 and the quantification of each compound is presented in Table 4.

Table 3: Retention time (Rt), wavelengths of maximum absorption in the visible region (λ max), mass spectra data, tentative identification of phenolic compounds and anthocyanins in lettuce waste samples

The results revealed the presence of 14 compounds distributed among the samples, of which six were phenolic acids and eight were flavonoids. The group of phenolic acids proved to be the most abundant in the extracts and the ones that were present in all lettuce varieties. Among them, five compounds were assigned as caffeic acid derivatives, showing characteristic UV–visible spectra with maximum absorption at 326 nm (peak 2), 327 nm (peak 5) and 328 nm (peak 7).

Compound 1 showed a deprotonated molecule at m/z 341, and main fragment ion at m/z 179 in MS^2^ [M – H − 162], evidencing the loss of a hexose moiety. Further fragmentation yielded m/z 135 in MS^3^, [M − H − 162 − 44], indicating the presence of caffeic acid.43 Therefore, peak 1 was assigned as caffeic acid hexoside. Compound 2 was identified as 5‐caffeoylquinic acid by comparison with authentic standards.

Compound 5 presented a molecular ion at m/z 295 and main product ions at m/z 179 [M − H − 116] and 133 [M − H − 162], which could be attributed to the loss of maloyl and caffeic acid moieties.44, 45 This fragmentation pattern matches the one found by Llorach et al.44 and Materska et al.45 in lettuce, corresponding to caffeoylmalic acid.44

Peak 7 was the most intense of those for caffeic acid derivatives in all the samples. Its deprotonated molecule at m/z 473 yielded the ions at m/z 311 and m/z 149 as base peaks in MS^2^ and MS^3^, respectively, suggesting the loss of two caffeic acid moieties (M − H − 162 − 162).46 Based on previous reports, compound 7 was assigned as chicoric acid (dicaffeoyltartaric acid).45, 46, 47, 48

Compound 8 showed a molecular ion at m/z 677, and main product ions at m/z 505 and 353 in MS^2^ and MS^3^, corresponding to the successive loss of two caffeoyl moieties (M − H − 162 − 162). Further fragmentation of m/z 353 in MS^4^ generated the ions at m/z 191, 179 and 173, which could be attributed to a caffeoylquinic acid.49 On the basis of these data, peak 8 was identified as tricaffeoylquinic acid.

Peak 6 showed a deprotonated molecule at m/z 337, m/z 191 as the base peak in MS^2^ (M − H − 146) and a minor fragment at m/z 163 (M − H − 174), corresponding to the losses of coumaroyl and quinoyl moieties.49 This fragmentation pattern indicates that peak 6 is 5‐*p‐*coumaroylquinic acid.49

Regarding flavonoids, the samples presented two kaempferol derivatives, five quercetin derivatives and one luteolin derivative.

Compound 9 showed a molecular ion at m/z 651 yielding a product ion at m/z 447, which could have resulted from the loss of an acetylhexoside moiety [M − H − 204]. Further fragmentation of m/z 447 gave product ions at m/z 284 and 285, suggesting the loss of another hexoside moiety [M − H − 204 − 162]. Therefore, compound 9 was identified as kaempferol‐acetylhexoside‐hexoside.

Similarly, compound 13 showed two absorption maxima in the UV–visible, 346 and 266 nm, characteristic of kaempferol derivatives. The deprotonated molecule [M − H] at m/z 461 and the product ion at m/z 285 suggested the loss of 176 Da, which was attributed to a glucuronide moiety. Thus, peak 13 was assigned as kaempferol‐*O‐*glucoronide.

Peak 11 showed a molecular ion at m/z 461 and the main product ion at m/z 285, which was attributed to luteolin after the loss of a glucuronide moiety. Moreover, this compound showed characteristic absorption spectra of luteolin with three absorption maxima in the UV–visible: 254, 267 and 347 nm. Thus, it was identified as luteolin‐*O‐*glucuronide. Glucuronide flavonoids have been reported in previous studies on lettuce.44, 45, 48 Specifically, luteolin‐*O‐*glucuronide was reported by Llorach et al.44 in different lettuce varieties, including Romaine, Iceberg and Lollo Rossa.

Six quercetin derivatives were tentatively identified in the samples. Compounds 3, 4 and 12 had molecular ions at m/z 725, 711 and 549, respectively. They showed MS fragmentations with common characteristics. Peak 12 ([M − H]^−^ at m/z 549) gave main product ions at m/z 505 [M − H − 44] in MS^2^, indicating a decarboxylation typical of malonyl residues linked to flavonoid glycosides.44 Further fragmentation in MS^3^ yielded m/z 301, which denotes the loss of 204 Da [M − H − 162 − 44 − 42], attributed to a glucose carrying the rest of the decarboxylated malonyl residue.44 The presence of quercetin as the aglycone can be stated due to the UV–visible absorption maxima (256 and 356 nm) and diagnostic fragments in MS^3^ (179, 151).50 Based on these data, compound 12 was assigned as quercetin‐3‐(6″‐malonyl)glucoside, which has also been reported in lettuce by other authors.44, 45, 46, 48

Peak 3 ([M − H]^−^ at m/z 725) gave a similar fragmentation pattern to peak 12, with product ion at m/z 681 after the loss of 44 Da in MS^2^. In the MS^3^ fragmentation, the base peak at m/z 505 denoted the loss of 176 Da, assigned as a glucuronide moiety,44, 48 followed by the further loss of 204 Da to give the aglycone at m/z 301. This type of fragmentation, in which the loss of a sugar gives the most abundant base peak different from the base peak of the aglycone, indicates that there is a glycosylation in more than one site.44 Therefore, compound 3 was identified as quercetin‐3‐O‐(6″‐malonylglucoside)‐7‐O‐glucoronide.

For compound 4, the UV–visible data revealed two absorption maxima, 349 and 255 nm, which are in the range found for glycosylated flavonoids. Similarly, compound 4 ([M − H]^−^ at m/z 711) showed the neutral losses of 44 Da in MS^2^ yielding m/z 667, and of 204 Da in MS^4^, giving m/z 301. This also suggests the loss of a malonylglucoside. The main fragment ion at m/z 505 in MS^3^ indicated the loss of a glucose moiety [M − H − 44 − 162]. In conclusion, peak 4 was identified as quercetin‐3‐O‐(6″‐malonylglucoside)‐7‐O‐glucoside.

Peak 10 presented absorption maxima at 256 and 350 nm, deprotonated molecule at m/z 477 and main product ion at m/z 301, denoting the neutral loss of 176 Da, which was attributed to a glucuronide moiety.44, 48 The fragmentation of m/z 301 in MS^3^ gave typical fragments of quercetin (m/z 151, m/z 179, m/z 257).50 Based on the data, this compound was identified as quercetin‐O‐glucoronide. Other authors have reported this flavonoid in lettuce.44, 45, 46, 48

Compound 14 had molecular ion at m/z 505 and fragments at m/z 301 [M − H − 204], suggesting the loss of a hexosyl moiety (162 Da) and an acetyl moiety (42 Da). Typical fragments for quercetin were also found in MS^3^ (m/z 179 and m/z 151). Peak 15 was therefore identified as quercetin‐*O‐*acetylglucoside.

These compounds were previously identified in some of the varieties studied. Some studies also showed that 5‐caffeoylquinic acid51 and chicoric acid1, 52 were among the major compounds. In these same studies we can prove the difference regarding higher concentrations of phenolic compounds in red lettuce varieties than in green varieties. The same is demonstrated in our study through the only red variety studied (Lollo Rossa).

Studies that investigated the phenolic compounds of green lettuce ‘Maravilla de Verano’ identified feruloylquinic acid as one of the major compounds, 257.353 and 564.9 μg g^−1^,54 and in our case this compound was not identified in any of the varieties.

Lollo Rossa variety showed the highest contents of phenolic compounds for both hydroethanolic extract (29.77 mg g^−1^; Fig. 3) and decoction (25.99 mg g^−1^). In contrast, Romaine variety showed the lowest contents with values of 0.26 and 0.22 mg g^−1^ for hydroethanolic extract and decoction, respectively. However, in both extracts of the Iceberg variety no phenolic compounds were detected. The differences in the content of compounds identified for the same sample mainly vary according to the solvent used. In the case of hydroethanolic extraction, it has an extraction capacity for a wide range of phenolic compounds since it is a polar solvent capable of extracting both polar and nonpolar compounds. Water can also be used as a solvent; however, the solubility of these compounds varies according to their chemical structure.55

Phenolic profile of hydroethanolic extract of Lollo Rossa variety, recorded at 330 nm (A) and 370 nm (B). The peaks are identified in Table 2.

The Lollo Rossa variety showed two anthocyanins, as detailed in Tables 3 and 4. Peak 1, with a molecular ion at m/z 449, produced a single MS^2^ fragment at m/z 287, indicative of cyanidin following the neutral loss of 162 Da, corresponding to a hesoxide moiety. Thus, peak 1 was designated as cyanidin‐O‐hexoside.56 Peak 2, presenting a deprotonated ion at m/z 535, exhibited a base peak at m/z 287 in MS^2^, suggesting the presence of another cyanidin derivative. Additionally, a minor intermediary fragment at m/z 449 ([M − 86]^+^) was detected in MS^2^ for this peak, indicating the loss of an 86 Da malonyl group. These data suggest the presence of an acylated anthocyanin. Given that the mass difference between this minor ion fragment and the aglycone corresponds to 162 Da, peak 2 was identified as cyanidin‐3‐(6″‐malonyl)glucoside.56 This compound has been previously detected in red lettuce56 and was characterized based on the presence of the observed minor fragment at m/z 449.

The most abundant anthocyanin in this sample was cyanidin‐3‐(6″‐malonyl) glucoside (0.706 mg g^−1^ extract), with the total anthocyanin content found in this variety being 1.20 ± 0.01 mg g^−1^ extract. Previous studies have also highlighted the presence of cyanidin, while another study reported the detection of 3‐O‐peonidinglucoside in four commercial varieties of Lollo Rossa – ‘Nestorix’, ‘Red sails’, ‘Revolution’ and ‘Romired’ – with quantities ranging from 0.95 to 2.60 mg (100 g)^−1^ dw.57 In the same study, total anthocyanin values ranging from 38.86 to 61.36 mg (100 g)^−1^ dw were reported using an Acquity UPLC H‐Class liquid chromatographic system equipped with an Acquity UPLC photodiode array detector. These findings align with those from other authors, who observed values of 44.77 mg (100 g)^−1^ dw.58 However, the values found in this study are higher, with a total anthocyanin content of 120 mg (100 g)^−1^ dw. The differences in anthocyanin profiles and quantities may be attributed to variations in extraction methods, solvents used and lettuce growing conditions.57

Bioactive properties

Antioxidant activity

The hydroethanolic extracts and decoction preparations of WL samples were evaluated for their ability to scavenge DPPH radicals, their potential to reduce ferricyanide ions to ferrocyanide and their ability to inhibit lipid peroxidation in pig brain tissue (Table 5). In terms of the TBARS assay, the decoction preparations of the Lollo Rossa and Frisée varieties exhibited the highest activity (EC_50_ values of 0.08 and 0.5 mg mL^−1^, respectively) compared to the other varieties, which is consistent with higher content of total phenolic acids (25.99 and 4.16 mg g^−1^ extract, respectively). The decoction preparation of the Iceberg variety showed the lowest capacity to inhibit TBARS formation, as indicated by higher EC_50_ values (3.1 mg mL^−1^). In the DPPH scavenging and reducing power assays, decoction extraction generally demonstrated superior activity compared to hydroethanolic maceration. The Lollo Rossa variety particularly stood out, registering concentrations of 0.104 and 0.2 mg mL^−1^, respectively.

The extraction process (e.g. hydroethanolic maceration) has some drawbacks, such as the possibility of solvent saturation not occurring and the loss of secondary metabolites and samples during the solvent renewal process (filtration).59

The antioxidant activity of different lettuce varieties has been reported by several authors. Mampholo et al.58 evaluated the antioxidant activity of L. sativa samples from both green and red varieties. Regarding DPPH assay, no significant variations were observed among the varieties. However, for the ABTS and FRAP assays, the extracts obtained from the red samples exhibited superior antioxidant activity, which is in agreement with our study. In another study comparing a green cultivar (Yanzhi) with a red one (Red Butter), the results demonstrated that the DPPH was higher for Red Butter (89.8%) compared to Yanzhi (77.62%).60 Llorach et al.61 also revealed that the byproducts (mainly outer leaves) of three different varieties of lettuce contain phenolic compounds related to their antioxidant capacity.

None of the samples showed toxicity potential against the non‐tumor cell line tested (Vero; epithelial cells derived from the kidney of the African green monkey, Chlorocebus sabaeus) (data not shown). There are no studies available about the toxicity activity of lettuce extracts.

Antimicrobial activity

The results of the antimicrobial activity of lettuce extracts and preparations are presented in Table 6. The antibacterial activity was analyzed against eight foodborne bacteria, namely Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Yersinia enterocolitica, Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus. For the decoction preparations, the Romaine, Iceberg and Little Gems varieties exhibited MIC values of 1.25 mg mL^−1^ against the Yersinia enterocolitica strain. The Lollo Rossa variety demonstrated the same MIC value against the Salmonella enterica strain. On the other hand, the hydroethanolic extracts demonstrated superior antimicrobial activity, as MIC values of 0.007 mg mL^−1^ were obtained for the same bacterial strain (Yersinia enterocolitica), demonstrating better antimicrobial activity than the positive controls used.

In a previous study,62 it is described that a methanolic extract of lettuce from the longifolia variety exhibits superior activity against both Gram‐positive and Gram‐negative bacteria, with a MIC of 2.5 mg mL^−1^. Another study63 reveals that an aqueous extract of lettuce variety crispa demonstrated even higher inhibition than that obtained for methanolic and petroleum ether extracts.

Table 6 also presents the results of the antifungal activity obtained with the hydroethanolic extracts and decoction preparations. Generally, both the decoction and hydroethanolic extracts yielded similar results for the tested fungi. The samples from the Curly‐leafed and Lollo Rossa varieties exhibited the lowest MIC values, ranging from 2.5 to 5 mg mL^−1^.

CONCLUSIONS

The present study highlighted the biochemical potential of waste and losses generated from various lettuce varieties. Lollo Rossa stood out from the other varieties by having a higher content of tocopherols (vitamin E), carotenoids and minerals. HPLC‐DAD‐ESI/MS analysis identified phenolic acids and flavonoids, with chicoric acid and 5‐caffeoylquinic acid being the most prominent. Additionally, quercetin and luteolin derivatives were detected, along with cyanidin derivatives among the anthocyanins. Overall, phenolic acids constituted the main class of compounds identified, except in the Lollo Rossa variety, where flavonoids were more abundant in both the hydroethanolic extract and the decoction preparations. The hydroethanolic extracts and aqueous preparations exhibited antioxidant capacity in DPPH, reducing power and TBARS assays. Moreover, they demonstrated efficacy against the tested bacterial and fungal strains.

Despite the promising results, some limitations should be considered. The study was conducted under controlled laboratory conditions, and the bioactivity of the extracts may vary when applied in complex food or biological matrices. In addition, the stability and bioavailability of the identified compounds were not evaluated, which may influence the effectiveness in real industrial applications. Further in vivo studies and scale‐up assessments are therefore necessary.

Nevertheless, the high content of bioactive compounds and the demonstrated antioxidant and antimicrobial activities suggest significant potential for industrial exploitation. Lettuce waste and losses, particularly from the Lollo Rossa variety, could be valorized as sustainable sources of natural antioxidants and antimicrobial agents. These extracts may find applications in the food industry as natural preservatives, in the nutraceutical and pharmaceutical sectors as functional ingredients, and in cosmetic formulations, contributing to waste reduction and the development of value‐added products within a circular economy framework.

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.

AUTHOR CONTRIBUTIONS

JPBR: Formal analysis, Methodology, Investigation, Data curation, Writing – original draft. TFFdS: Methodology, Software, Validation, Investigation, Data curation, Writing – ‐review & editing. DBR: Methodology, Software, Validation, Investigation, Data curation. LD: Methodology, Software, Validation, Investigation, Data curation. PM: Methodology, Software, Validation, Investigation, Data curation, Writing – review & editing. TCSPP: Methodology, Data curation. MBPPO: Visualization, Supervision. ÂF: Conceptualization, Supervision, Validation, Investigation, Data curation, Writing – review & editing. LB: Conceptualization, Validation, Investigation, Writing – review & editing, Visualization, Project administration, Funding acquisition.