Medicinal Fungus Ganoderma lucidum as Raw Material for Alcohol Beverage Production

MEDICINAL FUNGUS GANODERMA LUCIDUM AS RAW MATERIAL FOR ALCOHOL BEVERAGE PRODUCTION

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Sonja Veljović*,†, Ninoslav Nikićević†, Miomir Nikšić† *Institute of General and Physical Chemistry, University of Belgrade, Belgrade, Serbia †University of Belgrade, Belgrade, Serbia

6.1 Introduction In recent decades, there has been an increased demand for purchase and consumption of healthy food products. Spirits in their composition contain a slight amount of biologically active substances compared with wine and beer. The process of strong drinks production has a very important influence on their chemical composition, because during fractional distillation, which is the main phase, volatile components are partially separated (Pecić et al., 2016). Thus, various raw materials of plants or animal origin have been used as a source of bioactive compounds in spirits for many centuries. Even today in Asian countries, the alcohol spirits produced with poisonous snakes and scorpions can be found, as well as with seafood. The snake wine has mystical appearance, and additionally attracts attention due to the belief that it has magical and healing powers. Medicinal fungi are equally appreciated raw material with spiritual power for spirit production in the Asian countries (Halpern, 2007; Wasser, 2014). The fruit bodies of medicinal fungi have woody texture and in food industry are used in different forms such as alcohol or water extract, powder, syrup, and liquors (Berovič et al., 2003). In China and throughout the countries of the Orient, these fungi are soaked in whiskey or wine in order to produce spirit drinks which are traditionally used (Halpern, 2007). Maceration of these fungi in alcoholic beverages is achieved by increasing the effective composition and dissolution of the biologically active substances compared to extraction in an aqueous solution. Spirits also improve blood circulation enhancing curative effect (Leskošek-Čukalović et al., 2010; Nikšić et al., 2001). Alcoholic Beverages. https://doi.org/10.1016/B978-0-12-815269-0.00006-4 © 2019 Elsevier Inc. All rights reserved.

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According to the previous reports, the most notable medicinal fungi used in spirit production are Ganoderma lucidum (Kim et  al., 2004; Pecić et  al., 2016; Wang et  al., 2013; Zhao et  al., 2015) and Trametes versicolor (Zhang et al., 2015). In addition, the producer examined the possibility of using a variety of edible mushrooms, such as Lentinus edodes (Shiitake) (Lin et  al., 2010), Cordyceps militaris (Yang et  al., 2016), and Paecilomyces japonica (Lee et al., 2002). In the previous study, it has been established that some types of mushrooms can be used as replacement for yeast Saccharomyces cerevisiae in the process of fermentation in the production low-­alcoholic beverages (Okamura et al., 2001). Since they contain the enzyme alcohol dehydrogenase, these edible fungi are a rich source of fiber, proteins, and vitamins, such as thiamine and riboflavin, and even increased functionality of beverages. Fungus G. lucidum contains very much appreciated bioactive compounds, which influence the bioactivity and also sensory characteristics of alcoholic beverages. Thus, this fungus is marked as the most notable raw material for the production of alcohol beverages.

6.2  Ganoderma lucidum The economically significant species commonly used in spirits production is G. lucidum. More than 100 brands of different products based on G. lucidum can be found in the world market (Lai et al., 2004). The demand for this mushroom grows year after year and is estimated at several thousand tons per year. The annual sale of G. lucidum products is estimated to be more than 2.5 billion US dollars (Li et al., 2013). In markets, especially in eastern countries, numerous products enriched with compounds from the fruiting body, mycelium, and spore of this mushroom are available for purchase (Zhou et al., 2012). Ganoderma lucidum (Fig.  6.1) belongs to a genus Ganoderma of polypore macrofungi (Kirk et  al., 2011). According to the current mushroom taxonomy and based on the latest scientific knowledge, this species has the scientific name G. lucidum (Curtis: Fr.) P. Karst. The importance of this fungus is also shown by the fact that this mushroom had a countless number of trivial names. G. lucidum, the mushroom with many names such as Reishi, Mannentake, Ling Zhi, is one of the most famous medication used in preventing and treating various diseases for over 4000 years in the Asian countries (Boh, 2013). The ancient Chinese people called this mushroom Ling Zhi (ling qi or ling chi), meaning “spirit plant.” The Chinese characters for Ling Zhi consist of three logographic characters: one means “shaman,” one “praying for,” while the third means “rain.” In Japan, this fungus is called Reishi or Mannentake, which means “10,000-year mushroom” and “the fungus of immortality.” The Latin name of this mushroom,

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Fig. 6.1  Fruit bodies of Ganoderma lucidum.

G.  lucidum, originates from the Greek words: gan—glossy, derma— skin and lucidum—brilliant (Halpern, 2007; Wasser, 2005). All names describe this fungus as remarkable Oriental elixir. Ling Zhi was recorded in the oldest Chinese Shen Nong Materia Medica written about 2000 years ago (Wang et al., 2012). Furthermore, it is reported in Chung Hua Pen Tsao (Chinese Materia Medica), where the 365 Chinese medicinal ingredients were divided into three levels: upper, middle, and lower (Lee et al., 2003). On a superior level, G. lucidum takes the first place with ginseng. In order for an ingredient to be at a superior level, it had to have extremely high medical qualities, as well as the absence of side effects when used over a longer period of time (Halpern, 2007). In accordance with traditional Chinese medicine, different types of G. lucidum have different tastes and therefore have an influence on different organs. Depending on the color and the shape of the fruiting body, G. lucidum is divided into following six different types, blue, red, yellow, white, black, and purple, each used for different purposes. The red varieties are the most appreciated of all types in Japan and the black ones in South China (Wasser, 2005). Since G. lucidum is rare in nature, the artificial cultivation has become necessary in order to meet the increasing market demands. Until now, different methods of cultivation have been successfully developed. The most commonly used are traditional methods of cultivation on wood or sawdust from different hartwood species (Ćilardžić et al., 2014).

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Over recent decades, the development of submerged mycelia cultivation in liquid media has abbreviated the growing period. This modernization improves the efficient production of highly valuable metabolites, such as polysaccharides and ganoderic acids (Boh et al., 2007; Feng et al., 2016; Xu et al., 2010; Zhanga et al., 2014).

6.3  Chemical Composition of G. lucidum Until now, over 6500 scientific papers have been published about the G. lucidum and related species, but more than half were written in the Chinese language (Adams et al., 2010; Baby et al., 2015). Although Ling Zhi was the main subject of many different studies, modern researches enable the investigation of a very wide pharmaceutical effects and evaluation of their bioactivity. Thus, the interest for this mushroom is even increased with every new study. The main constitutes of G. lucidum are proteins, fat, carbohydrates, and fiber (Mau et  al., 2001). Pecić (2015) analyzed the composition of the fruiting body of mushroom G. lucidum, and the results are shown in Table 6.1. The fruit body, mycelia, and spores of this fungus contain more than 400 different bioactive compounds, which belong to a wide variety of bioactive molecules, such as triterpenoids, polysaccharides, triterpenes, sterols, lectins, nucleotides, steroids, fatty acids, and proteins/peptides (Batra et al., 2013; Ferreira et al., 2015; Yuen and Gohel, 2005). The beneficial health effects of Ling Zhi are mainly attributed to bioactive effects of triterpenes, polysaccharides, and peptidoglycan (Boh et al., 2007; Zhou et al., 2007). Modern studies also recognized phenolic compounds as secondary metabolites with important bioactive compounds (Ferreira et al. 2009; Veljović et al., 2017). Biologically active compounds have to be soluble in an alcoholaqueous multicomponent mixture in order to be a homogeneous part

Table 6.1  Chemical Composition of G. lucidum Chemical Composition

G. lucidum

Moisture (%) Dry matter (%) Crude protein (g/100 g) Crude fat (g/100 g) Carbohydrate (g/100 g) Ash (g/100 g)

10.39 ± 1.22 89.61 ± 9.89 10.33 ± 1.15 1.13 ± 0.11 68.10 ± 5.15 0.86 ± 0.05

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of the liquor—hence, the triterpene and phenolic compounds are the most valuable compounds, which are soluble in strong alcoholic beverages. Polysaccharides are best dissolved in aqueous solution, and due to the partial content of water, beverages contain them only in small quantities. Therefore, the special attention will be dedicated to phenols and triterpenes, which are the homogeneous part of the liquor.

6.3.1 Triterpenes Terpenes are a naturally widespread group of plant metabolites, whose carbohydrate skeleton is composed of one or more isoprene C5 units. Triterpenes are their subclass, composed of six isoprene units and are a common product of the synthesis of many plants during their growth and development (Galor et al., 2011). In general, triterpenoids have a very complex highly oxidized structure with a molecular weight of 400–600 kDa (Zhou et al., 2007). Until now, 240 secondary metabolites have been isolated and identified from fungus G. lucidum, including C30 ganoderic acids (112), other C30 lanostanes (55), C27 lucidenic acids (27), other C27 lanostanes (18), C24 lanostanes (3), meroterpenoid (1), steroids (23), and benzofuran (1) (Baby et al., 2015). Ganoderic acids, highly oxygenated C30 lanostane-type triterpenoids, are the abundant secondary compounds (Qin et al., 2016). Kubota et al. (1982) first isolated ganoderic acids A and B from the epidermis G. lucidum and presented these ganoderic acids as a new highly oxidized triterpenes. Triterpenes can be isolated from the fruit body, spore, mycelium, and cultural medium of Ling zhi (Wasser, 2005). The triterpene composition of the fruiting body varies depending on the place and conditions of growth and preliminary studies indicate that spores contain significantly higher amounts of ganoderic acids compared with other parts of the fungus (Min et al., 2000). The extraction of triterpenes is usually carried out by using a solvent of methanol, ethanol, acetone, chloroform, ether or by means of some of the mixtures of these solvents. The extracts are further purified by various separation methods, including liquid chromatography with normal and reverse phases (Chen et al., 1999; Su et al., 2001). Most triterpenes have extremely bitter taste, and most of them are ganoderic acids (Kim and Kim, 1999). Nisitoba et al. (1988) performed the division of triterpenes based on their bitter taste into • intensively bitter triterpenes: lucidinic acid A and D1, ganoderic acids A, J, and C1, lucidones C and A • slightly bitter triterpenes: lucidinic acid I, ganoderic acids B, C2, and K • very slightly or not bitter triterpenes: ganoderic acid D, lucidinic acid B, C, E1, G and H, ganolucidinic acid D and C, lucidone B

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By the intensity of bitterness, the order of terpenoid is as follows: lucidic acid D > ganoderic acid C > lucidone A > lucidic acid A > ganoderic acid B > lucidic acid B, C, E. The bitterness of triterpenes is emphasized in subsequent taste and is relatively long lasting. Also, the compounds of this group have a fairly low-sensitivity threshold compared to quinine sulfate and naringenin (Nisitoba et  al., 1985). The bitter taste of this mushroom attracted attention and contributed to its traditional use in the form of tonics. Depending on different functional groups and by the type of side chains, the structural skeleton of ganoderic acids can be classified into three groups. This division includes a large number of oxidative modifications in the structures of these compounds, and this phenomenon is especially noticed on the following carbon atoms: C-3, C-7, C-15, and C-22 (Xu et al., 2010). So far, it has been found that the only producers of ganoderic acids are species of the genus Ganoderma. Scientific research has confirmed a large number of biological effects of various ganoderic acids, and we will list those that have been scientifically proven (Xu et al., 2010): • antitumor activity (-U, -V, -H, -E, -F, -T, -K, -B, -D, AM1, -A, -H, -Me, -W, -X, -Y) • anti-HIV-1 activity (-α, -β, -C1, -H, -GS2) • antihypertensive activity (-Y, -F, -H, -B, -D, -K, -S) • antihepatotoxic activity (-R, -S) • antiinflammatory activity (-A, -F, -DM, -T, -Q) • Inhibition of histamine release (-C, -D) • hypocholesterolemic activity (-Me, -Mf, -Y) • anticomplementary activity (-Sz) • antioxidant activity (-A, -B, -C, -D) • antinociceptive activity (-A, -B, -G, -H) • antiaging activity (-B, -C2, -G)

6.3.2  Phenolic Compounds The phenolic compounds of medicinal fungi, including G. ­lucidum, represent one of the most valuable groups with bioactive effect (Ferreira et al., 2009; Heleno et al., 2015; Nowacka et al., 2015). Until now, phenolic compounds have attested a many bioactive effect, such as antioxidative, anticarcinogenic, and antiinflammatory properties which are mostly related to the potential health-promoting benefits against human health risks such as hypertension, obesity, cardiovascular diseases, diabetes, and cancer (Thai et  al., 2014). Despite this fact only few studies were conducted to determine the content and composition of phenolic compounds in this fungus. Most of the detected phenolic compounds of commercial mushrooms belong to phenolic acids (Dubost et al., 2007; Kim et al., 2008). The amino acid

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l-phenylalanine or l-tyrosine is a precursor for the synthesis of phenolic acids through the shikimate pathway (Heleno et al., 2015). Kim et  al. (2008) studied the content of phenolic compounds in edible and medical mushrooms in Korea, including G. lucidum, cultivated in Korea. The total reported phenol content was 162 μg/g dw. More than 30 phenolic compounds were analyzed. The following phenolic compounds, gallic acid, pyrogallol, 5-sulfosalicylic acid, protocatechuic acid, catechin, benzoic acid, myricetin, quercetin, kaempferol, hesperetin, formononetin, and biochanin, were detected in G. lucidum. Furthermore, the phenolic compounds were evaluated in phenol extracts, made from fruiting body, spores, and mycelium produced in different media (Heleno et al., 2012). Total phenol content in phenol extracts was ranged from 2.5 to 12.3 μg/g, and phenolic compounds detected in extracts were the p-hydroxybenzoic, p-coumaric, and cinnamic acid. Gallic acid (1103 μg/g) was the only detected phenolic compound in methanol extract of wild growing G.  lucidum collected from Fruška gora, Serbia. However, its content was significantly higher than described by the other studies (Karaman et al., 2010). In a recent study, the phenolic compounds of wild and cultivated G. lucidum from Serbia (GS) and China (GCN) were determined by author Stojković et al. (2014). The content of protocatechuic, cinnamic, p-hydroxybenzoic, and p-coumaric acids was analyzed. Both samples contained protocatechuic and cinnamic acids, while phydroxybenzoic and p-coumaric acids were only found in GCN and GS, respectively. Protocatechuic acid was the most abundant in the GCN with 2.87 mg/100 g dw. The ethanol extracts of G. lucidum produced with different particles size and time extraction were analyzed by HPLC-DAD (Veljović et al., 2017). Detected phenolic compounds can be classified into phenolic acids (gallic acid and trans-cinnamic acid) and flavonoids (quercetin, kaempferol, hesperetin, and naringenin). Veljović et al. (2017) also examined total phenolics content (TPC) of ethanol extracts produced with different extraction parameters, time (1, 15, and 30 days) and particle size (milled and chopped). TPC were ranged from 8.6 ± 1.0 to 13.9 ± 0.3 g/100 g gallic acid equivalents (GAEs). The lowest content of PC was found in the sample produced with milled fungus extracted 30 days, thus the transfer of soluble compounds from inside the small fungi particles to alcohol water solution is intensive, and extraction period is shorter. In case of the chopped G. lucidum sample, total phenolic compound transfer from inside the particle was slower, but longer extraction had influence on increasing its content. The obtained results showed that the extraction procedure had an important effect on the TPC. The notable studies on G. lucidum extracts were conducted by Kozarski et al. (2012) and Heleno et al. (2012) in order to determinate

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the TPC. In the research of Heleno et al., TPC in phenolic and polysaccharide extract of G. lucidum fruit body and spore, the reported values were 2.86, 5.5, 1.5, and 4.3 g/100 g GAE, respectively (Heleno et al., 2012). TPC of hot water extracts of G. lucidum was found to be 3.3 g/100 g (Kozarski et al., 2012). While, Rawat et al. (2013) evaluated the effect of used solvent for extract production on TPC of wildly collected G. lucidum from central Himalayan hills of India. TPC in methanolic and aqueous extracts of G. lucidum was found to be 9.2 and 8.4 mg catechol equivalent/gm dry weight of G. lucidum, respectively.

6.3.3  Other Constituents Polysaccharides are considered to be one of main bioactive compounds isolated from fungus G. lucidum. Since polysaccharides are a prominent ingredient of water extracts, and they are not soluble in spirits, the adequate attention will not be attended to these valuable compounds. The fruiting body, spores, and mycelia, or separation from the broth of a submerged liquid culture of G. lucidum can be used as a source of polysaccharides (Wasser, 2005). The majority of identified polysaccharides with bioactive effect are composed of (1-3) and (1-6)-α/β-glucans, glycoproteins and water-soluble heteropolysaccharides (Nie et  al., 2013; Ferreira et al., 2015). Ganoderma ­polysaccharides demonstrated to possess a wide specter of bioactive effects, including anticancer, neurological effects, immune modulation, ­immunomodulatory, antitumor, antioxidant, hepatoprotective, and antihypertensive activities (Nie et al., 2013; Bishop et al., 2015). Medicinal mushrooms in their composition have a lower percentage of proteins compared with edible mushrooms that abound with them. About 7%–8% of proteins are present in G. lucidum (Mau et al., 2001), but some proteins have an important bioactive effect. The most famous active protein is Ling zhi 8 (LZ8) and was detected from the mycelium extract, and in subsequent studies it was found to have an immunosuppressive effect and the prominent anticancer capability (Wu et al., 2015). An antifungal protein of 15 kDa was isolated from the fruit body of G. lucidum and was named ganodermin (Wang and Ng, 2006). Proteins are also found in complexes where they are bound to polysaccharides, creating the peptidoglycan complex. G. lucidum also contains organic germanium, due to the ability of this mushroom to concentrate it. The fruiting bodies of wild growing G. lucidum contained a relatively higher content of this element (0.5 mg/kg dw) compared with the one cultivated in China (0.13 mg/kg dw) (Marek et al., 2017). The content of this element is interesting because of the fact that in small quantities it has a beneficial effect on immunity, antitumor, antimutative, and antioxidant activity (Kolesnikova et al., 1997; Marek et al., 2017).

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6.4  Therapeutic Application of Fungus G. lucidum Many world cultures consider mushrooms to be important components of traditional medicines. The old Chinese proverb reads: “Food and medicine have the same origins” (Klaus et al., 2008). Traditionally, the use of fungi for medical purposes is particularly present in the countries of the Far East. Today, more than 150,000–160,000 species of mushrooms are currently known, of which 2000 can be used in the diet, and about 700 species are found to have different therapeutic properties (Wasser, 2014). Modern analyses determined the chemical composition and confirmed their therapeutic effect on consumers. Due to phytotherapeutic and pharmacological effects, the use of mushrooms in the diet and treatment increases every year worldwide. The market of medicinal mushroom dietary supplements has a value of more than 18 billion US dollars per year (Wasser, 2014). In addition to edible mushrooms, medicinal mushrooms also include decay wood mushrooms G. lucidum and T. versicolor. The most commonly used edible fungi with medical and functional properties are species from the genera Lentinus, Auricularia, Hericium, Grifola, Flammulina, Pleurotus, and Tremella, while the nonedible mushrooms, Ganoderma and Trametes, are used only for their medical properties (inseparable due to their rough and hard texture and bitter taste) (Smith et al., 2002). The fruit body of G. lucidum in China and the eastern countries was recommended as a panacea for the treatment of all types of illness due to the knowledge based on long-term use, oral tradition and cultural practices. In traditional medicine, it has been successfully used in the treatment of chronic hepatitis, arthritis, hypertension, insomnia, bronchitis, hyperlipidemia, neoplasia, asthma, gastric ulcer, atherosclerosis, diabetes, long-term fatigue, and other diseases (Lai et al., 2004). Knowing the reputation of this healing mushroom, scientists have been intensively exploring its medicinal properties over the past decade. A number of studies have proven numerous pharmacological effects. G. lucidum has immunomodulatory and antiinflammatory activity, acts as an anticholesterol and analgesic, has hemopreventive, antitumor, radioprotective, antibacterial, antiviral (including anti-HIV), hypolipidemic, hepatoprotective, sleep promoting, antiaging, antioxidant and hypoglycemic properties, positive effects on diabetes, etc. (Halpern, 2007; Dong and Han, 2015). The main pharmacological effects of G. lucidum ethanol extract are shown in Fig. 6.2. G. lucidum can be used in various forms. It is traditionally used as an addition to alcoholic beverages, hot beverages, or soups. Lingzhi products may be produced from fruiting bodies, spore powder

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G. lucidum

Alcohol extraction

Filtration

Ethanol extract of G. lucidum

Bioactive compounds

Terpenoids

Anticomplementary Antiinflammatory Antioxidant Antiplatelet aggregation Antiviral Cytotoxicity Enzyme inhibitor Hepatoprotective Hypolipidemic Hypotensive

Phenolic compounds Antioxidant Antitumor Antineoplastic Anti-HSV-2 Antimutagenic Antifungal Chemopreventive Neuroprotective

Fig. 6.2  The main pharmacological effects of Ganoderma lucidum ethanol extract.

fermentation mycelia, or fermentation broth. On the market, these products are sold as nutraceuticals and pharmaceuticals (Li et  al., 2016). Several products made of G. lucidum were subjected to clinical trials and are commercially available in the form of syrups, injections, tinctures, in the form of bolus injections (powdered) and capsules. The dose of tincture (20%) recommended for use is 10 mL 3 times daily, in the form of tablets 1 g tablet 3 times daily and syrup 4–6 mL daily. It is recommended that dried G. lucidum (200–300 g) be dipped in hot water and be consumed as a drink—three to five times a day (Wasser, 2005; Zhuang et al., 1993). The long-term use of G. lucidum mushroom does not have toxic effects on the body, but in some people oral use of powdered extract of 1.5–9 g per day can cause increased sensitivity of the organism, resulting in temporary symptoms of drowsiness, thirst, frequent urinating, abnormal sweating, and rash (Soo, 1996).

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6.5  Alcohol Beverages Produced With Medicinal Fungus G. lucidum 6.5.1  Different Materials of G. lucidum Used in Alcohol Beverage Production The raw material used for production of distillate depends on the local tradition and available raw materials in a particular climate. In Asian countries, cereal distillates and wine are dominantly used as an alcohol medium, while European countries use fruit brandies produced from different fruits. Primary aromatic compounds which originate from the raw material are the most important for the authenticity and uniqueness of spirits (Pecić et al., 2012a; Tešević et al., 2005). Considering the traditional knowledge and scientific data of modern researches, fungi can be added to beverages in one of the following ways: • during fermentation • macerated in alcoholic media in a defined time period • added in the form of an aqueous extract • added as an ethanol extract • added as distillate Some types of fungi can be used to produce ethanol because they are found to have alcohol dehydrogenase that can replace yeast S. cerevisiae in the production of low alcoholic beverages, which is traditionally used for fermentation. At the same time, edible mushrooms are a rich source of fiber, proteins, and vitamins, such as thiamine and riboflavin, and the resulting beverage also increases functionality. Оkamura et al. (2001) examined the possibility of using mushrooms such as Pleurotus ostreatus, Flammulina velutipes, and Agaricus blazei in the production of cereal wines. It was found that the largest amount of alcohol was contained in the wine produced from P. ostreatus fungus. Modern production requires standardization of final products, and for this practical reason the extracts are commonly used in production. As we mentioned, fungus G. lucidum is a rich source of bioactive compounds among which the most numerous are polyphenols, β-glucans, triterpenoids, etc. (Paterson, 2006; Veljović et al., 2017). In addition, it consists of volatile aromatic compounds which influence the sensory characteristics such as taste, smell, and color. Several efficient techniques are commonly used for selective separation and concentration of biologically active compounds of plant and fungi. Extracts used for the production of strong alcoholic beverages are produced by one of the following processes: infusion, maceration, percolation, ultrasonic extraction, distillation, vacuum microwave hydrodistillation, and supercritical fluid extraction (Azmir et  al., 2013;

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Tonutti and Liddle, 2010). These techniques include conventional extraction techniques, such as maceration, infusion, distillation, and percolation. In addition, nonconventional extraction techniques, which are more environmentally friendly, reducing operational time, and provide better yield and quality of extract, such as ultrasonic extraction, supercritical fluid extraction, and vacuum microwave hydrodistillation, are used. Due to the constant growth of the necessary quantity of G. lucidum extract for world food and pharmaceutical production, investigations aimed at developing new, faster, and more efficient innovative extraction methods are very intensive. For the development of new products with increased functional characteristics, the most commonly used are extracts in the form of tinctures, concentrates, syrups, or powders. Among all the techniques used in the production of strong alcoholic beverages, the most commonly used method for extracting aromatic and bioactive components of fungi is maceration in ethanolaqueous solution (Buglass et al., 2011; Śliwińska et al., 2015). It is the oldest and most abundant extraction technique in the production of alcoholic beverages. The maceration is a conventional process that is carried out with alcohol-aqueous solutions (usually 40%–60%) at room temperature, thus the thermosensitive bioactive compounds will be preserved (Tonutti and Liddle, 2010). This method is equally important for small homemade production and for industrial production, since it represents an inexpensive way of extracting important secondary metabolites from fungi tissue. For the efficient maceration of medicinal fungi, a longer period of time is required, since their fruit bodies are tough and corky. Maceration is traditionally done by placing plants in a closed vessel, which is dipped by distillates or alcoholic drinks. In order to improve the extraction efficiency, occasional mixing is performed. The extraction efficiency for bioactive compounds mainly depends on the choice of solvents (Cowan, 1999). Therefore, the use of solvent in this solid-liquid extraction will affect the transfer of extractive substances into the solution, mostly due to the polarity of targeted compounds, and will define the chemical composition and sensor profile of the final product. Since alcoholic beverages are alcoholic-aqueous mixtures, after the addition of aqueous extracts in which polysaccharides dominate, precipitation may occur and the blur may appear which is not desirable in the production of these beverages. According to earlier studies, the antioxidant components of the polyphenol nature are more soluble in polar than in nonpolar solvents, and the most suitable solvents for the extraction of polyphenolic compounds are methanol or ethanol (Cowan, 1999; Oreupoulu, 2003).

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Ethanol is more advisable, because it has no toxic effect compared with methanol and can be used in the food and pharmaceutical industry without further purification procedures. In addition, the use of ethanol is also one of efficient approaches for the extraction of G. lucidum triterpenoids, besides water, methanol, chloroform, dichloromethanol, etc. (Cowan, 1999). The goal of producing G. lucidum mushroom extract is to concentrate the colored and biologically active compounds, so that the extract can be added in small quantities, which speeds up the production process. For the production of ethanolic extracts of G. lucidum mushrooms used for rapid maturation of strong alcoholic beverages, a 60% solution of ethanol was used mainly. In previous studies, Nikšić et al. (2001) found that extracts of this mushroom made from 60% ethanol can be used as a more effective supplement for rapid maturation of alcoholic beverages compared with extracts made from 40%, 50%, and 70% ethanol. Thus, Veljović et al. (2017) investigated influences of particle size (chopped and minced) and extraction time (1, 15, and 60  days) on the physicochemical characteristics of G. lucidum ethanol extracts. Fragmented fungi were extracted by using 60% ethanol. In addition, the effect of produced extracts on the color and chemical composition of grain brandy was determined. The produced extracts were added in different concentrations, from 2% to 50%, to grain brandy. The used extract of G. lucidum was not sufficiently efficient since, in order to produce grain brandy with the same color intensity as maceration of 1% in this medium, it is necessary to add 10%–15% depending on the used fungus extract. By adding large amounts of extract, the intensity of the color increases with the tested samples. Based on the results, it can be concluded that the dosage of G. lucidum is very large and the use of untreated mushrooms is more effective. It can be concluded that vacuum evaporation results in degradation or loss of colored substances. So it is necessary to investigate the use of different conventional and nonconventional extraction methods in the production of more efficient G. lucidum extract for accelerating aging of brandies in order to preserve color of compounds.

6.5.2  Alcohol Beverages With G. lucidum As a result of modern research on the medicinal properties of mushroom G. lucidum, there are an increasing number of products that contain it, including strong alcoholic beverages. Today, there are a large number of patents for the production of strong drinks in which G. lucidum mushroom has been used. The Google Patent Searcher can find over 23,000 patents granted for the production of this mushroom.

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A large number of them refers to the production of soft drinks (tea and coffee), but also alcoholic drinks such as beer, strong drinks, etc. In recent decades, there have been an increasing number of patents for the production of strong alcoholic beverages to which G. lucidum is added, either alone or as a component of herbal mixtures. Although there are a large number of patents, there are a small number of written works in which the effect of mushrooms on the characteristics of strong drinks is examined. In one of the few scientific papers, Kim et al. (2004) examined the effect of mushroom G. lucidum on the quality and functional characteristics of traditional rice wine, yakje. With the increase in the amount of added mushrooms, the intensity of bitterness and grass aroma also increased, and the intensity of the aroma of alcohol was reduced. Vukosavljević et al. (2009) examined the antioxidant activity of the plant liquor Bitter 54, in which was added the extract of G. lucidum as a 55th ingredient. By comparing the antioxidant capacity of Bitter 55 and the commercial pharmaceutical extract, it is concluded that the antioxidative activity of Bitter 55 is significantly higher. Another valuable study was conducted in order to define the recipe and production process of compound beverage produced with G. lucidum and Ziziphus jujuba by Wang et al. (2013). Fruit Jujube (Z. jujuba Mill.) is native to China, where it has been traditionally used as an edible or medicinal plant for more than 3000 years (Pu et al., 2017). The results showed that the addition of these raw materials remarkably affects the quality. The optimal ratio of raw materials was: G. lucidum juice to Z. jujuba juice was 1∶2, sugar 6%, citric acid 0.2%. Fungus G. lucidum is also coupled with medlar in order to produce distillate which, blended with quality Kouzi Liquor, is used for the production of G. lucidum health wine. The low-alcohol wine (25% degree alcohol) had specific taste and high functional quality (Gao, 2004). In Asian countries, a mixture of mushrooms is used in the production of strong alcoholic beverages. Zou et  al. (2003) used fungus Hydnum erinaceus and ginseng, besides G. lucidum, to produce health wine with unique taste and improve functionality. Many other herbs, such as Huang qi, Shan yao (Dioscorea opposita), Wu wei zi (Schisandra chinensis), and Rou cong rong (Cistanche deserticola or Cistanche tubulosa) can also be used in combination with fungus G. lucidum to prepare herbal wine (Dong and Han, 2015). For the production of the Chinese plant brand Essenshu, over 30 types of fungi and herbs are used. Some of them are Cordyceps, Ginseng, G. lucidum, etc. For the production of the strong drink of the WEILISHEN brand, 58 animal and plant species are used. The recipe was designed by a doctor from the Qing Dynasty (1738). The most common production procedure of special brandy with G. lucidum is presented in Fig. 6.3.

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Special brandy with high phenolic and terpenes content, improved bioactivity and distinctive sensory profile

G. lucidum Extraction Alcohol base

Liquid extract

Filtration Special brandy

Polysaccharides

Fig. 6.3  The production procedure of special brandies with Ganoderma lucidum.

6.6  Chemical Content of Alcohol Beverages With Addition of G. lucidum Aromatic components, their content, sensory properties and sensitivity thresholds are the most important for the quality and authenticity of distilled drinks. The most commonly used method for exploring the volatile aromatic complex of strong alcoholic drinks is gas chromatography in combination with mass spectrometry (Wisniewska et al., 2015). By direct injection of the distillate, it is possible to determine more than 50 compounds with a content of 0.1–1000 mg/L, while using special extraction methods, this number can be increased to over 1000 (Christoph and Bauer-Christoph, 2007). By analyzing these compounds, a type of beverage and country of origin can be determined, which is of great importance in the production control and the prevention of abuse (Tešević et al., 2005). Compounds that contribute to the sensory characteristics of distilled beverages can be divided into four groups: • primary aromatic components • secondary aromatic components • tertiary aromatic components • quaternary aromatic components (Pecić et al., 2012a; Tešević et al., 2005). Primary aromatic components are volatile compounds with characteristic sensory properties derived from raw materials used for fermentation, which significantly contribute to the formation of a typical flavor of beverages. Aromatic compounds of fruits and grapes are not evenly distributed in the fruit mass, but are at the highest concentration in the composition of the skin. The process of producing strong

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alcoholic beverages should be defined in such a way that the primary compounds that determine the specific flavor of the product are preserved. Fruits contain several hundred aromatic compounds, which united form general aromatic matrix (Śliwińska et al., 2015). Aromatic components originating from fruit vary depending on the variety, geological formations, climatic conditions, and conditions of breeding and technological maturity of the fruit (Nikićević and Tešević, 2010). Secondary aromatic components arise in the process of alcoholic fermentation of carbohydrate raw materials. In addition to ethanol and carbon (IV) oxides, which are the main products of alcoholic fermentation, there is also a large number of other volatile compounds (aldehydes, ketones, higher alcohols, organic acids, esters), by-products of fermentation, which are found in strong alcoholic beverages in relatively low concentration (0.5%–1%) (Christoph and Bauer-Christoph, 2007). Tertiary aromatic substances arise in complex chemical processes during the distillation of strong alcoholic drinks, and strongly depend on the condition of distillation process. Distillates are multicomponent alcoholic-aqueous mixtures whose components during the aging react with each other, dissolve, evaporate or become absorbed, therefore their concentration changes during the process (Tešević et  al., 2005). Different compounds react with one another, so during aging, the concentration of fatty acid ethyl esters increases, while the content of esters of other alcohols decreases due to transesterification. Also, evaporation of the aldehyde and acetal buildup occurs. The compounds formed during the maturation process are quaternary aromatic compounds. If maturation occurs in wooden casks, certain volatile compounds such as cis- and trans-β-methyl-γ-octalactone (viscous lactones), vanillin, guaiacol, eugenol, cresol, etc. will be extracted (Christoph and Bauer-Christoph, 2007). After maturation in wooden barrels, colorless distillates change color in different tones, from yellow to brown. Part of the extracted polyphenol from the wood is decomposed under the influence of atmospheric oxygen, which results in a change in color and a reduction in fatigue (Balcerek et al., 2017). Thus, the aromatic complex of the beverage can be enriched with extractable wooden compounds during the maturation of the distillate. Furthermore, in the Far East, medicinal plants and fungi are traditionally added to strong drinks. Fungi used for the production of strong alcoholic beverages are primarily enhanced by their sensory characteristics with quaternary aromatic compounds. Among mushrooms the most appreciated used is G. lucidum, which contains triterpenic acids that give bitterness to drinks. Therefore, extractable compounds of fungi and herbs have valuable influence on the aroma of spirits defining their sensory quality. After extraction, during aging process extractable compounds suffer conjugation reactions causing

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several changes in their initial structure. These changes in their structure and concentration of G. lucidum compounds additionally affect the aroma of spirits, and even more influence the bioactivity of initial G. lucidum compounds. The preceding chapter states that mushrooms or their extracts are most often added after completion of fermentation and that the extractive compounds of fungi belong to quaternary aromatic compounds. In production, fungi can also be used for fermentation of raw materials, and then lead to the formation of secondary aromatic coagents, but smallvolume products are primarily used for the production of soft drinks.

6.6.1  Aromatic Compounds of Fungus G. lucidum All classes of fungi have representatives producing aromatic compounds, including G. lucidum, only possible exception is Myxomycetes fungi. Basidiomycetes are the most important producers of aromatic components because they possess a developed enzymatic system that allows their catabolism. The aromatic profile of a particular type of fungus varies depending on the medium used, the growth conditions, the genetic variation, and the sensory sensitivity of the tester. The characteristic aromatic components of fungi can be divided into two groups: nonvolatile and volatile components. The volatile components define the smell of mushrooms. So far, over 300 different easily volatile organic components have been isolated, which are the most common complex mixture of individual compounds with mild concentrations (Korpi et al., 2009; Pennerman et al., 2015). Nonvolatile aromatic components form flavor, consisting of a large number of compounds in low concentrations (Jong and Birmingham, 1992). Although they are used in the production of food products and change their aromatic complex, the testing of volatile components of mushrooms has become actual only in recent years. So far, the focus of the research has been primarily on components with a therapeutic effect. Only the scarce available data are listed on the aromatic compounds of mushrooms. The most important volatile compounds of fungi are terpenes, aromatic alcohols, aldehydes, ketones, hydrocarbons with eight carbons and their derivatives. These compounds form their smell, but they also affect the flavor of strong alcoholic drinks in which they participate. Volatile hydrocarbons C8 are formed by the oxidation of free linoleic acids by lipoxygenase. The 1-octen-3-ol compound was first identified as the main flavoring compound in Tricholoma matsutake mushroom in 1938, and it has a unique fragrance on the ground and sweetness (Wang et al., 2014). This compound is also known as “mushroom alcohol,” and it has an extremely low (0.01 ppm) odor threshold in humans (Pennerman et al., 2015).

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According to studies of volatile aromatic compounds of G. lucidum, Chen et al. (2010) detected 58 components, including 7 alkenes, 2 aromatic hydrocarbons, ether, 12 alcohols, 5 alkanes, 8 aldehydes, 2 acids, 7 esters, and 3 furans. The main volatile components of the mycelia flavor of G. lucidum are 1-octen-3-ol, ethanol, hexanal, 1-hexanol, sesquirosefuran, 3-octanol, and 3-octanone. Taşkın et al. (2013) analyzed the flavor of volatile components of G. lucidum samples collected in the Province of Mersin (Turkey) during 2010–11. The study identified 18 components of flavor. The main components are alcohols 1-octen3-ol, 3-octanol, 1-octanol, 2-ethyl-1-hexanol, which account for about 48.05% of the aroma composition. The valuable study was conducted by Ziegenbein et al. (2006) in order to determine the compounds of the essential oil of G. lucidum. Overall, 65 compounds were identified. The major constituents in the essential oil were trans-anethol (9.1%), R-(−)-linalool (4.4%), S-(+)-carvone (4.4%), and α-bisabolol (2%). The main components of the essential oils were monoterpenes with floral and spicy odors. In addition, the essential oil contained an important ratio of the fatty acids, such as n-hexanoic acid (2.1%), n-tetradecanoic acid (1.4%), linoleic acid (1.1%), and n-pentadecanoic acid (0.5%). The fatty acid is an important precursor for a wide range of ethyl esters, which provide a pleasant fruity and floral fragrance of the spirits (Karagiannis and Lanaridis, 2002). Esters represent the largest group of aromatic ingredients of spirit with mostly pleasant aromatic characteristics. They are most important for the sensory profile of beverages and are mainly found in concentrations that exceed their sensitivity thresholds (Christoph and Bauer-Christoph, 2007). The influence of the fungus G. lucidum (40 g/L) on the aromatic profile of special brandies produced with G. lucidum from distillate made from wine distillate, plum, grape, and grain brandy was investigated by Pecić (2015). The aromatic profiles of special grain, plum and grape brandy, and wine distillate after 60 days of extraction consisted of 15, 41, 38, and 24 compounds, respectively. In the process of maturation due to the large number of reactions many compounds detected in the used brandies are not determinations in the composition of the samples after the extraction of the mushroom. Considering the aroma compounds by groups of analyzed special brandies, the esters and higher alcohols were the predominant volatiles, followed by acids, terpenes, phenols, and carbonyl compounds. The first two groups, produced during the alcoholic fermentation, play an important role in the flavor of spirits. Due to the addition of mushrooms and the aging process itself, it can be concluded that there are significant changes in the composition of all analyzed samples. After the addition of G. lucidum, the content of fatty acid increases significantly, as opposed to previous studies in which alcohols were marked as the most abundant components of the fungus aroma. However, the fatty acids were an important constituent of G. lucidum essential

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oil (Ziegenbein et  al., 2006). Only decanoic acid was detected in all special brandies samples. Hexadecanoic acid is the most prevalent in special grain brandy, which is in line with the research by Taşkin et al. (2013). Triterpene alcohols, eugenol, α-terpineol, nonanol, and linalool, are important primary components of plum brandy (Tešević et al., 2005). These compounds were detected at an insignificantly reduced or at the same concentration in the special plum brandy with added mushroom. Based on the results of the research carried out by author Pecić (2015), it can be concluded that extracted volatile organic compounds (VOCs) of this fungus had major influence on the concentration of already existing components of spirit aromatic profiles than in their composition. Special brandy samples produced with different alcoholic base and the same quantity of mushrooms were compared, in order to compare the interaction of chemical compounds of different alcoholic media with fungal compounds. In all analyzed samples with added mushroom, alcohol hexanol was detected, which in the initial medium was contained only in the grape brandy. Furthermore, the concentration of hexanol in the sample of grape brandy was increased. Some compounds, such as isoamyl acetate, were not detected in special brandies after addition of fungus, although all initial distillates contained it. The content of the acids increases in all samples, and the addition of mushrooms also results in a change in the qualitative composition of the acids in the tested samples. Due to the reaction of acids and alcohols in the investigated media, esters that enrich the flavor of the spirits are formed. Although the concentration of isoamyl alcohol (3-methyl1-butanol) decreases in all special brandy samples, this compound with malty flavor was most dominant in all variety of special brandy. In an alcoholic aqueous mixture, a number of reactions take place during stabilization, among which is the formation of 1,1-diethoxyethane acetal in the reaction of acetaldehyde with ethanol, therefore in all analyzed samples the content of the aforementioned acetal increases and the scent is reduced. Furfural, with smoky and almond odor, is detected in all samples with added mushroom. Even more, in the brandy used as alcohol media, in which furfural was initially detected, there is an increase in its concentration. The characteristic quaternary compounds of aged brandy, such as eugenol and furfural, have been detected in the special brandies after extraction addition of fungus.

6.7  Biological Activities of Alcohol Beverages With Fungus G. lucidum Spirits in their composition contain a slight amount of biologically active substances. A recent investigation has shown that moderate intake of alcoholic beverages rich in phenolic compounds can have a

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positive effect on the consumer’s health (Arranz et  al., 2012). Fungi and herbs that are traditionally used for the production of spirits enrich primarily their sensory characteristics. Moreover, they change a chemical composition and potentially improve functional properties. The process of producing strong drinks influences their chemical composition (Pecić et  al., 2016). In addition, distillation process increases the ethanol content at the expense of the other constituents, which are present in a very low content (Spaho, 2017). Volatile compounds of spirits usually constitute the complex mixture in individually low concentration. Quality strong drinks must be homogeneous without blurring or sludge, so the final product is always stabilized by filtration. In order for biologically active compounds to be part of a homogeneous beverage, they must be soluble in a multicomponent alcoholic-aqueous mixture. As we emphasized earlier, special attention will be paid to phenols and triterpenes, which are soluble in strong alcoholic beverages. Polysaccharides are best dissolved in aqueous solutions, and because of the partial content of water, they are contained in beverages in small quantities. Recently, many studies have been done in order to investigate the possibility of using different forms of this fungus in food and pharmaceutical production, especially as pharmaceutical and nutraceutical. Because of their huge potential, an increasing number of these products have been included in the assortment of small craft shops, as well as large multinational companies worldwide.

6.7.1  Triterpenes of Alcohol Beverages With Fungus G. lucidum Medicinal fungus G. lucidum contains a large number of triterpenes, especially lanostanoids. Many biological activities of G. lucidum terpenoids are well established and reported. It has been reported that they possess diverse and potentially significant pharmacological activities, such as antitumor, antiinflammation, antioxidant, antimicrobial, anti-HIV-1, hepatoprotection, antihypertension, cholesterol reduction, blood fat reducing effects, as well as inhibiting platelet aggregation (Liu et al., 2015; Nguyen et al., 2014; Qu et al., 2017; Xia et al., 2014). In the studies of Pecić et al. (2016), the content of triterpenoid acids in grain brandy samples was analyzed with HPLC-DAD/ ESI-ToF-MS analysis. In addition, the influence of different concentrations of G. lucidum (10, 25, and 40 g/L) and different extraction times (7, 21, and 60 days) on the content of triterpenoid acids in special grain brandies was investigated. The analyzed special brandies samples contained 15 triterpenoid acids (Table 6.2). The total triterpenoid acids contents strongly depended on the extraction parameters (2.69–4.40 mg/100 g),

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Table 6.2  Taste and Bioactivities of Triterpenic Acids Detected in Grain Brandies With G. lucidum Compounds

Taste

Bioactivity

References

Ganoderic acid A

IBT

Xu et al. (2010), Duru et al. (2015), and Keypour et al. (2010)

Ganoderic acid B

MBT

Ganoderic acid C2

IMT

• Antitumor • Antioxidant • Antinociceptive • Antiinflammatory activity • Farnesyl protein transferase (FPT) inhibition • Effect of on adipocyte differentiation in 3T3-L1 cells • Anti-HIV-1 activity • Antitumor • Antihypertensive • Antioxidant • Antinociceptive • Antiaging • Anticholesterol • Anti-HIV-1 • Hepatoprotective activity • Antiinflammatory activity • Antivirus activity • Anticholinesterase • Antihypercholesterolemic activity • Antiaging • Antihistamine • Antihypercholesterolemic • Antinociceptive • Antihistamine

Xu et al. (2010), Wasser (2005), and Gao (2004) Xu et al. (2010), Keypour et al. (2010), and Duru et al. (2015)

Ganoderic acid C6

Ganoderic acid D

Ganoderic acid F

NBT

• Antitumor activity • Antihypertensive • Antihistamine • Antioxidant • Histamine release inhibition • Antitumor activity • Antihypertensive • Angiotensin convertix enzyme inhibition • Inhibitory effect on Epstein-Barr virus activation

Xu et al. (2010), Wasser (2005), Duru et al. (2015), Keypour et al. (2010), and Gao (2004)

Xu et al. (2010), Gao (2004), and Keypour et al. (2010)

Xu et al. (2010), Baby et al. (2015), and Duru et al. (2015)

Continued

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Table 6.2  Taste and Bioactivities of Triterpenic Acids Detected in Grain Brandies With G. lucidum—cont’d Compounds

Taste

Ganoderic acid G

Ganoderic acid J Ganoderenic acid D

IBT

Lucidenic acid A

IBT

Lucidenic acid E Lucidenic acid D2 Lucidenic acid LM1 12-Hydroxy-ganoderic D Elfvingic acid A

Bioactivity

References

• Antiaging • Antinociceptive • Antiinflammatory activity (5)

Xu et al. (2010) and Duru et al. (2015)

• Antitumor activity • Antihypertensive • Inhibition of histamine release • Antioxidant • Antitumor • Anticholinesterase • Inhibitory effect on Epstein-Barr virus activation • Antiinvasive

Xu et al. (2010) and Wasser (2005)

• Inhibitory effect on Epstein-Barr virus activation

Baby et al. (2015)

• Citotoxic

Duru et al. (2015)

Zhou et al. (2006), Baby et al. (2015), and Duru et al. (2015)

IBT, intensely bitter taste; SBT, slightly bitter taste; NBT, no bitter taste.

but the ratio of bitter triterpenoids was similar in all analyzed samples. In the analyzed samples with an increase in concentration of extracted fungi, the extraction time necessary for the complete extraction of triterpenoid acids is increased proportionally. The most abundant triterpenoids were ganoderic acid A, the content of which depends on the extraction conditions from 0.528 to 0.781 mg/100 g. In previous studies, it has been proven that ganoderic acid A has a wide range of medical effects, antinociceptive, antioxidative, cytotoxic, hepatoprotective, and anticancer activities (Cao et al., 2017).

6.7.2  Phenolic Compounds and Antioxidative Capacity of Alcohol Beverage With G. lucidum A recent study has focused on the investigation of antioxidant activity of mushrooms and their products, including G. lucidum (Ferreira et  al., 2009; Nowacka et  al., 2014). Addition of fungi enriched the

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­ istillate with bioactive compounds, and the great majority of them d showed the antioxidant activity. However, newer investigations have reported that phenolic compounds of G. lucidum provide the greatest contribution to the antioxidant activity (Ćilardžić et al. 2014; Saltarelli et al., 2015). The influence of this fungus on the antioxidant activity and TPC of the alcoholic beverage was exhibited in many previous studies. The effect on the Korean yakja, Serbian fruit brandies, and herb brandies was investigated. Pecić (2015) examined the influence of extracting parameters (concentration, extraction time) and different alcoholic media on the antioxidant activity and TPC of special brandy produced with G. lucidum. Mushrooms at the concentration of 10, 25, and 40 g/L were added to samples of plum brandy, grape brandy, grain brandy, and wine distillate, with extraction time of 7, 21, and 60 days (Pecić, 2015; Pecić et al., 2016). The total phenolic content of 36 samples was determined according to the Folin-Ciocalteu spectrophotometric method. The total antioxidant capacity was evaluated by using DPPH, FRAP and TEAC methods. TPC of distillates used for special brandies production was insignificant, and the plum distillated contained only 3.02 g/L GAE. Addition of fungi results in a statistically significant increase in the content of polyphenols in all samples of special brandy, ranging from 34.3 to 141.00 mg/L GAE. The antioxidant activity of analyzed brandies was ranged 0.102–0.641 mM TE, 0.212–1.661 FRAP units, and 1.977– 3.963 mM TE analyzed by DPPH, FRAP, and TEAC methods, respectively. It has been found that in special brandies, the content of total phenols and antioxidant capacity increases proportionally with increasing concentration of added mushrooms. The extraction time for the samples with the same concentration of added mushrooms had a different effect on the TPC and the antioxidant capacity compared to the medium used. The optimum extraction time for the same concentrations differed from the applied medium. By analyzing the correlation between results of the TPC and the antioxidative capacity using FRAP, DPPH, and TEAC methods of analyzed special brandies with added fungus, a high correlation was found (rTPC-FRAP = 0.9702, rTPC-DPPH = 0.9618, rTPC-TEAC = 0.9462). It can be concluded that the content of total phenolic compounds has an important influence on the antioxidative capacity of special brandy samples. The antioxidant activity of special brandy samples can be increased with addition of different plant material, such as fruits, spice, and herbs. It is well established that plants possess the wide spectrum of bioactive compounds with improved antioxidant activity. However, phenolic compounds are marked as their most important group with

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antioxidant activity (Do et  al., 2014). Hence, the herb extracts were used as valuable ingredient of the herb brandies and special brandies with G. lucidum recipes (Pecić, 2015; Pecić et al., 2012b). Other notable studies were conducted in order to investigate antioxidant capacity of strong alcoholic beverages, including commercial herbal liqueur “Bitter 54” and small-scale product “Bitter 55” by Gorjanović et  al. (2010) and Vukosavljević et  al. (2009). “Bitter 54” (35% vol of alcohol) is a natural product made from the extracts of 54 aromatic herbs and fruits, and “Bitter55” (Ganoderma bitter) is a natural product made from Bitter 54 and medicinal mushroom G. lucidum (1 % w/v) at Faculty of Agriculture, University of Belgrade, Serbia. In the bottle of Bitter 54 a slice of mushroom was added and extracted for 30 days at room temperature. The content of total phenolic compounds in alcoholic beverages was determined by the Folin-Ciocalteu procedure. TPC of these samples was 477.9 and 445.1 mg/L GAE for the Bitter 54 and Bitter 55, respectively. The added concentration of fungi did not affect the TPC; Bitter 55 had even lower quantity of TPC. Since the sample of bitter is saturated with the bioactive compounds, perhaps for more completed extraction of PC the extraction time must be prolonged. The antioxidant (AO) capacity of these samples was measured by DPPH and hydrogen peroxide scavenging activity (HPS) methods. The AO capacity of Bitter 54 and 55 was 0.1754 and 0.1811 mL/mL, and 31.7 and 29.7% measured by DPPH and HPC methods, respectively. Addition of fungus G. lucidum (1%) also did not improve the antioxidant activity of Korean wine yakja (Kim et al., 2004). Consequently, it can be concluded that the fungus has a greater effect on the brandies antioxidant capacity. Vukosavljević et al. (2009) also examined the AO capacity of Bitter 54 and 55 by DPPH methods. It was found that these samples possessed stronger capacity than commercial bitter liquors from local supermarket.

6.7.3  Other Functionality of Alcohol Beverages With Fungus G. lucidum Lingzhi herb wine is traditionally used for balancing the body and slowing down the aging process (Dong and Han, 2015). In addition, this alcoholic beverage can be consumed for memory enhancement and body strengthening. Lingzhi wine produced with ginseng is recommended for the effective treatment of phthisis chronic cough. Newer research studies have shown that wide variety of G. lucidum chemical compounds possesses significant anticancer potential. In particular, polysaccharides are marked as main compounds with this effect, but recent studies have shown that phenolic compounds (Heleno et  al., 2014; Vaz et  al., 2012) and triterpenes (Patlolla et  al., 2012; Qu et al., 2017) are also an important contributor. In addition,

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these compounds of G. lucidum extract have a synergistic effect (Liu et al., 2002). Thus, the antiproliferative effect of the lyophilized sample of plum brandy enriched with compounds of G. lucidum and herbs extract was evaluated (Table 6.3; Pecić, 2015). The study was performed in several human neoplastic cell lines (HeLa, A549, LS174) and transformed endothelial cell line EA.hy 926. Based on the obtained results it can be concluded that the effect of the sample is dose dependent and time dependent. The most effective effect was on HeLa cells for which the IC50 value for 24 h was 212.7 μg/mL. With increasing duration, the IC50 value decreases, so it is established that it is necessary a smaller amount of sample in order to achieve the same effect. Based on the IC50 values, the effect of the T3 sample on the cell lines was ranked as follows: HeLa > FemX > EA.hy 926 > A549. In one of the pioneer researches, the functionality of Korean traditional wine yakju and G. lucidum yakju (1% of added fungus) was compared. The influence of fungus G. lucidum on functionality was examined by using following assays: angiotensin-converting enzyme (ACE) inhibitory activity (%), fibrinolytic activity (U), superoxide dismutase (SOD)-like activity (%), tyrosinase inhibitory activity (%), nitrite scavenging activity (%), and electron-donating ability (%). The ACE inhibitory activity of G. lucidum GL-1 yakju was increased compared with that of yakju brewed without G. lucidum. Based on the chemical analyses, the increase is directly dependent on the content of the ganoderic acid K. However, the fibrinolytic activity and antioxidant activity of G. lucidum GL-1 yakju were very low, while tyrosinase inhibitory activity and nitrite activity were not determined in either sample. This research estimated that fungus G. lucidum significantly increased the functionality of Korean traditional wine yakju.

Table 6.3  Results of the MTT Assay Presented as IC50 Values Obtained After 24 and 48 h Treatments IC50 (μg/mL) Cell Line

24 h

48 h

EA.hy 926 A549 FemX HeLa

305.3 ± 8.9 376.0 ± 7.8 240.9 ± 4.5 212.7 ± 8.9

204.0 ± 3.7 177.1 ± 8.5 110.5 ± 3.8 123.8 ± 8.4

Cell lines: Human cervical carcinoma (HeLa), human alveolar basal adenocarcinoma (A549), human colon carcinoma (LS174), endothelial cell line EA.hy 926.

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6.8  Sensory Characteristics of Alcohol Beverages With G. lucidum Over the last few years, there have been an increasing number of beverage products in whose production unusual raw materials, such fungi, are used (Sampaio et al., 2013). In the past, the majority of these products belonged to the group of nonalcoholic beverages (tea and coffee), but recently the production of alcoholic beverages, such as beer, spirits, etc., has constantly increased. The main goal of these current researches is to enable an expanding market and to present consumer’s acquisition of different flavors, in order to attract new world markets. It is essential to analyze the physicochemical characteristics of spirits enriched with fungus and also their influence on the sensory characteristic of final product. The use of modern analytical methods in order to analyze the chemical composition does not give us a final judgment on sensory acceptability of the product. Final assessments of the sensory quality of strong alcoholic beverages produced with G. lucidum are obtained as reverse information from trained and experienced expert evaluators, and from consumers. Sensory evaluators of drink characteristics employ universal and understandable terms using natural senses of vision (color and purity), smell (aroma/flavor), taste, and touch (texture and temperature) (Buglass and Caven-Quantrill, 2011). Medicinal fungus G. lucidum is a valuable raw material for production of special spirits, since it is a rich source of terpenoids with bitter taste. As we mentioned earlier, triterpenoids are divided into three groups based on intensity of bitterness (Nisitoba et al., 1988). In the production of alcohol spirits more important groups are those with intensively bitter and with moderately bitter taste than the group with very little bitterness (or no bitterness). Owing to the valuable medicinal properties, no bitter terpenoids are appreciated compounds of final spirits. In the previous study, it was found that the particle size of used fungi had important influence on the sensory characteristics. Sensory analysis of brandy produced with different G. lucidum particle size (milled and chopped) found that the samples of special brandies produced with milled fungus had an unpleasant, bitter aftertaste, probably due to smaller particles which have more intensive transfer of bitter compounds. In addition, it was found that extracts made by using chopped mushrooms contained a higher amount of extracted triterpenic acids. Therefore, chopped G. lucidum was used for the production of special brandies (Pecić et al., 2016). Over the past few decades, many research groups have been studying ways to improve the sensory and functional quality of Korean traditional rice wines and liquors. Kim et  al. (2004) investigated the possibility of development of a new functional G. lucidum yakju. The yakju samples were brewed with different amounts (0.1%–2.0%) of

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G. lucidum in the mash. The sensory characteristics were evaluated by using the quantitative descriptive analysis (QDA) profile for odors and tastes of analyzed samples. The sensory acceptability of analyzed samples can be ranged: no addition yakju, >0.5% G. lucidum yakju and 1.0% G. lucidum yakju. The samples with 2.0% G. lucidum were not acceptable due to overstated bitter taste. Sensory characteristics of Linzhi brandies were determined by using a modified model of positive ranking by Pecić et  al. (2016). The common quality parameters were evaluated: clearness, color, distinction, odor, and taste. In this evaluation a brandy sample may have a maximal score of 20 points (Tešević et al., 2005; Pecić et al., 2016). Linzhi brandies were produced from different alcohol media (plum brandy, grain brandy, grape brandy, and wine distillate), and with different concentration (10, 25, and 40 g/L G. lucidum) and time extraction (7, 21, and 60 days). The final sensory evaluation of 36 analyzed samples was between 16.20 and 18.26, which is a very good result. The wine distillate was the most suitable for special brandy production, because extracted compounds of fungi were the most compatible with the aromatic profile of wine distillate and they improved its sensory quality. The increase in extraction time, for special brandies produced from wine distillates with the same amount of fungus, had a positive effect on the sensory characteristics of analyzed samples. The concentration of added fungus did not have a significant influence on the sensory characteristics of analyzed samples. In addition, G. lucidum compounds did not have a significant effect on the sensory characteristics of special brandies produced from plum brandy. The extraction parameters have strongly influenced the sensory characteristics of special brandies, so in the production of special brandies they have to be carefully designed. The increased extraction time had a positive influence on the sensory characteristics of special brandies, because it influenced the decrease in compounds with an unpleasant, bitter aftertaste, while the increase in extraction time had a negative effect on the sensory characteristics of the samples produced with the same concentration of added G. lucidum. Another important conclusion established by this research is that the physicochemical characteristics of alcoholic medium used for Ganoderma brandies production have a significant influence on their sensory evaluation. The samples produced from plum and grain brandy were evaluated with lower final sensory score compared to the samples produced from wine distillate and grape brandy. However, the aromatic profile of special grain brandy is with the smallest fraction esters, which influences the pleasant aroma of strong beverages. Furthermore, the content of bitter triterpenes analyzed in grain brandies samples did not have a significant effect on the sensory evaluation. Many detected VOCs, although in trace, have a significant influence on the sensory characteristics. Based on the results, grape brandy, and

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wine distillate are the most suitable alcohol media for special brandy production, because extracted G. lucidum compounds are better correlated with compounds of their aromatic profiles.

6.8.1  Color of Alcohol Beverages Produced With Fungus G. lucidum The color of spirit is one of most important organoleptic attributes that directly affects consumers’ acceptance and has a pivotal influence on the consumers’ selection (Martins et al., 2016). The use of natural color is the marketing brand and recent trend in food industry, in order to reduce the artificial food colorant (Rodriguez-Amaya, 2016). In order to accelerate and standardize the process of maturation of aged brandies and liqueurs, the common practice is to use extracts of oak or herbs in the industrial production. Pecić (2015) investigated the possibility of using extract of G. lucidum to accelerate the aging of distillates. Furthermore, the influence of different concentration of fungus G. lucidum extract on the color intensity of grain brandy was investigated and compared with the color intensity of special brandies produced with added mushroom. In the research, six different extracts produced with 40 g/L of chopped and milled G. lucidum were used. Since the chopped fungus had no aftertaste, the influence of different concentration is presented in Fig. 6.4. Extracts were added in various

10 E1

9

E2

E3

Color intensity (CU)

8 7 6 5 4 3 2 1 0 0

5

10

15

20

25

30

35

40

45

50

Concentration of added extract (%)

Fig. 6.4  The influence of Ganoderma lucidum extracts on the color intensity of unaged grain brandy. (E1, E2, and E3—ethanol extract of chopped Ganoderma lucidum (40 g/L) during 15, 30, and 1 days, respectively.)

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Fig. 6.5  Herb brandy with added a slice of Ganoderma lucidum, special brandy with added a slice of G. lucidum, and plum brandy.

percentages of 2%–50%. The most efficient was extract E1 produced with 40 g/L of the fungus extracted for 15 days. The influence of G. lucidum compounds on the color of spirits was also investigated by Pecić et al. (2016). At the initial stage, all brandies used as alcohol media were colorless, but adding of the fungus G. lucidum and/or herbs changed the color of produced spirits, which can be seen in Fig. 6.5. With increasing concentration, the added mushrooms in the samples increase the intensity of the color in all alcoholic media (wine distillate, grain, plum, and grape brandy). The color intensity of special brandies produced with different content of G. lucidum strongly depended on the extraction time and the concentration of added fungi. Even more, the influence of time extraction was more significant with addition higher content of fungus, and this parameter had an insignificant effect on the intensity of color of the samples with 1%. The extraction time of the samples made by adding 2.5% of the fungus is more significant effect on the color intensity especially for the special brandy produced from grape brandy and wine distillate as an alcohol medium (Fig. 6.5). The strong correlation was found between the results of color intensity and total phenolic content of special brandies produced with fungus G. lucidum (Pecić et al., 2016). The correlation (r = 0.9618) indicates that phenolic compounds had an important effect on the color intensity. Thus, increasing of total phenol content will have a positive effect on the intensity of the color of special brandies.

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As previously mentioned, G. lucidum ethanol extracts consisted of phenolic compounds (Veljović et al., 2017). Based on the classification, detected kaempferol and quercetin are the flavonols. The most abundant detected phenolic compounds were naringin and hesperetin that belong to flavanones that are dyed substances, yellow to orange. In a recent work, Saltarelli et al. (2015) analyzed flavonoids profile of G. lucidum strain, and identified quercetin, morin, and myricetin. The results of CIElab analyses demonstrated that the addition of fungi increases the intensity of parameter a*, thus increases the intensity of yellow color (Pecić et al., 2016). Caramel Class I and II, artificial colorants, are commonly used for the standardization of the color of aged brandies. According to recommendation of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), an acceptable daily intake (ADI) of Class I caramel color has been established as “not specified”; and of Class II as 0–160 mg/kg body weight (Sengar and Sharma, 2014). Since the ADI of products of G. lucidum has not been established, and extractible compounds of fungi G. lucidum changed the color of unaged brandy, a different form of G. lucidum can be innovative replacement for this food colorant. Since the fruit body is expensive, G. lucidum waste in the process of extracts production can be used as the alternative source of this colorant.

6.9 Conclusion In recent decades, we have witnessed a shift in consumer habits and a growing focus on the purchase of “healthy” products. With the development of multinational companies, “healthy” products with traditional taste for certain regions have become available to consumers around the world. Because of their characteristics, mushrooms are highly regarded as culinary delicacies, but also as an important raw material for the production of various food and therapeutic products. Medicinal mushrooms, especially G. lucidum, are interesting raw materials for the production of strong alcoholic beverages, because they are a rich source of aromatic and bioactive substances. Due to the high potential, the number of these products is increasing, which is included in the range of small craft stores, but there is also a significant number of sales in global trade networks. Since many innovators and scientists focus their research mostly on finding the most convenient raw materials for developing new G. lucidum products, and also on the investigation of their bioactivity, the future research must be additionally expanded on the study of the bioavailability of these compounds. In addition, the future research may include the mycelium as innovative raw material for alcohol beverage production.

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Acknowledgments This work was performed within the framework of the research Projects Nos. III 46001 and III 46010 supported by the Ministry of Education, Sciences, and Technological Development, Republic of Serbia.

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