**Use of the Versatility of Fungal Metabolism to Meet Modern Demands for Healthy Aging, Functional Foods, and Sustainability**

**Jacqueline A. Takahashi 1,\* , Bianca V. R. Barbosa <sup>1</sup> , Bruna de A. Martins <sup>1</sup> , Christiano P. Guirlanda <sup>2</sup> and Marília A. F. Moura <sup>2</sup>**


Received: 31 August 2020; Accepted: 27 September 2020; Published: 15 October 2020

**Abstract:** Aging-associated, non-transmissible chronic diseases (NTCD) such as cancer, dyslipidemia, and neurodegenerative disorders have been challenged through several strategies including the consumption of healthy foods and the development of new drugs for existing diseases. Consumer health consciousness is guiding market trends toward the development of additives and nutraceutical products of natural origin. Fungi produce several metabolites with bioactivity against NTCD as well as pigments, dyes, antioxidants, polysaccharides, and enzymes that can be explored as substitutes for synthetic food additives. Research in this area has increased the yields of metabolites for industrial applications through improving fermentation conditions, application of metabolic engineering techniques, and fungal genetic manipulation. Several modern hyphenated techniques have impressively increased the rate of research in this area, enabling the analysis of a large number of species and fermentative conditions. This review thus focuses on summarizing the nutritional, pharmacological, and economic importance of fungi and their metabolites resulting from applications in the aforementioned areas, examples of modern techniques for optimizing the production of fungi and their metabolites, and methodologies for the identification and analysis of these compounds.

**Keywords:** fungi; secondary metabolites; metabolomics; NTCD; additives; functional foods; nutraceuticals; sustainability; healthy aging

#### **1. Health and Modern Food Demands**

Health concerns have always existed among humans. Although some conditions and diseases cannot be avoided yet, the manifestation of several non-transmissible chronic diseases (NTCD) with high prevalence in patients over 60 years of age such as diabetes, cardiovascular, and neurodegenerative diseases can be delayed by adhering to a healthy lifestyle, which among other factors, is directly correlated to eating habits. The physiological effects associated with the consumption of certain foods are thus becoming very popular. Several types of diets and foods such as the fat-free diet, low-carb diet [1], Mediterranean diet [2], and soy-based diet [3] have been adopted in the quest for healthy aging. Several effects of NTCD have also been postponed through calorie restriction diets in animal models [4]. However, prolonged caloric restriction in humans generates undesirable effects; thus, alternative ways of preventing NTCD have been sought through the development of drugs, foods, and/or nutraceuticals that have both health-promoting and anti-aging effects, without causing adverse effects [5]. There is an

increasing trend to combine the use of nutraceuticals with pharmacotherapy, even among individuals with non-aging-related diseases.

As oxidative stress is among the metabolic factors and pathways most related to cell aging, the consumption of nutraceuticals and functional foods with antioxidant activity has increased. Antioxidants can benefit the human body by directly or indirectly neutralizing reactive oxygen species (ROS), modulating metabolic pathways and gene expression, and activating mechanisms of cellular stress and autophagy that delay aging through pathways unrelated to ROS [5]. The nutraceutical market has already reached market values up to USD 117 billion [6]. Nutraceuticals can be classified into several categories based on the level of innovation and area of application (Table 1) [7,8]. Another trend associated with health improvement and NTCD prevention is that of consuming natural foods or foods containing natural, rather than synthetic, additives such as natural flavoring agents, acidulants, and colorants. Several people also prefer vegetarian and vegan diets, which involve restrictions in food and additives of animal origin to different extents; such diets are mainly motivated by the environmental impacts of livestock farming and animal welfare and have prompted studies on the possible effects of these diets on health [9] and increased the market demand for new vegetarian and vegan food products.

Filamentous fungi are capable of responding to different demands for the development of functional foods, nutraceuticals, and bioactive substances that can be used as medicines or in the food industry, either through the use of their biomass or the metabolites produced by them. A comprehensive review of the various aspects of fungal biotechnology and industrial applications was recently published by Meyer et al. [10].

The consumption of fungi as food mainly involves the consumption of mushrooms (Ascomycota and Basidiomycota phyla), which are enjoyed worldwide, sometimes as delicatessen or gourmet products. However, the role of fungi in human and animal health extends much further than the recognized health benefits of mushrooms [11]. Many fungal species are commercially available as supplements or nutraceuticals, and the fungal metabolites produced by these organisms including many non-Basidiomycota species as functional foods have multiple pharmacological activities. Examples of fungal species used as functional foods include many species of microscopic filamentous fungi that are easily cultivated under ex situ scalable conditions such as some well-known *Penicillium*, *Aspergillus*, and *Fusarium* species. However, lesser known species such as *Ashbya gossypii* also play an important role in the production of food additives such as riboflavin (vitamin B2) **(1)** (Figure 1) [12]. Other important fungal products associated with health improvement are enzymes such as β-galactosidase, which hydrolyzes lactose from dairy foods, is produced by filamentous fungi such as *Trichoderma* sp., and is helpful for lactose-intolerant individuals [13].



This review discusses the usefulness of metabolites produced by fungi for food and pharmaceutical purposes closely linked to health improvement and the prevention of NTCD, respectively, as lead compounds, additives, nutraceuticals, supplements, and functional ingredients. The health benefits of fungal metabolites are highlighted. Cutting-edge tools for yield improvement and thus the scaling up ω

γ‐linolenic acid

of fungal metabolite production as well as the means for ensuring a successful circular economy in this area are also discussed.

**– – Figure 1.** Chemical structures of some fungal-derived food additives **(1–4, 22, 51–55)**.

#### **2. Natural Food Additives from Fungi**

Prompted by the growing evidence for the association between natural compound intake and health, there is a demand for the replacement of synthetic food additives with natural products. Fungal metabolites feature many properties that have been explored for such replacement. This section presents the potential of fungal metabolites as food additives, focusing on their versatility as coloring agents, producing species, and recent achievements and challenges in this area.

Fungi provide several food additives and technology adjuvants such as organic acids, colorants, and fatty acids including some ω-3 and ω-6 class fatty acids, which are essential for human metabolism. Citric acid **(2)** and fumaric acid **(3)** (Figure 1), metabolites of *Aspergillus niger* and *Rhizopus oryzae*, respectively, are outstanding food additives that are industrially produced. Citric acid **(2)** and gluconic acid **(4)** (Figure 1) are fungal products with the highest commercial-scale production [24]. Citric acid **(2)** has a number of applications as an antioxidant, preservative, acidulant, pH control agent, and flavor regulation agent, with market numbers predicted to reach USD 3.6 billion in 2020 [24].

The harmful effects of synthetic food colorants on human health such as attentiondeficit/hyperactivity disorder, asthma and allergies, cancer, and neurological disorders have accelerated the search for natural substitutes. The use of the yellow coloring compounds tartrazine, quinoline, and sunset as well as the red coloring compound amaranth [25] has been regulated and supervised by the World Health Organization. In addition, sustainability issues have contributed to the decreased acceptability of non-biodegradable synthetic colorants that are difficult to remove from effluents, causing toxic effects on plants, bacteria, algae, fishes, and crustaceans [26]. Fungal pigments and dyes have emerged as alternatives to these synthetic food additives [27].

Food dyes are coloring materials soluble in food substrates, while food pigments are insoluble in food and need to be carried by vehicles that bind to the food instead [28] (ACS, 2020). Food colorants are pigments or dyes approved for use as food additives [29] (FDA, 2017). A wide spectrum of natural colors can be obtained from metabolites of fungi of different genera such as *Eurotium*, *Fusarium*, *Monascus*, and *Penicillium*, isolated from marine and terrestrial environments and extracted by techniques considered environmentally friendly including those employing ultrasound, pressurized liquid, microwaves, and pulsed electric field [30]. The fungal metabolites have a wide color range, which can be represented by carotenoids such as lycopene (red) **(5)**, β-carotene (yellow-orange) **(6)**, and astaxanthin (pink-red) **(7)**; azafilones such as monascorubrin (orange) **(8)** and ankaflavin (yellow) **(9)**; and the quinine derivatives alizarin (purple-red) **(10)** and bikaverin (red) **(11)** (Figure 2). Arpink Red **(12)** (Figure 2), an anthraquinone produced by *Penicillium oxalicum*, has been approved by the Codex Alimentarius for use as a food colorant in meat products, dairy, confectionery, ice cream, and alcoholic and non-alcoholic beverages [31,32]. Naftoquinone hydrosoluble metabolites (purple color) were produced by a soil-originated strain of *Fusarium oxysporum* using a simple culture medium containing glucose, ammonium sulfate, and salts [33]. Crystalline neoechinulin A **(13)** (ivory color), neoechinulin B **(14)**, and cladosporin **(15)** (all having a yellow color) (Figure 2) are described as metabolites of *Eurotium amstelodami*, *Eurotium herbariorum*, and *Eurotium rubrum* isolated from outdoor and indoor samples in Canada and grown in medium containing sucrose, yeast extract, and salts [34].

The filamentous fungus *Monascus purpureus* is one of the first species that was used to produce natural colorants. It is traditionally consumed in Asia in fermented red rice, which is important in Chinese cuisine, and used in folk medicine as a regulator of digestive and circulatory functions [35,36]. Some species of the genus *Monascus* produce the secondary metabolite monacolin K **(16)** (Figure 2), which consists of a lactone with a free hydroxyl-acid moiety. Aside fromits coloring property, monacolin K **(16)** has a high antioxidant activity and is marketed as a hypocholesterolemic drug known as lovastatin. The pigments of yellow, orange, and red color produced by the *Monascus* genus are considered safe for human consumption [37].

The development of mutant strains of *Monascus* has enabled an increase in available pigments including monashin **(17)** (Figure 2), which is obtained through the mutation of the enzyme polyketide synthase and has an antioxidant activity [35]. The monascin **(18)** (Figure 2) (yellow) produced by *M. purpureus* after activation by the transcription factors DAF-16/FOXO increased the production of superoxide dismutase and thermal shock protein HSP16.2, improving survival in a worm model [38]. Monascin **(18)** also reduced non-alcoholic fatty liver disease and increased AMPK levels and γ 1α co-activator of the receptor activated by peroxisome proliferator in mice [39]. In another study, a new azaphylone, monapurpureusone **(19)**, and a new brownish natural product, monapurpureusin **(20)** (Figure 2), were obtained from a mutant strain of *M. purpureus* cultivated in fermented rice extract. Both compounds presented superoxide radical scavenging activity (EC<sup>50</sup> = 176.2 and 271.2 µM, respectively), with monapurpureusone **(19)** superior to the control gallic acid (237.1 µM), and anti-inflammatory activity (IC<sup>50</sup> = 27.5 and 24.9 µM, respectively), with both compounds superior to the control quercetin (35.9 µM) [36].

Red and orange pigments were produced by a strain of *Talaromyces albobiverticillius* isolated from a marine environment at pH 6.5, and their colors were shown to be dependent on the fermentation period (198.6 and 229.0 h, respectively) [40]. Optimal conditions for the production of pigments were also described for *F. oxysporum* (red color, rate C:N = 9, blue LED light, and absence of co-culture) and *Aspergillus chevalieri* (yellow color, rate C:N = 20, glucose as the carbon source, UV and red light, and co-cultivation with *Kluyveromyces marxianus*) [41]. The production of natural food colorants such as melanins, azaphilones, flavins, phenazines, and quinines by filamentous fungi has also been reported for Basidiomycetes [28].

The replacement of synthetic colorants with fungal pigments and dyes enables the production of safer and healthier foods. Compared with plant sources of natural pigments, fungal sources are more economically attractive, considering the relative ease of yield maximization by manipulating fungal fermentative parameters instead of relying on seasonal factors, as may occur in the production of pigments and dyes of plant origin.

**Figure 2.** Chemical structures of some colored fungal secondary metabolites **(5–20) –** .

Nevertheless, some challenges need to be considered for regulating new food-related colorants such as the possibility of interactions with the food matrix, which causes undesirable sensorial changes, loss of color stability, and contamination by toxic substances such as mycotoxins. Some alternatives proposed for these problems are based on the controlled release of the colorant using microencapsulation as well as the use of nanoformulations to eliminate undesirable aromas and flavors [30]. Spray-drying microencapsulation was successfully used in broth fermented by three species producing yellow dyes, *Aspergillus keveii*, *Penicillium flavigenum*, and *Epicoccum nigrum*. Encapsulation with three adjuvants (maltodextrin, modified starch, and gumarabic) provided pigment retention above 70% [41]. With respect to problems linked to the production of mycotoxins such as citrinin **(21)** (Figure 3) produced by *Monascus*, strategies vary from changes in cultivation and fermentation conditions to disruption of genes encoding the production of mycotoxins in question to create non-mycotoxin-producing mutant strains [30].

**Figure 3.** Chemical structures of some mycotoxins **(21, 29)** and classes of mycotoxins **(30, 31)** frequently produced by fungal species.

The use of fungal metabolites as natural colorants contributes greatly to the development of healthier foods. Nonetheless, several other additives of fungal origin such as substances related to taste and pH (acidulants, flavorings, and sweeteners), texture (thickeners and emulsifiers), and increased shelf life (antioxidants and preservatives) have been studied. Some examples can be found in Table 2. The application of some of these additives including exopolysaccharides is not restricted to the food industry. Fungal exopolysaccharides produced by species such as *Phellinus linteus*, *Ganoderma lucidum*, *Fusarium* sp., *Pleurotus* spp., *Inonotus obliquus*, and *Aureobasidium pullulans* have been employed to aid moisture retention in confectionery, increase the viscosity and crystallization of sugar, and as stabilizers, emulsifiers, and thickening agents [42,43]. These compounds are also of great interest to textiles, food, cosmetics, and pharmaceutical industries and are also important in agriculture as preservatives, bioherbicides, and microbicides.

β



β β β β β ββ β β β β β β Metabolic engineering of microorganisms has been successfully employed for producing 42 out of the 316 food additives from numerous species of fungi and bacteria currently approved by the European Union [44]. Using this technique, the production of glutamic acid, a metabolite capable of providing "umami" flavor to food, though fermentation with *Corynebacterium glutamicum* was optimized. The production of malic acid **(22)** (Figure 1), an acidulant used in food and beverages, was increased after overexpression of the genes encoding its precursors in the species *S. cerevisiae*, *Aspergillus flavus*, *Aspergillus oryzae,* and *A. niger* [51,52]. Metabolic engineering can also be used to introduce heterologous routes for enzyme production in microorganisms (e.g., to produce enzymes of plant origin) [51]. Changes in the molecular structures and colors of microbial pigments were made using the engineering system CRISPR-Cas9, which cleaves specific microbial DNA sites and introduces changes in one or more target genes [30]. For instance, *Escherichia coli* was used for the production of β-carotene **(6)** through genomic editing to introduce insertions, deletions, and substitutions in regions of the lacZ, galK, and ldhA genes using the CRISPR-Cas9 system [53].

β β β A yeast species, *Yarrowia lipolytica*, was engineered by introducing genes for the production of β-ketolase and β-hydroxylase of seaweed and bacteria origin, increasing the yield of astaxanthin **(7)** (285 ± 19 mg/L; 47% of total carotenoids) [54]. This was a great achievement, given the lower productivity of astaxanthin **(7)** by yeasts like *Xanthophyllomyces dendrorhous* (10.2 mg/L) [55] and microalgae like *Haematococcus pluvialis* (84.8 mg/L) [56] and *Chlorella zofingiensis* (12.5 mg/L) [57]. Astaxanthin **(7)** has a high market value (USD 2500–7000/kg), 5–20 times higher than that of β-carotene **(6)** produced by the microalga *Dunaliella salina* (USD 300–500/kg) [54,58].

Overall, tools such as genetic manipulation based on transcriptomic analysis, induction of mutations, cloning, and insertion of heterologous plasmids into species with well-known genomes have enabled the production of large quantities of metabolites by previously non-producing species [59]. The elucidation and manipulation of the different stages in the transcription and secretion of amylases, xylanases, and cellulases in filamentous fungi has enabled their overexpression [60]; these enzymes are widely used in bakery products to improve the quality of dough through hydrolysis of long-chain carbohydrates and non-starch polysaccharides (cellulose and arabinoxylans).

It was hypothesized that enzymes produced by *A. oryzae* and *A. niger* could cleave the anti-nutritional factors present in flour, in addition to hydrolyzing proteins and carbohydrates into smaller molecules and thus making them more accessible for digestion and releasing phenolic compounds from the matrix. However, the fungi used the amino acids present in the flour for their own metabolism, which negatively affected the protein quality of the final product [61]. A new fermented food product was thus developed using stale bread as a substrate for the fungus *Neurospora intermedia*, which converted 65% of the starch into 21% of protein, in addition to supplementing the final product with minerals and vitamins; although there was a reduction in the amounts of proline, glutamic acid, and phenylalanine, the overall amino acid composition was improved [62].

#### **3. Benefits, Research, and Industrial Applications of Fungal Metabolites**

The scope of the pharmacological activity of fungal metabolites seems to be as endless as the structural diversity. However, several issues in this area must be addressed such as the extent of in vivo effects. The wide range of beneficial biological effects of fungal metabolites can be related to the prevention and treatment of NTCD, and some such effects have identified several potential compounds for developing new drugs.

Terrein **(23)** (Figure 4), for instance, is a secondary metabolite biosynthesized in high concentrations (537.26 ± 23.42 g/kg crude extract) by *A.terreus* [63]. The anti-inflammatory and antioxidant properties of terrein **(23)** were reported in in vitro studies [64]. These important medicinal properties and the high initial yield of this metabolite allow the large-scale production and technological development of the *A. terreus* crude extract for the prevention of some age-related NTCD.

Compounds presenting cytotoxicity against tumor cells form one of the most important classes of fungal metabolites. The world incidence of cancers is high, with cancer affecting one in five people at some point in life. The number of cancer cases registered in 2018 was 18.1 million people, a number that could double in 2040 [65]. Cancer is a multifactorial disease that affects people regardless of age, gender, or origin. As an NTCD, cancer is worrying in several ways, since working-age people can be affected, treatment is costly and long, mortality rates are high, and individuals often have health issues even after cure.

Many reports on fungal metabolites with cytotoxic activity show the vast arsenal of molecules that fungi provide to combat cancer. Table 3 provides examples of antitumor metabolites produced by fungi. Species from different genera or secondary metabolites produced by them sometimes present inhibitory power more pronounced than or relatively close to that presented by standard compounds. This was observed for hypocriol A **(24)** and F **(25)** (Figure 4), isolated from the strain *Hypocrea* sp. [66], and for sesquiterpene strichocaranes E **(26)** and F **(27)** (Figure 4), isolated from the entomopathogenic fungus *Isaria fumosorosea* [67], in comparison to the standard cisplatin. Many human tumor cell lines can be inhibited by fungal metabolites such as breast (MDA and MCF-7) [67], cervical and lung [66], colorectal [63], gastric and liver [68], and pancreatic [69] cell lines. The majority of compounds

presented in Figure 4 are terpenes with different degrees of hydroxylation as well as linear and cyclic nitrogen-bearing compounds with free NH groups.

**Figure 4.** *Cont*.

**Figure 4.** Chemical structures of metabolites **(23–28, 32–41, 43–50) – – –** and class of bioactive compounds **(42)** produced by fungal species.


#### **Table 3.** Cytotoxic activity of some fungal secondary metabolites against several human tumor cell lines.

Note: ni = not informed.

Despite the huge number of fungal bioactive metabolites, there is controversy regarding the consumption or development of new drugs from metabolites biosynthesized by toxin-producing fungi. Terrein **(23)** and agmatine **(28)** (Figure 4)are examples of biologically active metabolites produced by *Aspergillus*, a genus often associated with the production of mycotoxins [71,72]. However, species from *Aspergillus* and *Fusarium* genera, known sources of toxins such as zearalenone **(29)**, trichothecenes **(30)**, and fumonisins **(31)** (Figure 3), have shown a biotechnological potential beyond the production of mycotoxins. For example, azaanthraquinone derivatives, 7-desmethyl-6-methylbostrycoidin **(32)**, and 7-desmethylscorpinone **(33)** (Figure 4), isolated from *F. solani* cultures, showed significant activity against tumor cell lines (Table 3) [69].The mycoprotein "Quorn", a popular fungal food for human consumption, is produced using mycelia of *Fusarium* sp. [72]. Therefore, disregarding the potential of species from mycotoxin-producing genera is unnecessary in the research and industrial development of food products and medicines.

Cancer and neurodegenerative diseases are pathologies targeted by antioxidant therapies, not only for treatment but also for prevention, as proven by pre-clinical, clinical, and epidemiological studies [73]. Although compounds and foods with antioxidant activity have been targeted by many studies [5,74], some brief considerations are worthwhile. Despite encouraging data from several screenings pointing to a very significant number of fungal products with antioxidant activity, it is important to note that most of the experiments in these studies were not applied to tissues and organ systems, and the pharmacokinetic aspects of the absorption of these substances by the human organism were not evaluated [74]. Thus, the straightforward extrapolation of the results of these screenings to in vivo human applications is not possible [75].

Conversely, the in vitro antioxidant profile of a natural product can be improved. This was observed in a study in which three strains of *M. circinelloides* (CBS 277.49, WJ11, and CBS 108.16) were grown for different time periods (three, five, and seven days) in different culture media (standard Kendrick medium and Ratledge and modified Kendrick and Ratledge media (MKR), with nitrogen deficiency) and evaluated for phenolic compounds and antioxidant capacity. The total phenolics (TPC) and flavonoids (TFC) were improved for CBS 277.49, while CBS 108.16 produced a higher amount of condensed tannins (TCT). The ethanol extract obtained from CBS 277.49 (five days of growth in MKR medium) presented the best results regarding neutralization of the2,2′ -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS+) radical, copper reducing power, and ferric reducing power. The latter had a higher ferric reducing power than those of the standards BHT and α-tocopherol [76].

Despite the relevance of cancer as an NTCD, several other diseases linked to aging limit a healthy life as people age. Accumulated dietary deficiencies throughout life and loss of immunity increase mortality rates associated with non-fatal diseases such as infections. Chronic inflammation is another worrisome condition that increases the risk of developing various pathologies such as hypertension, diabetes, and neurodegenerative diseases [77]. Although early diagnosis and measures to avoid aging-related pathologies and their consequences are essential, drugs for existing diseases continue to be required. Table 4 presents the structures of fungal metabolites associated with the treatment or control of infections, inflammatory and neuroinflammatory conditions, and Alzheimer's disease and examples and extents of the biological activities of these compounds.


#### **Table 4.** Examples of non-cytotoxic biologically active fungal secondary metabolites.

The use of epigenetics for the production of new secondary metabolites through the activation of biosynthetic routes involving transcriptional genes previously silenced is an approach to increase and diversify the production of bioactive secondary metabolites by fungi. The use of a mutant strain of *Fusarium graminearum* lacking H3K27 methyltransferase after removal of the secondary metabolism repressor *kmt6* gene was reported as being advantageous for obtaining 22 metabolites, three of which had not been previously identified to be produced by this species: N-ethyl anthranilic acid **(34)**, N-phenetylacetamide **(35)**, and N-acetyltryptamine **(36)** (Figure 4). The production of the double mutant *kmt6fus 1*, with elimination of the production of compounds of the class fusarins, enabled the discovery of two new sesquiterpenes, tricinolone **(37)** and tricinolonic acid **(38)** (Figure 4) [84].

#### **4. Mushrooms as Functional Foods for Preventing Aging-Related Non-Transmissible Chronic Diseases (NTCD)**

The intake of edible mushrooms as functional foods associated with health improvement has been widely described [85]. Some metabolites obtained from mushrooms have been introduced into the market as they have antitumor properties or immunostimulants such as the polysaccharide lentinan produced by the edible mushrooms *Lentinula edodes* (Shiitake) and *G. lucidum*. *G. lucidum* latter is consumed in traditional East Asian medicine as bitter tea (hot aqueous extraction) and can also be obtained as a dry powder. The pharmacological properties of several mushroom metabolites have been demonstrated such as those of polysaccharides produced by *G. lucidum*, which were able to increase the survival of worms by activating the transcription factors DAF-16/FOXO [86].

*Hirsutella sinensis* produces polysaccharides with prebiotic properties related to insulin resistance and diabetes control [87,88]. Spermidine **(39)** (Figure 4) produced by several species [89], proved capable of prolonging the life of mice by reducing histone acetyltransferase EP300 [90], while epidemiological evidence indicates that spermidine **(39)** intake may also contribute to the reduction of human mortality [91]. Considerable amounts of the metabolite lovastatin **(40)** (Figure 4), a drug registered as low-density lipoprotein (LDL) cholesterol lowering, were detected in the widely consumed mushrooms *Agaricus bisporus* (30.79 mg/100 g dry weight), *Cantharellus cibarius* (67.89 mg/100 g dry weight), *Imleri abadia* (6.21 mg/100 g dry weight), and *L. edodes* (0.95 mg/100 g dry weight) [92].

However, the most prominent example of a mushroom with nutraceutical properties may be *Cordyceps militaris*, a rare and naturally occurring entomopathogenic medicinal mushroom in the Himalayan Mountains, Tibet, Nepal, and India. Studies have reported that consuming *C. militaris* extracts significantly increases glucose metabolism, thereby decreasing the glucose level in the blood. In addition, consumption of this mushroom provides protection against diabetic nephropathy [93]. *C. militaris* produces polysaccharides in the fructification body that are active toward α-glucosidase [94] and have immunomodulatory activity; this suggests their incorporation into functional foods and dietary supplements [95]. Anti-adipogenic activity was reported for a fermentation mix containing strawberry, silkworm pupae, and *C. militaris* [96]. Several nutraceutical products containing *Cordyceps* are available in the global market. Among the benefits claimed by their manufacturers are the promotion of mental health and benefits to the vascular system *("Cordyceps* active"), cognition support ("Mushroom Plus"), anticancer and antioxidant activity ("Bhutan *Cordyceps* Tea"), strengthening of the cardiovascular system ("MRM *Cordyceps*CS-4 Strain"), and immune system support ("MycoNutri *Cordyceps* Organic") [97]. Cordycepin **(41)** (Figure 4) is the main metabolite produced by *C. militaris* and is very effective in reducing the accumulation of LDL, total cholesterol, triglycerides, and hyperlipidemia caused by high-fat diets [98]. Cordycepin **(41)** has several pharmacological properties such as anti-inflammatory, immunomodulatory, antioxidant, anti-aging, anticancer, antiviral, cardio, and hepatoprotective properties, among others [97]. This range of activities results in health effects that may help in postponing aging-linked NTCD. *C. militaris* also has high nutritional value and contains proteins, phenolic compounds, steroids, and lectins [99].

Another mushroom species with high functional and nutraceutical potential of high market value is *Agaricus subrufescens* (synonymy *Agaricus blazei* and *Agaricus brasiliensis*) [100]. It is commercialized in several countries such as Brazil (brand name "Sun mushroom"), China (Ji Song Rong), and Japan (Himematsutake) [101]. Sun mushroom contains polyphenols and polysaccharides and is known to decrease oxidative stress and prevent NTCD; it is indicated to have antioxidant, antitumor, anti-inflammatory, and immunomodulatory properties [102].

Another class of bioactive compounds of increasing prominence found in edible mushrooms includes antcins **(42)** (Figure 4), steroids that contain an ergostane-type skeleton and are produced by *Antrodia* species such as *Antrodia cinnamomea* and *Antrodia salmomea*. Studies suggest that these compounds are promising agents in the treatment of cancer, inflammation, diabetes, and diseases resulting from oxidative stress, among others. The aforementioned species have been historically used in communities of Taiwan for treating various diseases such as diarrhea, abdominal pain, hypertension, dermatological irritation, and intoxication by food, alcohol, and drugs [103]. A study tracking 36,499 middle-aged and elderly Japanese men over an average of 13.2 years found a positive relationship between regular mushroom consumption and decreased incidence of prostate cancer [104]. Table 5 summarizes the classes of compounds and health benefits from some mushroom species cited in this section.

**Table 5.** Mushroom-originated compounds and health benefits related to non-transmissible chronic diseases (NTCD).


#### **5. Toward a Sustainable Production of Fungal Metabolites**

As the demand for preventive medicines, nutraceuticals, new drugs, food additives, and other health-related products of natural origin grows, the need for scaling up is also increasing. For bioactive metabolites of plant origin, efforts to increase production may be slow because production sometimes dependent on the seasonality of plants and a long plant growth period. Thus, efforts to increase the production of bioactive compounds have been directed toward microorganism-based options, such as metabolic engineering. Modifications in the culturing of fungi have been successful in the yield improvement of biomass and bioactive compounds. Moreover, special consideration is being given to endophytic fungi, especially those able to produce metabolites biosynthesized by their host plants. Table 6 shows some interesting examples of bioactive compounds produced by endophytic fungi after optimization of fermentation conditions. The good outcomes in this area were exemplified by Torres-Mendoza et al. [105] who reported, from 2001 to 2019, 224 patents related to metabolites from endophytic fungi applied to agricultural, biotechnology, pharmaceutical, and food industries, most of which used species from the *Aspergillus*, *Fusarium*, *Trichoderma*, *Penicillium*, and *Phomopsis* genera.


#### **Table 6.** Bioactive compounds produced by endophytic fungi under different fermentation conditions.

ni = not informed.

Yield enhancement in microbial production is one of the most challenging issues, but good progress has been reported. The production of fungal metabolites can also be optimized by co-cultivation with other fungal or bacterial species and variation of chemical parameters, such as composition of the culture medium, and physical parameters, such as temperature, pH, stirring speed, intensity and color of light, and oxygenation. The metabolic modulation resulting from these approaches depends on the fungal species; thus different fungi have been explored to produce fungal metabolites with new industrial applications around the world [38].

Biosynthesis of the natural polyamide agmatine **(28)** by *A. oryzae*, a fungus generally recognized as safe, was described during the fermentative process of sake production (Japanese rice wine) [118]. Optimization of the initial yield of agmatine **(28)** produced by *A. oryzae* in the presence of *S. cerevisiae* in a solid state (3.5 mM agmatine) was achieved by varying some fermentative parameters. At pH 5.3, the production of agmatine **(28)** increased to 6.3 mM. An increase of over 100% in the initial productivity was obtained by adding l-lactic (pH 3.0, 8.2 mM agmatine), succinic acid (pH 3.5, 8.7 mM agmatine), and citric acid **(2)** (pH 3.2, 8.3 mM agmatine) in the fermentation medium. Therapeutic evidences indicate that agmatine **(28)** is a promising lead compound against several NTCD that affect the central nervous system, such as Alzheimer's disease [119]. In 2019 only, the number of people affected by dementia was estimated at 50 million, with the worrying forecast of this number to be trebled by 2050 [120]. Therefore, new molecules for the treatment of Alzheimer's disease and other types of dementia are highly desired. The pharmacological properties and yield of agmatine **(28)** in fermentation by *A. oryzae* suggest the possibility of its incorporation into nutraceuticals [119]. In excess, agmatine (**28)** may present toxicity and can enhance the toxic action of other biogenic amines, such as histamine and tyramine, produced by the decarboxylation of amino acids by food-fermenting microorganisms. Therefore, determining a proper intake for agmatine **(28)** is necessary to mitigate risks and allows for the numerous aforementioned health benefits [16].

The preference for faster, less expensive, and green approaches has sped up research on bioactive fungal metabolites. Some promising analytical tools, such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS), have been useful for addressing some bottleneck problems in this area, such as fungal identification. Genetic and morphological methods, although very efficient, require expertise, time, and resources that may be saved by the use of analytical instrumentation [121]. Such improvements start during screening steps, in the initial laboratory prospection of relevant species and promising fermentative conditions, and continue to the production steps, with the use of alternative substrates for fungal growth, such as agro industrial residues, towards a circular economy. Thus, extracts intended for screening are prepared on a reduced scale, using lower amounts of reagents, such as solvents, during extraction. In addition, modern analytical tools coupled with hyphenated techniques have been recently used for the analysis of a greater number of extracts, without the need for prior isolation of fungal metabolites from extracts. Biochemometrically derived fingerprints obtained by gas chromatography, high-performance liquid chromatography, MS, and nuclear magnetic resonance spectroscopy (NMR), together with statistical analysis also enable the establishment of straightforward associations between metabolite content and biological activities detected for a large number of crude extracts [70,122].

Although MS is the most widely used technique for this kind of analysis [123], NMR, in particular <sup>1</sup>H NMR metabolomic analysis, has gained ground by enabling the nondestructive identification of molecules in complex mixtures originating from various biological materials, such as plants and fungi extracts, foods, biological fluids, and tissues [124,125]. NMR is comparable to MS in criteria, such as efficiency, speed, reproducibility, and ease of sample preparation; however, it has advantages in terms of sample recovery and isotope detection. It is also independent of ionization potential and does not require complex internal standards [126]. Bi-dimensional biochemometric NMR evaluation of the crude extract of a marine-derived strain of *P. chrysogenum* enabled the attribution of the anti-proliferative activity of the extract against breast cancer cell lines to ergosterol **(43)** (Figure 4; Table 3), a structural metabolite common in fungi [70]. In addition, other metabolites of the ergostane

class, ergosterol peroxide **(44)**, 3β, 5α, 9α, 14 α-tetrahydroxy-ergosta-7,22-dien-6-one **(45)**, and 3β, 5α, 9α-trihydroxy-ergosta-7,22-dien-6-one **(46)** (Figure 4), were detected as metabolites of the edible mushroom *V. volvacea* and proved to be active against human cancer cell lines (Table 3) [68].

An interesting application of NMR-based metabolomics for the comparison and origin identification of edible mushrooms was reported. Multivariate analyses, such as PCA, were used to compare the chemical profiles of species, enabling the differentiation of *Kuehneromyces mutabilis* and *Hypholom acapnoides,* and the direct identification of 17 secondary metabolites in their extracts. Statistically significant differences were observed inthe1H NMR data in the variation of composition based on the collection site and restrictedness of metabolites in *K. mutabilis*. Upon comparing *H. capnoides* and *K. mutabilis*, a higher diversity of sugars was observed in *H. capnoides*, while *K. mutabilis* presented significantly higher amounts of some organic compounds, such as fumaric acid **(3)**, showing the versatility of the <sup>1</sup>H NMR technique for comparing fungi in terms of metabolites [127].

Other sophisticated approaches combining UPLC−ESI-TOF/MS, differential off-line LC−NMR, and quantitative1H NMR (qHNMR) analysis were used to identify d-Phe-l-Val-d-Val-l-Tyr **(47)**, d-Phe-l-Val-d-Val-l-Phe **(48)**, and cis-bis(methylthio)silvatin **(49)** (Figure 4) in the aerobic fermentation of *Penicillium roqueforti* with and without L-Tryptophan enrichment. Another metabolite, roquefortine C **(50)** (Figure 4), was identified as an antimicrobial agent against *Bacillus subtilis* and *E. coli* [126]. <sup>1</sup>H NMR-based metabolomics, applied in a study on extracts of 11 species of edible mushrooms, identified dry *Pleurotus geesteranum* and *Hericium erinaceus* and fresh *Pleurotus sapidus* as the most prominent species in terms of metabolite production. In addition, the use of <sup>1</sup>H NMR led to the detection of more than 100 different metabolites of interest to the food industry including trehalose **(51)**, mannitol **(52)**, and glucose **(53)** (Figure 1). The analysis also showed high levels of carbohydrates and proteins, in addition to considerable amounts of vitamins A **(54)** and C **(55)** (Figure 1) and amino acids as nutritional compounds [14]. Therefore, NMR-based metabolomics has been successful in selecting fungi with outstanding potential for further technological development. Metabolomic tools have also been applied for the evaluation of organoleptic properties and interference of external factors with food quality [128] and nutrient quantification such as in the determination of the nutritional contents of 11 *Capsicum annuum* cultivars in terms of ascorbic acid (vitamin C) **(55)** [129]. In the near future, once there is sufficient data in this area, these useful metabolomic tools could be applied in several meta-studies such as for the determination of vitamin C **(55)** content in edible mushrooms. One study [130] reported vitamin C contents between 31.16 ± 0.93 (*Calvatia gigantea*) and 108.11 ± 3.22 (*Lepis tagilva*) mg/kg dry matter. In another study [14], the vitamin C contents ranged from 0.5 to 111.4 mg/100g dry weight. The highest levels of vitamin C were produced by the species *D. indusiata* and *A. subrufescens* at 111.4 and 69.7 mg/100 g dry weight, respectively [14], values close to the daily levels of vitamin C **(55)** recommended in some countries (75 to 110 mg) [131].

Metabolomic approaches have also been applied to the quantification of aflatoxins in industrialized baby food [132], to determine adulteration and its effect on the safety of nutraceuticals [133], and for differentiating extracts of various biomaterials such as crops [134] and fungi [14].

While metabolomic tools provide speed for research, technological development for future industrial applications of fungal metabolites must consider that the modern economy is increasingly challenged to transform traditional processes into sustainable production chains, minimizing waste, and reusing biomass for applications in the food and drug industries, even for well-established processes such as citric acid **(2)** production. The global market value of citric acid **(2)** was estimated to increase to USD 3.6 billion in 2020 and maintain an annual growth of 5% until 2025. Despite the successful industrial experience for citric acid **(2)** production, research to increase sustainability in bio-refineries is ongoing, opening new possibilities such as clustering-related fermentation production including olive oil and wine to reach models for circular bioeconomy [135].

Fortunately, the production of nutraceuticals and functional foods from biomass, extracts, and fungal metabolites greatly addresses the challenge of sustainability improvement, allowing the incorporation of low-cost, underutilized, and abundant materials into the industrial fermentation

process. Many agro-industrial residues available all over the world are sources of peptides, prebiotic dietary fibers, and hydrolyzed or smaller organic molecules such as phenolics, carotenoids, and tocopherols, among other classes. These features make them good materials for fungal cultivation as well as interesting substrates for the production of bioactive components from macromolecules [59,136]. For example, fermentation of pomegranate bark residue by *A. niger* resulted in the production of citric acid **(2)** with reduced production costs, while adding value to a residue usually directed for disposal [137]. High yields (7.8 to 14.4 g/L) of dry biomass, with protein contents ranging from 33.8 to 50.8%, and ethanol were recovered from the fermentation of effluents from the wheat starch industry by the fungi *A. oryzae* and *R. oryzae*. This kind of process contributes to the development of a circular economy, because the large volume and high chemical demand of oxygen in effluents from the starch industry represent an environmental burden and an additional cost as they create a need for effluents to be treated before being discarded [138]. The filamentous fungi *Actinomucor elegans* and *Umbelopsis isabelin* were simultaneously used for the enrichment of white grape-producing bagasse, a residue with little nutritional attractiveness. The fungi were able to increase the γ-linolenic acid and carotenoid contents, improving the nutritional content of bagasse, and thus creating the possibility of reintroducing bagasse into the production chain as a low-cost functional food for humans [139].

#### **6. Conclusions**

Fungi and their metabolites have important industrial applications in high-value-added products and have potential for the development of nutraceuticals that can contribute to the prevention of NTCD and improve health, especially in terms of human aging. In addition, fungi are suitable in the production of natural food additives such as colorants and stabilizers that have lower health risks than synthetic food additives, and bioactive metabolites for pharmacological use such as enzymes, statins, and antitumor agents. Fungal antioxidants have applications in both food preservation and the combat of oxidative stress in the human body, with positive outcomes for several diseases such as cancer. The use of metabolic engineering techniques has facilitated the overcoming of some obstacles to explore the pharmacological potential of fungi, even those producing toxic substances such as some species of the genera *Monascus*, *Aspergillus*, and *Fusarium*. Modern approaches have been successfully utilized to evaluate the interference of additives derived from fungi with the organoleptic properties and quality of food. Strategies currently available for scaling up metabolite production include direct genetic alteration with tools such as CRISPR-Cas9 and gene recombination. Research on the use of agro-industrial byproducts for sustainable fungal fermentation has shed light on its remarkable economic importance to the production of natural additives, food, drugs, and nutraceuticals. Further in vivo antioxidant activity studies of fungal metabolites are still scarce; however, new insights are required to expand the use of metabolites from filamentous fungi to improve human health in the 21st century.

**Author Contributions:** All authors participated in the layout of this review. J.A.T., B.V.R.B., B.d.A.M., C.P.G., and M.A.F.M. identified the literature for review and drafted the manuscript. In addition, J.A.T. conceived and critically revised the manuscript, B.V.R.B. and B.d.A.M. provided the tables and figures, and C.P.G. and M.A.F.M. formatted the document. All authors have read and approved the final version of the manuscript.

**Funding:** The authors gratefully acknowledge financial support from the Brazilian Agencies Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG PPM-00255-18); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq Grants 304922/2018-8, 141601/2018-3, 142517/2018-6, 121994/2019-8); the National Institute of Science and Technology–INCT BioNat/CNPq (Grant 465637/2014-0); and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)–Finance Code 001.

**Acknowledgments:** We thank Michel Almeida for kindly preparing the graphical abstract illustration.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


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### *Review* **Selenium Biofortification of Crop Food by Beneficial Microorganisms**

### **Yuanming Ye, Jingwang Qu, Yao Pu, Shen Rao, Feng Xu and Chu Wu \***

College of Horticulture & Gardening, Yangtze University, Jingzhou 434025, China; y.m.ye2020@gmail.com (Y.Y.); qujw@yangtzeu.edu.cn (J.Q.); puyao456@163.com (Y.P.); raoshen1989@163.com (S.R.); xufeng198@126.com (F.X.) **\*** Correspondence: wuchu08@yangtzeu.edu.cn; Tel.: +86-716-806-6262

Received: 30 March 2020; Accepted: 26 April 2020; Published: 3 May 2020

**Abstract:** Selenium (Se) is essential for human health, however, Se is deficient in soil in many places all around the world, resulting in human diseases, such as notorious Keshan disease and Keshin–Beck disease. Therefore, Se biofortification is a popular approach to improve Se uptake and maintain human health. Beneficial microorganisms, including mycorrhizal and root endophytic fungi, dark septate fungi, and plant growth-promoting rhizobacteria (PGPRs), show multiple functions, especially increased plant nutrition uptake, growth and yield, and resistance to abiotic stresses. Such functions can be used for Se biofortification and increased growth and yield under drought and salt stress. The present review summarizes the use of mycorrhizal fungi and PGPRs in Se biofortification, aiming to improving their practical use.

**Keywords:** selenium; biofortification; transporters; mycorrhizal fungi; plant growth-promoting rhizobacteria (PGPRs)

### **1. Introduction**

At present, it is widely accepted that selenium (Se) possesses multiple physiological functions in various biological systems as an integral part of a range of proteins containing Se. Therefore Se is important for human health. However, Se distribution in the earth's crust is greatly uneven, ranging from 0.005 mg·kg−<sup>1</sup> in Finland to 8000 mg·kg−<sup>1</sup> in Tuva-Russia [1]. Se deficiency has been reported in many places all around the world including China, North America, New Zealand, Australia, Sweden, and Finland [2–5]. Some notorious diseases are directly related to Se deficiency, such as Keshan disease and Keshin–Beck disease, two endemic diseases related to Se deficiency. Keshan disease was first prevalent at alarge scale in 1935 in Keshan county, Heilongjiang province, China. Keshan disease generally occurs in children and women of childbearing age and its symptoms are related to impairment of cardiac function, cardiac enlargement, and arrhythmia [6]. Although the main factor was not determined for the disease in etiology, it was closely related to Se because it was found that there was an obvious Se deficiency in local soil, and Se supplementation could partly control the disease. An investigation analyzed some physiological parameters, including blood Se level, glutathione peroxidase-1 (GPx-1) activity, and variance at codon 198 in *GPx-1* gene, and found that the main risk factors for the disease were low GPx-1 activity, Keshan disease family history, and living in an endemic area [7], suggesting that Keshan disease is closely related to low GPx-1 activity. Kaschin–Beck disease is an osteoarthropathy, which manifests as severe dysarthrosis of joints, shortened fingers and toes, and in severe cases dwarfism. In China, the disease is prevalent in the Tibetan Plateau [8,9]. An investigation carried out by Zhang et al. [8] showed that the levels of environmental Se were very low, and Kaschin–Beck disease in the Tibetan Plateau was much severe with decreasing environmental Se under the Se-deficient condition, suggesting the relationship between Kaschin–Beck disease and Se deficiency in the Tibetan Plateau. In addition, Se is related to other human diseases and health, such as

cancer [10–13], muscle disease [14], and healthy aging and longevity [15–17]. Therefore, it is essential to maintain Se homeostasis in human body [18,19]. It was estimated that Se intake of >900 µg·day−<sup>1</sup> is harmful, and intake of <30 µg·day−<sup>1</sup> is not enough [20]. Some data have shown that over 800million people all around the world might suffer from Se deficiency [21–26]. Therefore, sufficient dietary Se uptake is important for human health.

Acquired Se is converted into some proteins thatcontain at least one of the two amino acids (i.e., selenocysteine (SeCys) and selenomethionine (SeMet)) as a key component (i.e., selenoproteins). Human health and diseases are related to selenoproteins, and selenocysteine is regarded as the 21st proteinogenic amino acid. The human genome encodes about 30 selenoproteins. In the article written by Reeves and Hoffmann [27], they described functions of selenoproteins in detail. Among the selenoproteins in human, glutathione peroxidases (GPxs) seem to be more important, because they include eight proteins (GPx1–GPx8) having antioxidant properties with multidimensional roles in living cells, ranging from H2O<sup>2</sup> homeostasis to regulation of apoptosis [28]. Therefore, enough Se uptake is essential for functional maintenance of these selenoproteins. Since Se is deficient in many places all around the world, Se fortification in food is necessary. In view of high toxicity of selenite and selenate, Se biofortification is relatively bio-safe. Organic Seleno-compounds act as potential therapeutic and chemo-preventive agents that function as antioxidants, enzyme modulators, antitumor, antimicrobials, antihypertensive agents, antivirals, and cytokine inducers [29]. Organic seleno-compounds are provided with crop food [30–35], vegetables [36–42], fruits [43–45], and even nuts [46–48]. Therefore, how to increase concentrations of organic seleno-compounds in these plants is of significance for improvement of dietary Se acquisition by human being.

Se biofortification may be carried out by multiple ways, such as application of Se fertilizers on leaves [49–53] and in soil [52,54,55]. Se-enriched organic fertilizers are also applied. For example, Bañuelos et al. [56] used Se-enriched *Stanleya pinnata* to cultivate Se-enriched broccoli and carrots, and found that more than 90% of organic Se was converted to inorganic selenate and selenite. Se foliar application seems to be most effective way to fortify Se uptake in most arable crops [52,57]. However, a contrary result was observed by Lyons et al. [58]. They found foliar application was less efficient than application to soil at planting (at application rates of 40 and 120 g·ha−<sup>1</sup> , respectively) in Australian trials. The agronomic application of Se fertilizers are more expensive and short-term solutions, especially in large-scale fields. Relatively, agronomic Se biofortification with beneficial microorganisms (BMOs) is a more inexpensive and long-term solution, especially in poor places and Se-rich places, such as Enshi, Hubei province, China [59] and Pineridge Natural Area, a seleniferous site west of Fort Collins, CO, USA [60].

In the present article, we focus on the roles of beneficial microorganisms in Se biofortification and our aim is to improve use of beneficial microorganisms in practice.

#### **2. Improvement of Se Biofortification by BMOs**

Symbiosis of plants with BMOs is helpful for plant growth and to increase in micronutrition uptake and resistance to abiotic and biotic stresses. Based on the characteristics of BMOs, BMOs can be used for Se biofortification. BMOs, including mycorrhizal fungi (endo- and ectomycorrhizal fungi), root endophytic fungi (REFs), and PGPRs, are popular in biofilmed biofertilizers. Arbuscular mycorrhizal fungi (AMFs) are preferential to colonize in roots of angiosperms, and ectomycorrhizal fungi are popular in gymnosperms. Most REFs possess a wide range of plant hosts.

#### *2.1. Arbuscular Mycorrhizal Fungi*

Arbuscular mycorrhizal fungi are used for Se biofortification because of their ability to enhance nutrition uptake of their host plants (Table 1). Functions of mycorrhizal fungi have been the primary focus of research, especially those involved in phosphate uptake. The genomes of these fungi encode some high-affinity inorganic phosphate transporters and some of them have been isolated and identified [61–65]. On the other hand, *in planta*, some symbiosis-specific phosphate transporters

can be induced by symbiosis with mycorrhizal fungi [61,66–72]. Thus, the interaction between plants and mycorrhizal fungi strengthens phosphate uptake and transportation to host plants [73–77]. Similarly, there are some sulfate transporters encoded by genomes of mycorrhizal fungi, such as sulfate transporters GBC38160.1 and GBC25943.1 and sulfate permeases PKY50973.1 in arbuscular mycorrhizal fungus *Rhizophagus irregularis*, sulfate transporters EDR02618.1 and EDR02177.1, and sulfate permeases EDR11271.1 and EDR00466.1 in ectomycorrhizal fungus *Laccaria bicolor*. Since Se and sulfur (S) belong to the same element family (VI-A), the chemical properties of Se are very similar to S. Se is absorbed as selenate or selenite, which is metabolized via the sulfur assimilation pathway in plants, leading to biosynthesis of SeCys, SeMet, and other Se isologs of various S metabolites [78–82]. Se can be transported by sulfur transporters to host plants, just like phosphate transported between mycorrhizal fungi and their host plants, such as the high-affinity sulfate permease [83] and the high-affinity sulfate transporters Sultr1:1 and Sultr1:2 [84–86]. The two sulfate transporters are proton-sulfate symporters, such that for every molecule of selenate entry into root cells, three protons are taken up. Sulfate transporters function in Se accumulation in food crops. Wheat genotype 'Puelche' is the most Se-tolerant and has the greatest Se accumulation among the three wheat genotypes studied (i.e., 'Puelche', 'Tinto', and 'Kumpa'), such that its Se accumulation was related to the strongest transcript level of the sulfate transporter TaeSultr4.1 in roots [87]. In addition, other transporters also take part in Se transport, such as silicon transporters in rice [88] and tomato [89], phosphate/orthophosphate transporters in wheat [90], rice [91–93], tomato [89], and yeast (*Saccharomyces cerevisiae*) [94,95], and monocarboxylates transporters in yeast (*S. cerevisiae*) [96]. Thus, it is reasonable to explain the experimental results that plant availability of selenate and selenite was influenced by the competing ions phosphate and sulfate [97,98]. Competition between phosphate and Se uptake led to decrease in Se accumulation translocation coefficients, and Se concentrations in wheat roots, stems, leaves, and spikes when phosphate fertilizers were applied to selenite fertilized soil [99]. However, a different case occurred. An investigation was carried out on sulfate and selenate uptake in *Astragalus* species (two Se hyperaccumulators *A. racemosus* and *A. bisulacatus* and two closely related non-accumulators *A. glycyphyllos* and *A. drummondii*), and results showed that sulfur deficiency increased Se accumulation, and increased Se supply increased sulfate accumulation in both root and shoot tissues [100]. In certain *Astragalus* species, the high expression of sulfate transporters led to enhanced ability of Se uptake and translocation, and therefore contributed to the Se hyperaccumulation trait. At present, except for sulfate and phosphate transporters, it is not clear whether other transporters have their homologous proteins in mycorrhizal fungi. If these homologous proteins occur in mycorrhizal fungi, they could mediate Se transport to host plants. On the other hand, decreases in sulfate bioavailability and mycorrhizal symbiosis enhanced expression of sulfate transporters, resulting in increase in ability to absorb sulfate and consequent uptake of Se [101–104]. Similarly, Se deficiency also enhances expression of sulfate transporters, resulting in an increase in Se uptake, and mycorrhizal symbiosis also enhances Se uptake.


**Table 1.** Arbuscular mycorrhizal fungi (AMFs) and root endophytic fungi (REFs) often used for Se biofortification.


**Table 1.** *Cont.*

Some evidence supports the role of mycorrhizal fungi in enhancing Se uptake in plants. Wheat seedlings were inoculated with *Glomusversiform* or *Funneliformis mosseaein* hydroponic culture medium for eight weeks, the two arbuscular mycorrhizal fungi significantly increased selenate and selenite uptake by wheat root, but they did not show effect on uptake of SeMet [105]. Meanwhile, compared to non-mycorrhizal roots, mycorrhizal roots showed significantly higher *V*max for selenate and selenite uptake (179.6 vs. 55.93 nmol·g <sup>−</sup>1DW·h −1 for selenate and 1688.0 vs. 860.3 nmol·g <sup>−</sup>1DW·h −1 for selenite). Higher Se accumulation was carried out through up-regulating the expression of three genes encoding sulfate transporters, i.e., TaSultr1:1, TaSultr1:3, and TaSultr2:1, in the mycorrhizal roots, especially TaSultr1:1. In mycorrhizal roots with G. *versiform* and *F. mosseae*, the relative expressions of *TaSultr1:1* gene was significantly up-regulated by 2.18-fold and 2.12-fold, respectively. Garlic (*Allium sativum* L.) is an important condimental species. This species ispopular all around the world because of its diallyl disulfide, a component of garlic, which can inhibit proliferation of various cancer cells (e.g., colon, lung, and skin cancer cells) and WEHI-3 leukemia cells [115–117]. Garlic is used for Se biofortification with mycorrhizal fungi. A survey of applying selenate fertilizer and mycorrhizal fungus *Glomus irtraradices* to soil was conducted, and the results showed mycorrhizal addition increased the Se uptake of garlic by10-fold to 15 µg·g <sup>−</sup>1DW, and fertilization with selenate and amendment of mycorrhizal fungi strongly increased the Se concentrations in garlic to around 1% [55]. Further analyses showed that the amendment of soil with the mycorrhizal fungus and/or selenate increased selenate concentrations in garlic, but did not affect distribution of detected Se species in garlic. In Se-contaminated soil, mycorrhizal fungi inoculation increased Se accumulation of plants. Alfalfa, maize, and soybean seedlings were cultivated in the soil contaminated with different levels of Se, and results showed that mycorrhizal fungi inoculation decreased Se accumulation in roots and shoots of all the plants at low Se levels (0 or 2 mg·kg−<sup>1</sup> ), but increased Se accumulation in alfalfa shoots and maize roots and shoots at Se level of 20 mg·kg−<sup>1</sup> [112]. Contrary results were observed on ryegrass (*Lolium perenne* cv. 'Barclay') [111]. Their results showed that Se concentrations in roots of ryegrass were not affected by mycorrhizal inoculation with the AMF *G. mosseae*, but mycorrhizal inoculation significantly reduced Se concentrations in shoots [111], further decreasing Se uptake in whole plants. Lettuce (*Lactuca sativa* L.) is one of the most consumed leaf vegetables in some places around the world because of its good properties, such as high levels of antioxidants (such as carotenoids, polyphenols, ascorbate, α-tocopherol) and dietary fiber [118–120], thus it is suitable for Se biofortification to enhance dietary Se consumption. When two lettuce cultivars 'Batavia Rubia Munguia' (BRM) and 'Maravilla de Verano' (MV) were treated with Se compounds (selenite, organic Se compounds SeU and SeCH3) and AMFs (a mixture of *Rhizophagus intraradices* and *Funneliformis mosseae*), their growths were continuously improved by AMFs, except for BRM under treatment of SeCH<sup>3</sup> [106]. The positive effect of AMFs on plant biomass was different among lettuce cultivars and forms of seleno-compounds, and BRM lettuce plants showed the highest mycorrhizal efficiency index (MEI) under treatment of SeU, MV lettuce plants with the highest MEI under SeCH3, suggesting that the two lettuce cultivars possessed preference for different seleno-compounds when they were inoculated with AMFs. Meanwhile, AMFs inoculation significantly affected mineral accumulation in

the leaves of BRM lettuce. In general, mycorrhizal inoculation significantly increased levels of macro and micronutrients, but significantly reduced Se levels in leaves of BRM lettuce. Significant interaction occurred about Se levels in shoots of BRM lettuce between seleno-compounds and AMFs inoculation. Similar status occurred on MV lettuce. Under treatment of selenite, AMFs inoculation reduced Se concentrations in leaves of MV lettuce. In contrast, under treatment of organic seleno-compound SeCH3, MV lettuce never accumulated detectable levels of Se in leaves, regardless of whether they were inoculated with AMFsor not. Treatment of organic seleno-compound SeU slightly increased Se concentrations in leaves of MV lettuce without AMFs inoculation [106]. Other research showed similar results [108]. All the results suggest that combination of seleno-compounds and AMFs inoculation does not increase Se levels in lettuce leaves, although it increases levels of some macro- and micronutrients and antioxidants. Therefore, some AMFs are not suitable for Se biofortification in lettuce. Of course, other AMFs should be chosen to examine their role in Se biofortification in lettuce under treatment of seleno-compounds. At present, it is not clear whether lettuce symbioses with some ectomycorrhizal fungi. Thus, more research is necessary for Se biofortification in lettuce.

Consversa et al. [107] investigated the effect of Se fern application and AMFs (*Rhizophagus intraradices* and *Funneliformis mosseae*) inoculation on Se biofortification for two years, such that Se fern application was carried out on green asparagus (*Asparagus o*ffi*cinalis* L.). Their experimental results showed that Se levels in non-mycorrhizal *A. o*ffi*cinaliscv*. 'Grande' plants increased in trial A1 as exogenous selenate levels increased. Under selenate treatment of 75 and 125 g·ha−<sup>1</sup> , Se concentrations in spears increased 4.7 and 6.4-fold on a dry weight basis compared to control, respectively. Similar results occurred in trail B1. In trail B1, Se concentrations in spears were significantly affected by the interaction between Se amendment and AMFs inoculation. In spears of plants without Se amendment, Se levels were similar in mycorrhizal and non-mycorrhizal plants. All the results suggest a combination of Se amendment and mycorrhizal fungi greatly improve Se biofortification in *A. o*ffi*cinalis* and the combination should be recommended in field by large scale. However, contrary results have also been observed. When the AMF *Glomus mosseae* was used for inoculation with alfalfa (*Medicago sativa* L. cv. 'Chuangxin'), maize (*Zea mays* cv. 'ND108′ ), and soybean (*Glycine max* cv. 'Zhonghuang No. 17′ ), mycorrhizal inoculation significantly decreased Se concentrations in roots with the highest reduction for alfalfa (50–70%), while it was less than 40% for maize and soybean, Se concentrations in shoots decreased by 7–38% for mycorrhizal treatment, and the difference caused by inoculation influence was insignificant among the plant species [112]. When Se was added at the levels of 0 and 2 mg·kg−<sup>1</sup> , the total Se accumulation in roots and shoots of all the three plant species were lower in mycorrhizal than in non-mycorrhizal treatment, while the opposite pattern was observed in roots of maize and shoots of alfalfa and maize when Se was applied at 20 mg·kg−<sup>1</sup> [112]. These results show negative effects on Se accumulation in these plant species when low levels of exogenous Se were added.

In addition, some ectomycorrhizal fungi can accumulate Se in their fruit bodies [121–123], suggesting their ability to acquire Se. Some of these ectomycorrhizal fungi are edible, thus they are used for biofortification of Se in fruit bodies. Few investigations on the role of REFs in Se biofortification have beencarried out (Table 1). In general, REFs, especially members of the genus *Trichoderma*, can colonize roots of some host plants, thus they can be widely used for Se biofortification. At present, there are not reports on roles of dark septate fungi in Se biofortification of food crops.

Taken together, mycorrhizal inoculation might increase Se accumulation in some crop species, leading to Se biofortification of crops. For some crop species, more investigations are needed, especially for interactions between mycorrhizal fungi and crop species. For the abovementioned negative effects of G. *mosseae* on Se accumulation in alfalfa, maize, and soybean, more mycorrhizal fungi and root endophytic fungi should be used to investigation.

#### *2.2. Se Biofortification by PGPRs*

Plant growth-promoting rhizobacteria (PGPRs) are popular in improving nutrition uptake, plant growth, and resistance to abiotic stresses [124–127]. Some of them possess the ability to solubilize phosphate in soil. Such ability could be used for Se biofortification (Table 2), because in some soil agrotypes, such as volcanic Andisols in southern Chile, Se bioavailability is very low. On the one hand, Se can form stable complexes with clays and/or can be strongly absorbed onto oxy-hydroxides of aluminum, iron, or manganese, and remain low in terms of bioavailability to plants [128–130]. On the other hand, oxyanions of Se, i.e., selenite and selenate, are bioavailable to plants. When selenate and selenite are supplied to soil, they are rapidly reduced to insoluble forms (e.g., Se–metal ion complex), leading to their low bioavailability (less than 10% only). The Se fertilizers that are not acquired by plant roots readily after application are not bioavailable to plants in the next season or the next year [131]. Thus, Se re-solubility in soil is very important. Although there are no report concernsregarding Se-solubilizing PGPRs at present, some seleno-bacteria have been studied [30,35,109,132,133]. Trivedi et al. [35] isolated and identified some endophytic seleno-bacteria from the various tissues of *Ricinus communis* plants and molecular identification analyses showed that they were *Paraburkholderia megapolitana*, *Alcaligenes faecalis*, and *Stenotrophomonas maltophilia*. Among the three bacteria, *P. megapolitana* was most effective in improving the growth of *Glycine max* plants under drought and enhancing Se biofortification which was 7.4-fold higher compared to control. The synergistic effect on Se biofortification and increased drought tolerance is important for plants grown in arid and semi-arid places with Se deficiency. A great number of people all around the world are dependent on wheat as their main component of diet, thus it is important to fortify Se in wheat grains [30]. Many studies have been carried out on Se biofortification in wheat. Durán et al. [109] evaluated the effects of Se acquisition by wheat plants through the co-inoculation of native seleno-bacteria strains *Stenotrophomonas* sp. B19, *Enterobacter* sp. B16, *Bacillus* sp. R12, and *Pseudomnas* sp. R8, both individually and in mixture, as a seleno-nanosphere source with AMF *Glomus claroideum*. They found that Se concentrations in plant tissues in inoculated plants were significantly higher than those of un-inoculated controls.Meantime, regardless of presence of AMF *G. claroideum*, Se concentrations in grains of wheat plants inoculated with *Enterobacter* sp. B16 were higher than those of plants inoculated with the rest of the microbial strains. In addition, PGPRs showed their synergistic role in improving Se concentrations with AMFs. When plants were inoculated with the seleno-bacteria strains and *G. claroideum*, Se concentrations in grains were 23.5% higher than those in non-mycorrhizal plants. The synergisms might be related to the relationship between seleno-bacteria strains and AMFs, because the seleno-bacteria could acquire more nutrition from the hyphae of their neighboring AMFs or ectomycorrhizal fungi [134–137]. Moreover, Durán et al. [132] isolated two Se-tolerant endophytic bacteria *Acinetobacters* sp. E6.2 and *Bacillus* sp. E5. They studied production of seleno-compounds (SeMet and seleno-methyl-selenocysteins (MeSeCys)) by the two bacteria, but they did not study the effects of the two bacteria on Se biofortification. Co-application of Se fertilizers and seleno-bacteria sometimes leads to changes in bacterial population. When Se-tolerant bacteria and Se amendment were supplied to wheat in Andisols, Se amendment stimulated population growth of two bacterial groups (*Paenibacillaceae* and *Brucellaceae*), but inhibited other bacterial groups (Clostridia, *Burkholderiales*, *Chitinophagaceae*, and *Oxalobacteraceae*) [133]. Meanwhile, Se concentrations in roots and leaves of wheat plants inoculated with Se-tolerant bacterial strains *Pseudomonas* sp. R8 and *Stenotrophomonas* sp. B19 were significantly higher than those of the un-inoculated controls. Higher Se biofortification is related to the Se tolerance of the two bacteria, because higher Se concentrations in roots and leaves were also observed when wheat plants inoculated with *Stenotrophomonas* sp. B19 were grown at concentrations of 5 and 10 mM of selenite, compared to those grown at 2 mM [133]. The results suggested that Se in seleno-bacteria could be transferred into their host plants. Effects of other Se-tolerant bacteria on Se biofortification were also investigated. When wheat plants were inoculated with two Se-tolerant bacterial strains *Bacillus cereus* YAP6 and *Bacillus licheniformis* YAP7, Se concentrations in the stems of the Se-treated wheat plants were increased up to 375%, and Se concentrations in kernels increased up to 154% of those in un-inoculated Se-treated wheat plants [34]. Meanwhile, the *Bacillus* strains can produce auxin, leading to increased number of leaves and greater biomass and shoot length [34]. When wheat plants were inoculated with *Bacillus pichinotyi* in the presence of selenate, they posed significantly higher biomass, shoot length, and spike length compared

to un-inoculated plants [33]. Meanwhile wheat plants inoculated with *B. pichinotyi* had significantly higher Se concentrations in wheat kernels (167%) and stems (252%), compared to un-inoculated plants. Overall, greater biomass means higher Se biofortification, which is important for crops cultivated by large scale in field. Rhizobia not only fixes nitrogen, but also helps Se accumulation. Data from Alford et al. [138] showed rhizobia significantly increased shoot biomass and Se accumulation in shoots of the Se-hyperaccumulator *Astragalus bisulcatus* and the nonhyperaccumulator *A. drummondii*. The dual roles of rhizobia are of significance for organic Se production.


**Table 2.** Plant growth-promoting rhizobacteria (PGPRs) often used for Se biofortification.

Interestingly, volatile organic compounds (VOCs) released by PGPRs improve Se biofortification of plants. VOCs from *Bacillus amyloliquefaciens* BF06 significantly increased photosynthesis and growth of *Arabidopsis* plants and these VOCs led to an obvious increase in expressions of some genes encoding sulfate transporters and Se concentrations in plants [141]. VOCs released by *B. amyloliquefaciens* could not increase Se biofortification of *Arabidopsis Sultr1:2* mutants. All the results suggested sulfate transporters with high expression mediate Se uptake, as shown above. Meanwhile, the results indicate an unknown mechanism that PGPRs improves Se biofortification. The question is inevitable, how do the VOCs improve expression of sulfate transporters?

Taken together, Se amendment could improve population growth of some Se-tolerant bacteria; if these bacteria show synergistic effect on Se biofortification, they could be mixed in some biofilmed biofertilizers specific to certain crops and vegetables, thus, their combinative amendment along with Se fertilizers become a Se biofortification tool in sustainable agriculture [52,145].

#### **3. Concluding Remarksand Perspectives**

Since Se is essential for human health, Se biofortification must be carried out in Se-deficient places by various ways on food crops, vegetables, fruits, and nuts. Foliar and soil fertilization are effective for enhancing Se accumulation in crops. However, the two ways are expensive for large-scaled food crops, especially in poor places. Moreover, the effect of the two ways is short-term and they easily cause area source pollution. BMOs improve Se uptake and accumulation in food crops. Therefore, combination of BMOs and soil fertilization is a good approach to Se biofortification of crop food. At present, for Se biofortification by BMOs, there remain some questions to resolve. The first relates to the synergisms among these beneficial microorganisms. Biofilmed biofertilizers often include many BMOs. As they can compete for nutrition from their common host plants, some of them are possibly antagonistic. Therefore, before they are mixed in biofilmed biofertilizers, the synergism should be examined in detail. The second relates to Se biofortification and phytoremediation. Phytoremediation of Se is popular in Se-rich places. The plants harvested in phytoremediation could be used as the organic source of Se. However, attention must be paid to the fact that there are possibly other heavy metals in the harvested plants. In addition, transgenic plant technology has been used for phytoremediation and Se biofortification. Since some people are very sensitive to genetically modification of crop plants, application of transgenic plants for Se biofortification should be careful. The third relates to theuse of Se hyperaccumulators. Some Se hyperaccumulators, such as *Stanleya pinnata* and *Astragalus bisulcatus* and *Cardamine enshiensis*, should be paid more attentions, especially *C. enshiensis*, because it is edible and can be directly used in food. The forth relates to the use of Se nanoparticles. Relatively, Se nanoparticles are less toxic and more eco-friendly for both humans and the environment. More researches are necessary for use of Se nanoparticles, especially for the production of Se nanoparticles using plants and fungi. The fifth relates to increased plant resistance to abiotic stresses. Exogenous Se compounds and seleno-bacteria synergistically improve plant resistance to abiotic stresses, thus the synergism should be well used for plant resistance to abiotic stresses, especially drought and salt stress. Finally, the sixth relates to functions of root endophytic fungi and dark septate fungi. The two types of fungi possess ecological functions similar to mycorrhizal fungi, and they often colonize many plant species. However, very little attention has been paid to them.

**Author Contributions:** C.W. and F.X. planned the whole article and outline, and provided some references. C.W. and J.Q. revised the second manuscript. Y.Y. wrote the rough manuscript; Y.P. and S.R. reviewed the rough manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by National Natural Science Foundation of China (grant numbers 31870378); Fujian University Key Laboratory for Plant-Microbe Interaction (grant numberPMI2018KF2).

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


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