1. Introduction
Kargil is a district in the Ladakh Union territory of India, lying in the northwest Himalayas. The district has a peculiar arctic climatic condition coupled with desert climate; therefore, it is called a cold desert. It is connected to the rest of the country through Zoji La pass, which serves as its lifeline. However, the pass remains closed for an average of 150 days during winter because of heavy snowfall [
1,
2]. Due to road inaccessibility, Kargil faces acute shortage of essential food commodities, especially during winter. Moreover, a combination of climatic factors such as harsh temperatures, dropping to as low as −40 °C in winter to scorching heat in summer; low precipitation; intense solar irradiance; high-velocity winds; orographic barriers; and edaphic conditions including immature, coarse-textured, high pH, and highly permeable soils, makes the region an inhospitable terrain with sparse diversity and low productivity of vegetation [
3]. Since the growing period is short and cultivation during winter is not possible except in greenhouses, vegetables remain a rare item in diet leading to malnourishment [
3,
4]. Nevertheless, the region constitutes an important home for a diverse group of high-altitude wild macrofungi [
5], yet their availability is limited to a few months (June to September), as the sporadic fructification occurs only after slight showers of rain. Due to aforementioned factors, exploitation of the already available food resources, such as wild edible mushrooms, is a favourable option to achieve food security for the natives.
Wild mushrooms have received considerable interests and propelling expansion in many parts of the world [
6]. In Asian countries, mushrooms have always been highly prized for their value as food and source of medicines. They find use in indigenous therapies, particularly in traditional Chinese medicine [
7]. Only in recent decades have European countries showed interest in studying their impacts on human health [
8]. Consequently, an arsenal of studies evaluated the biochemical characteristics of mushrooms and revealed their potential as food supplements [
9,
10,
11]. Although a decent body of literature advocates for the nutritional benefits of various species of mushrooms the world over, the same set of findings cannot be conveniently and rigidly extrapolated to species from different geographical regions, since the biochemical constituents are contingent on the environment in which these mushrooms flourish [
12,
13,
14].
The most consumed mushrooms worldwide include
Agaricus bisporus,
Pleurotus ostreatus,
Lentinula edodes, and
Volvariella volvacea. They are easily cultivable and are characterised by culinary features and high nutritional value [
15,
16]. In addition to those that are commonly available in the markets, wild mushrooms are gathered and consumed by indigenous communities from across the globe [
17]. The exploitation of mushrooms for culinary purposes as well as pharmaceutical demands have reached new heights, thus presenting them to be a paramount food component [
18]. In recent years, there has been growing interest to domesticate wild mushrooms that are considered to have potential in the functional food market [
19]. Wild mushrooms have a long association with humankind. Owing to their flavour, texture, and desirable taste, they have an established position in international markets and command higher prices than cultivated mushrooms [
19,
20]. Consequently, mushrooms represent an attractive frontier in health sector. They have been reported as therapeutic foods for illnesses associated with unhealthy lifestyles, such as hypertension, hypercholesterolemia, atherosclerosis, non-alcoholic fatty liver disease, and even various types of cancer [
21]. Contemporary research has validated unique properties of bioactive compounds extracted from numerous species of mushrooms used in treating and reducing the severity of various medical conditions, including COVID-19 [
22,
23]. Mushrooms contain a wide variety of polysaccharides that are often cited for their immunomodulatory, antioxidant, antidepressant, and anticancer properties, among others [
24,
25,
26]. Clinical trials have substantiated mushrooms such as
P. eryngii and
A. bisporus as a favourable choice for “healthy snacking” suitable for individuals with an unhealthy metabolism and personalised nutritional needs [
27,
28,
29]. Thus, wild edible mushrooms have become remarkably important in our diet.
Multitudinous studies have documented indigenous knowledge of wild mushrooms of the northwest Himalayas [
30,
31,
32]. Although some regions of Kargil serve as a rich repository of mushrooms, the practice of adding wild mushrooms in diets is uncommon, with the exception of nomadic tribals and Nepali migrants, due to lack of knowledge on mushrooms’ invigorating benefits. Furthermore, studies on mushrooms of Kargil pertaining to diversity are apparently in their pioneer state, and biochemical characterisation is untouched. In this regard, two wild edible mushrooms,
L. drassinus Verma et al. (endemic to Kargil) and
L. controversus (Pers.) Pers., collected from Kargil, were evaluated for their nutritional and nutraceutical properties.
Lactarius, a genus within the
Russulaceae family, exhibits an ectomycorrhizal relationship with species of
Salix,
Populus, and
Betula [
33,
34,
35]. Distribution of
L. controversus spans across three continents, namely, Europe, North America, and Asia. Notably, the majority of the reports are from Europe (the Netherlands, the United Kingdom, Spain, Belgium, Serbia, Norway, Greece, Hungary, Romania), followed closely by North America (United States of America and Canada) [
35,
36,
37,
38,
39,
40,
41]. In Asia, reports of
L. controversus occurrence has been documented in Kazakhstan, Turkey, and India [
33,
42,
43,
44,
45]. There are limited studies on
L. controversus, wherein chemical profile, antioxidant, antibacterial, and cytotoxic activities have been studied [
41,
42]. On the other hand,
L. drassinus is a recently identified species reported from Drass, Kargil, by Verma et al. [
46]. Hence, no biochemical investigation has been performed per se. Considering the emerging use of wild culinary mushrooms, the present study assessed (i) metabolite profile based on gas chromatography-mass spectrometry (GC-MS); (ii) nutritional value: total protein, carbohydrates (total available carbohydrates, total soluble sugars, reducing sugars, non-reducing sugars, and starch), vitamins (C, B
3, B
6), minerals, macroelements (N, P, K, S, Mg, Ca), microelements (Na, Cu, Zn, Fe, Al, Ni), and antinutrients (phytates and tannins); and (iii) nutraceutical properties: total phenol, total flavonoid, ultra-performance liquid-chromatography-based quantification of phenols (gallic acid, chlorogenic acid, vanillin, ferulic acid, and cinnamic acid), flavonoids (quercetin dihydrate), and carotenoids (lycopene and β-carotene), and reducing power as well as antioxidant potential of
L. drassinus and
L. controversus from Kargil.
4. Discussion
Around the world, macrofungi are relished for their flavour, as well as nutritional and medicinal advantages. A great deal of work has been aggressively executed in this domain worldwide; however, nutritional investigation of mushrooms from cold desert areas, such as Kargil, has not been accomplished. The present study characterised the nutritional and nutraceutical potential of two undocumented wild edible mushrooms, L. drassinus and L. controversus, from Kargil.
Carbohydrates play a crucial role in the body by providing and storing energy, building macromolecules, and preserving protein and fat for alternative purposes [
72]. In contrast with previous studies on wild edible mushrooms [
73,
74], TAC were found in higher range in both the species studied. GC-MS analysis revealed that the
Lactarius spp. are rich in sugars, wherein their alcoholic derivatives such as glucitol, glycerol, arabitol, and erythritol are predominantly present. Owing to their well-recognised role in combating stress by redox homeostasis, a predominant presence of sugar alcohols could be perceived as a stress tolerance strategy adopted by the mushrooms to withstand the constant pressures of drought and low temperature [
75]. Pharmaceutically, sugar alcohols are used as a sugar alternative in diabetic foods and as an effective dietary approach as they contain a low number of calories and possess non-fermenting properties [
76,
77]. Moreover, high sugar composition in GC-MS profile was further supported by TSS estimated spectrophotometrically. The TSS in the two
Lactarius spp. were found to be higher than those reported for
Lentinus spp.,
Schizophyllum sp., and
Termitomyces sp. [
78]. RS levels, which are indicative of food quality, were found to be high. Similarly, NRS has been reported to increase under drought stress [
79], which explains their high accumulation in the studied mushrooms. In contrast to sugars and their alcoholic derivatives, starch concentration was found to be lower, making these macrofungi a healthy choice for dietary management of diabetes mellitus, as starch can potentially interfere with blood glucose levels [
80].
Alongside fats and carbohydrates, proteins are one of the three principal macronutrients in the human diet, and their deficiency is one of the world’s most acute nutritional problems [
81]. In this regard, mushrooms offer a protein-rich diet that could be a promising remedy against protein malnutrition problems. Mushroom proteins are reportedly known for their immunomodulatory properties and are thus considered to be a new class of bioactive proteins that have potential use as an adjuvant for tumour therapy [
9]. In this context,
L. drassinus and
L. controversus can be exploited as good sources of proteins as they exhibit higher concentration (almost twice) than previously reported values in
L. controversus from the Middle Black Sea Region of Turkey and
L. deliciosus from Macedonia, as well as other edible mushrooms including
Agaricus bisporus,
Flammulina velutipes,
Letinus edodes, and
P. eryngii, among others [
82,
83].
The presence of amino acids, particularly essential amino acids, is credited for the superior quality of mushroom proteins [
84]. In comparison,
L. drassinus was found to be richer in both essential and non-essential amino acids than
L. controversus. Amongst the amino acids identified, the non-essential amino acid 5-oxoproline was detected in the highest concentration. 5-Oxoproline plays an important role in forming the characteristic umami taste in mushrooms [
85]. Moreover, it possesses several bioactivities such as antibacterial, antioxidant, antidiabetic, and anti-inflammatory activities [
86], and it has been recently reported to be a potent antiviral candidate in the treatment of COVID-19, as well as Alzheimer’s disease [
87,
88]. Other main amino acid constituents detected in
L. drassinus were aspartic acid, glutamic acid, and lysine. Higher aspartic and glutamic acid concentrations correlated to the fact that they serve as the precursors from which the backbones of other amino acids are formed [
89]. Lysine, on the contrary, is an essential amino acid, generally found to be limited in most vegetable proteins. It was present in an appreciable amount in the examined mushrooms, analogous to reports from other studies [
90,
91]. However, it is noteworthy that these compounds were not detected in
L. controversus. Apart from their primary role as building blocks of proteins, amino acids are also known for their antioxidant potency [
92]. Therefore, a significant composition of the amino acids can be linked to the tolerance mechanism adopted by these mushrooms in response to cold stress [
93,
94].
Mushrooms have the potential to contribute immensely to the provision of both macro- and micronutrients. Elemental analyses revealed the presence of Ca, Mg, C, N, S, Zn, Ni, Cu, K, and Al in moderate to high concentrations [
95,
96]. Higher accumulation of mineral elements such as Cu, Fe, Zn, Ca, and Mg in the studied mushrooms insists that they should be included in the diet to combat mineral deficiencies. Although P and Na concentrations were in concordance with previous reports, mushrooms are generally considered to be low in the two minerals [
96,
97]. Low Na and high K in the investigated mushrooms indicates the importance of incorporating these mushrooms into the diet for impeding hypertension [
98]. The mineral values of these mushrooms can guarantee their healthy choice as supplementary foods to the population of Kargil and other states that predominantly rely on a cereal diet for mineral requirement.
It is notable that the two mushrooms investigated are not only rich in minerals, but also in terms of their bioavailabilities, which is attributable to low concentration of antinutrients, namely, phytate, tannins, and oxalate. Phytate forms stable complexes with mineral ions, such as Ca, Mg, Zn, and Fe, resulting in the formation of insoluble salts, which renders them unavailable for uptake in the intestines [
99,
100]. Oberleas and Harland reported that foods having a molar ratio of [Phytate]/[Zn] less than 10 showed substantial availability of Zn [
70]. Likewise, Hassan et al. reported [Phytate]/[Ca] and [Phytate]/[Fe] below 0.2 and 0.4, respectively, indicating adequate bioavailabilities of Ca and Fe [
71]. In this context, the estimated [Phytate]/[Fe], [Phytate]/[Ca], and [Phytate]/[Zn] in the tested mushrooms were lower than their critical values, indicating better bioavailabilities of Fe, Ca, and Zn. Considering the phytate content of mushrooms generally lower than that of green leafy vegetables [
101], mushrooms could be recommended as a superior food to combat nutrient deficiencies.
Tannins are one of the most studied antinutrients, whose composition above 10% of the total DW interferes with protein degradation and digestibility [
102,
103]. Tannins are also known as potent antioxidants [
104]. In the studied samples, although tannins were present in low concentrations compared with previously reported values in
Lactarius spp. [
105,
106], the higher antioxidant activities could have been due to the presence of other antioxidants such as phenols and flavonoids. Another antinutrient, oxalate, forms insoluble salts by binding to minerals, thereby increasing the likelihood of kidney stone formation [
107]. Interestingly, oxalates in the studied mushrooms were quite low for
L.
controversus when compared to the content reported in spinach (1.14%) and almonds (0.47%) [
108]. Biological interaction of Ca, Mg, and oxalate were found lower than the critical value (2.5) in the studied mushrooms [
70], indicating high availabilities of Ca and Mg. It can be deduced that in both the
Lactarius spp., the concentrations of phytates, tannins, and oxalates were insignificant in terms of interfering with mineral absorption and digestibility of proteins, and hence they should not affect their nutritional potential.
Fatty acids have varied functionalities in cells, ranging from structural “building blocks” of plasma membranes to facilitators of energy and signalling compounds [
109]. The assessment of saturated fatty acids of the two mushrooms diagnosed stearic acid and palmitic acid as the major contributor. Similar constituents of fatty acid have been reported in various mushrooms, given that the concentration differs from species to species [
110,
111]. High accumulation of unsaturated fatty acid is a feature of stress acclimation as they are known to lower phase-transition temperature and thereby assist in adapting to low temperature [
112]. Interestingly, though saturated fatty acid concentration was similar, that of unsaturated fatty acids varied between the two mushrooms. While only two unsaturated fatty acids, methyl linoleate and linoelaidic acid, were detected in
L. drassinus,
L. controversus was found to be rich in unsaturated fatty acids, especially linoleic acid (omega-6) and oleic acid (omega-9). In a clinical study by Richard et al., omega-3 and omega-6 fatty acids were reported to possess antioxidant activity in vascular endothelial cells, thereby subsiding inflammation and eventually reducing the risk of disease occurrence such as atherosclerosis and cardiovascular diseases [
113]. Moreover, the composition of unsaturated fatty acids was observed to be higher than that of saturated fatty acid in
L. controversus, testifying it as a legit source of healthy fats.
Ergosterol, a fatty acid derivative and fungal alternative to cholesterol, is crucial in regulating permeability and fluidity of fungal membranes [
114]. It was found to be present in higher concentration in comparison with other previous reports in other edible mushrooms (0.2–7.8 mg g
−1) [
115,
116]. Ergosterol is also a biological precursor of vitamin D
2, making these mushrooms a reserve to meet vitamin D intake for vegetarians, besides dairy and its products [
117]. Additionally, ergosterols are recognised for antitumour, anti-inflammatory, and antioxidant activities, as well as decreasing COVID-19 implications [
118]. The potential of mushrooms for these reasons needs to be sufficiently appreciated.
Deficiencies of vitamins represent a serious health issue on the global food map. On that note, the vitamin profile indicated higher concentrations of vitamins B
3 and B
6 and tocopherols than previous findings in wild edible mushrooms [
119,
120,
121]. Tocopherols, collectively called vitamin E, are fat-soluble vitamins best known for their antioxidant activities. Moreover, they are associated with cancer-preventive activities [
122]. Significant concentrations of vitamins in the mushrooms can, hence, be vouched for in redemption of vitamin deficiencies.
Numerous organic and mineral acids are present in the mushrooms studied, with malic acid, pyruvic acid, and fumaric acid as chief components. Besides primary metabolic activities, they are reported to be potent antimicrobial agents [
123]. Other functions include prevention of rancidity of fats and oils due to their synergistic effect with antioxidants, as well as being food acidity regulators and flavour enhancers [
124]. In the case of mineral acids, phosphoric acid was detected at a high concentration. It is often used in the food and beverage industries as an additive and acidulant. In view of pharmaceutical functions, it is known for lowering blood pH and has been used in the treatment of lead poisoning [
124].
The investigated mushrooms showed high concentrations of total phenol and flavonoids, comparatively higher than previously reported values in other wild mushrooms [
125]. The bioactivities of phenolics can be related to their metal chelating ability, lipoxygenase inhibition, and free radical scavenging capacity [
126]. Flavonoids are known to scavenge free radicals and terminate chain reactions taking place during the oxidation of triglycerides that reportedly play a protective role in diseases linked to oxidative stress, such as cardiovascular diseases and cancer [
127]. Congruent with studies on other edible mushrooms, phenols were found to be the major antioxidant in
L. drassinus and
L. controversus, followed by flavonoids [
128]. The profile of phenolic compounds puts emphasis on chlorogenic acid, which is an important dietary polyphenol playing several roles of therapeutic importance [
129]. In addition to its role as an antioxidant, chlorogenic acid has been reported to possess several bioactivities including antimicrobial, hepatoprotective, cardioprotective, anti-inflammatory, antipyretic, anti-obesity, and anticancer activities [
130]. Kalogeropoulos et al. [
131] and Heleno et al. [
120] reported lower concentrations of gallic acid, vanillin, ferulic acid, cinnamic acid, and quercetin dihydrate in wild edible mushrooms from the island of Lesvos (Greece) and Northeast Portugal in comparison to the mushrooms from Kargil in the present study. Thus, these mushrooms can be inferred as potential candidates for food supplementation, owing to their high phenolic contents. Furthermore, lycopene and β-carotene concentrations were estimated. They are the predominant carotenoids found in various mushrooms, both wild and cultivated [
132]. β-Carotene functions as antioxidants as well as provitamin A, and it plays key role as one of the vitamin A dietary sources [
133]. On the other hand, lycopene is a precursor for β-carotene synthesis and is known to be a potent antioxidant because of its unsaturated nature [
132].
A quantitative measure to correlate antioxidants with antioxidant potency was studied. As depicted by lower IC
50 values in assays of H
2O
2 scavenging activity and reducing power, both
Lactarius spp. correspond to high scavenging activities and enhanced reducing ability. Free radical scavengers inhibit oxidative lipid degradation, which otherwise can be deleterious to the cellular components and their functions [
134]. With the radical scavenging activity mediated by phenols, flavonoids, and carotenoids, these wild edible mushrooms can be recommended as natural healthy antioxidants.