Ganoderma lucidum — Medical mushroom as a raw material for beer with enhanced functional properties

Food Research International 43 (2010) 2262–2269

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Ganoderma lucidum — Medical mushroom as a raw material for beer with enhanced functional properties I. Leskosek-Cukalovic a,⁎, S. Despotovic a, N. Lakic a, M. Niksic a, V. Nedovic a, V. Tesevic b a b

Department for Food Technology and Biochemistry, Faculty of Agriculture, Nemanjina 6, 11080 Belgrade, Serbia Faculty of Chemistry, Department for Instrumental Methods of Chemical Analysis, Belgrade, Serbia

a r t i c l e

i n f o

Article history: Received 13 April 2010 Accepted 13 July 2010 Keywords: Beer Ganoderma lucidum Functional properties Terpenoids Sensory properties

a b s t r a c t Genoderma lucidum among all other cultivated mushrooms is unique for its medicinal, rather than nutritional value. A woody mushroom for centuries highly regarded in oriental folk medicine in recent years came in to focus of numerous pharmacological and medical researchers. The paper gives a brief overview of the obtained results and state of the art of knowledge about its bioactive components and pharmacological functioning indicating its possible use in brewing as a raw material for the production of beer with improved functional properties. It deals with the procedure for Ganoderma extract production, determination of main bioactive substance contents, procedure for beer enrichment and sensory evaluation of the final products. The consumer's acceptance test was carried out by 105 untrained young subjects. The results indicate that both male and female tasters evaluated the enriched beer similar or even better than initial commercially produced Pilsner beer. Male tasters especially showed great affinity for new sensory properties, particularly the body, liveliness, and taste. Female tasters evaluated both beers quite similarly. According to their opinion, beer with Ganoderma compared with initial beer differs statistically significantly better only by its bodiness. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction Various parts of higher plants and fungi have been in use for thousands of years. Their superior properties in treating different health problems and diseases have a long tradition in folk medicine, but they have attracted greater attention of conventional medicine in recent years as well. Increasing interest for natural and alternative therapies has brought them in focus of current researches. Plenty of scientific data about their properties and composition are now available giving the exact confirmation of the facts known for centuries. They have become very interesting as raw material for the pharmaceutical and cosmetic industries, but their potentials are much greater. They can be used in the food and beverage industry producing a wide range of products with defined functionality. One of them, which deserve special attention, is Ganoderma. Ganoderma lucidum, a woody Basidiomycotina mushroom belonging to the family of Ganodermaceae of Polyporales, is not edible but a remedy used in oriental medicine for longevity and health promotion since ancient times. Named as “Lingzhi” in China, “Youngzhi” in Korea, “Reishi” in Japan, and just “Ganoderma” in USA. It is highly regarded as a cure in the belief that it lowers the risk of cancer, liver, and heart diseases and boosts the immune system (Chen & Miles, 1996; Chang & Buswell, 1999; Wachtel-Galor, Tomlinson, & Benzie, 2004; Wasser &

⁎ Corresponding author. Fax: + 381 112199711. E-mail address: [email protected] (I. Leskosek-Cukalovic). 0963-9969/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2010.07.014

Weis, 1999). It is commercially cultivated and a diversity of remedies in the forms of tea, powder, and extracts are available on the market (Xuanwei et al., 2007). Produced from different parts of its fruit body, mycelia or spores are accepted as remedies, which can help enhance the body's immune system and improve metabolic functions. In recent times particularly in Western countries, there is an increasing interest to use it as dietary supplements and remedies in conjunction with or in place of allopathic medicine. The beneficial health properties of Ganoderma are contained in a wide variety of bioactive components present in the fruiting body, mycelium, and spores. The mushroom contains polysaccharides and triterpenes as the two major groups, then phenols, steroids, amino acids, lignin, mycins, vitamins, nucleosides, and nucleotides (Mizuno, Wang, Zhang, Kawagishi, & Nishitoba, 1995; Zhu, Chang, Wong, Chong, & Li, 1999; Adam, Odhav, & Bhoola, 2003; Gao, Chan, & Zhou, 2004a,b). Most fractions of identified polysaccharides and triterpenes have more than 100 compounds that are potent immune-modulators, antioxidants and/or chemo-preventive and tumoricidal (Gao, Tang, Gao, Lam, & Zhou, 2004; Min, Gao, Nakamura, & Hattori, 2000; Gao, Min, Ahn, Nakamura, & Lee, 2002). The importance of Ganoderma polysaccharides (Ganopoly) (including protein/peptide bound polysaccharides) in pharmaceuticals has a long history, and has received considerable attention in recent years (Lin, 2005; Chen, Huang, Luo, Luo, & Yang, 2005; Peng, Zhang, Zeng, & Kennedy, 2005; Peng, Zhang, Zeng, Xu, & Kennedy, 2005). The major bioactive polysaccharides isolated from Ganoderma species are glucans, β-1-3 and β-1-6 Dglucans. Ganopoly mainly consists of neutral polysaccharides of

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Fig. 1. LC/MS chromatogram of Ganoderma extract (a) and beer with Ganoderma extract (b).

glucose units; about one third of the polysaccharides consist of (1-3)β-D-glucan containing β-(1-6)-D-glucosyl branches. The groups of Ganoderma triterpenes have received attention because of their well-known pharmacological activities. Since the first isolation of two new triterpenes, ganoderic acids A and B, from the dried epidermis in 1982, more than 130 oxygenated triterpenes have been isolated (Huie & Di, 2004). These triterpenes could be divided into the C30, C27, and C24 compounds according to the number of carbon atoms and based on the structure and the functional groups. In general, triterpenoids have molecular weights ranging from 400 to 600 kDa and their chemical structures are rather complex due to their highly oxidized state (Gao, Yu, Li, & Wang, 2005). Several active proteins have been isolated from Ganoderma as well, while some other proteins exist in the polysaccharide-peptide complex. In early 1989, a novel protein ling zhi-8 (LZ-8) was isolated and its immunological properties have been described. It was shown to have mitogenic and immune-modulating effects on mice. Antifungal and other biologically active proteins have been isolated as well (Wang & Ng, 2006). Reported biological activities and pharmacological functions of Ganoderma include aspects of various effects on the physiological function of human organs and systems such as hepatitis, hypertension, hypercholesterolemia, asthma, allergies and even cancer (Pero, Amiri, Sheng, Welther, & Rich, 2005; Lin, Wang, Liu, & Chen, 2002). A variety of Ganoderma anticancer activities from prevention to treatment have been extensively and systematically reviewed (Gao et al., 2004a,b; Yuen & Gohel, 2005; Yang et al., 2005). Based on the consolidated scientific evidence available so far, it is accepted as a dietary food supplement with considerable potential to enhance the body's immune system with respect to become a new anticancer agent in different clinical therapies.

Beer as a fully natural product contains numerous healthpromoting ingredients. It can be a valuable source of many of the water-soluble vitamins and polyphenols, and is the only foodstuff, which contains hops, a plant with unique ingredients. From the functional point of view, the most important hops constituents are prenylated flavonoids xanthohumol and 8-prenylnaringenin or hopein, with a range of biological properties that may have therapeutic utility. Beer like this can serve as a promising basis for developing a wide variety of functional or dietary beverages and products with specific pharmacodynamic activity. These products can already be found on the market, but most of them are still in the experimental phase. There are “Epic Ales Terra — saurus” and “Shiimake” beers, in Belgium, enriched with Shiitake (Lentinula edodes) edible mushroom native to East Asia. The Shiitake mushroom has been considered a delicacy as well as a medicinal mushroom for a long time. At the Technical University of Munich has been developed “Xanthohumol beer” with a calorie and alcohol content similar to that of standard beer, but with 10 times the normal content of xanthohumol (Wunderlich, Zürcher, & Back, 2005). Medicinal herbs with a long tradition in folk medicine can be an adequate solution for beer enrichment as well (Leskošek-Čukalović, Jelačić, Nedović, Ristić, & Dordević, 2007), but there are a lot of different other possibilities. The aim of our study was to evaluate the possible use of Ganoderma extract for the production of beer with enhanced biologically active ingredients and acceptable sensory properties. 2. Material and methods G. lucidum was isolated from the collection of the Department of Microbiology, Faculty of Agriculture, University of Belgrade. Beer used

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Table 1 LC/MS data of Ganoderma extract and beer with Ganoderma extract. Retention time (min)

Bruto molecular formulae

Molecular mass (−/+) scanning mode

Compound

Ganoderma extract

Beer with Ganoderma extract

0.40 0.42

C6H12O7 C4H6O5

196.0582 (—) 134.0212 (—)

D-gluconic

+ +

+ +

0.54 0.95

C6H8O7 C6H10O5

+ +

+ +

1.84

C11H12N2O2

—

+

3.28

C11H21N3O5

192.0270 162.0525 162.0521 204.0896 204.0823 275.1477

—

+

Ganoderic acid E Ganoderic acid C6 Ganoderic acid G Ganoderic acid A Ganoderic acid C2 Lucidenic acid LM1 Ganoderic acid D

+ + + + + + +

tra tr — — — tr +

Lucidenic acid E

+

—

Ganoderic acid B Ganoderic acid H Absinthin Applanoxidic acid A Ganoderic acid F Ganoderic acid J Ganodermanontriol α-Linoleic acid Octadecenedioic acid (10E,12Z,15Z)-9hydroxy-10,12,15octadecatrienoic acid

+ + + + + + + + + +

+ + — tr. tr. tr. — + + +

(—) (—) (+) (—) (+) (+)

acid 2-Hydroxybutanedioic acid Citric acid 3,6-Anhydro-Lgalactose Tryptophan N-ethyl-L-glutaminylL-threonine

4.57 4.59 4.72 4.82 4.94 5.09 5.22

C30H40O7 C30H42O8 C30H44O8 C30H44O7 C30H46O7 C28H42O6 C30H42O7

5.36

C29H40O8

5.53 5.55 5.88 6.00 6.16 6.18 7.38 7.82 8.05 8.20

C30H44O7 C32H44O9 C30H40O6 C30H40O7 C32H42O9 C30H42O7 C30H48O4 C18H30O2 (+) C18H32O4 (+) C18H30O3

512.2728 530.2873 532.3019 516.3090 518.3242 460.2712 514.2919 514.2930 516.2784 516.2740 516.3073 572.2976 496.2814 512.2768 570.2820 514.2899 472.3552 278.2239 312.2297 294.2200

(+) (—) (—) (—) (—) (+) (—) (+) (—) (+) (—) (—) (—) (—) (—) (—) (+) (+) (−) (−)

294.2189 (+) tr. — in trace

in the experiment was from the commercial Pilsner type of domestic brand name. 2.1. Raw materials Ganoderma extract was produced using the percolation method in a percolator at room temperature (20 °C) according to the patent (Niksic, Leskosek-Cukalovic, & Despotovic, 2008/0120, 2008). Mushroom tissue was cut to pieces and mixed with an alcoholic solution of 70 vol.% of ethanol. Extraction was performed as a daily mixing with magnetic stirrer for 10 min and left to stand in a dry and dark place at room temperature (20 °C) for 21 days. After the extractions, the solution was filtered and concentrated in a vacuum to 1/5 of its initial content. Prepared extracts were added aseptically in concentration 0.1–1.5 ml/l of Ganoderma extract to commercially produce bottled Pilsner beer taking into account the sensory acceptability and values lower than (RDD 1.8–4.8 g) recommended daily doses (FDA, memorandum No 953-03 16, 1999, FDA No 955-0316, 2003). After injection of the Ganoderma extract, the bottles were immediately closed to mature at 5 °C for one day. 2.2. Analysis Ganoderma extract, initial beer, and beers with extracts were analyzed by LC/MS and 1H NMR methods. LC/MS analysis was performed on an Agilent MSD TOF coupled to an Agilent 1200 series HPLC, using Zorbax Eclipse XDB-C18 column (RR, 30 × 2.1 mm i.d., 3.5 μm). Mobile A phase was 0.2% formic acid in water, and mobile phase B was acetonitrile. The injection volume was 5 μl, and elution

was performed at 0.7 ml/min with gradient program (0–1.5 min 5% B, 1.5–10 min 5–95% B, 10–15 min 95% B, 15–16 min. Mass spectra were acquired using an Agilent ESI-MSD TOF. Working parameters were as follows: capillary voltage 4000 V, fragmentor voltage 140 V, nebulizer pressure 45 psig, drying gas 12 L/min, gas temperature 350 °C, mass range (100–1500) m/z, negative and positive ionization mode. Processing of data was done with the software Molecular Feature Extractor. All samples were analyzed by NMR immediately evaporated at reduced pressure at 45 °C. The 1H NMR (200 MHz) spectra were recorded on at 27 °C on a Varian Gemini 2000 spectrometer in DMSO. Chemical shifts are given on the scale relative to TMS as internal standard, operating at 200 MHz for proton spectra. 2.3. Sensory evaluation The consumer's acceptance test was carried out by 105 untrained subjects, 74 of which were male and 31 female, with the consumption frequency of one or more beers per week. Their age profile ranged from 20 to 25 years. The subjects did not have any formal training or experience in the description of beer flavor. The sensory evaluation was performed on the same day in the controlled rooms (temperature, noise, individuality of the tasters) so that unbiased results were obtained. The samples were presented in 50-ml plastic cups with lids, at 5 °C. The beer samples were rated using a 5 point scale. Tested were aroma, taste, body, bitterness, liveliness, and overall impression. The experimental results were analyzed with the statistical package STATISTICA v. 6. The data of the results of the evaluation of sensory properties are presented through basic indicators of descriptive

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Fig. 2. 200 MHz 1H NMR spectra of Ganoderma extract (a) and beer with Ganoderma extract (b).

statistics and graphically through box-plots based on the arithmetic mean, standard deviation and standard error. The statistical analysis was carried out on the correlation analysis, χ2-test, discriminate analysis, Levene's test, t-test for independent and dependent samples and Wilcoxon matched pairs test. 3. Results and discussion The tentative identification of the Ganoderma extract (a) and beer with Ganoderma extract (b) constituents (Fig. 1, Table 1) was performed by LC/MS technique. Exact mass measurements of pseudomolecular ions of analytes performed with a time-of-flight

(TOF) mass spectrometer in negative (−) and positive (+) polarity modes, enabled the determination of molecular formula. All identified compounds exhibited quasi-molecular ion. Molecular formula determinations were performed by Molecular Feature Extractor and Mass Profiler programs. The exact mass measurements were within acceptable limits. The deviations from calculated masses were below 2 ppm in all measurements. 1 Fig. 2 shows the 1D H spectrum of a Ganoderma extract and beer with Ganoderma extract recorded at 200 MHz. In the aliphatic region of the spectra of a Ganoderma extract (0–3 ppm), peaks from terpenic compounds mainly ganodermic acid, amino acids and fatty acids are observable. The mid-field region of both spectra (3–6 ppm) shows the

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Fig. 3. Box-plots for obtained sensory properties and overall impression of beer samples.

contribution of carbohydrates, which normally comprise fermentable sugars and dextrins in beer. In NMR spectra Ganoderma extract peaks in area 4–4.5 ppm contribution of terpenic compounds. The signals in the aromatic region (6–8.5 ppm) show the presence of aromatic amino acids, nucleosides, aromatic alcohols and polyphenolic compounds that give rise to underlying broad humps between 6.7 and 8.7 ppm. From visual inspection of the spectra shown in Fig. 2, it is clear that the spectral profiles of the samples of Ganoderma extract and beer with Ganoderma extract are very similar, although small changes may be noted in all regions in the relative proportion of some peaks. The triterpenes (Ganoderic acids and Lucidenic acids) and fatty acids, biologically active and important for expected pharmacodynamic properties, were the major groups of chemical compounds in Ganoderma extract and the enriched beer. The results of the consumer's acceptance test of given beer samples and the obtained mean grades of tested sensorial parameters are shown in Fig. 3. It is obvious that beer enriched with Ganoderma was well accepted. According to mean grades, it was evaluated similar or better than initial commercially produced Pilsner beer.

More information about experimental results is shown in Table 2. The obtained grades for particular sensory property and beer sample were in the range of 1.0 to 5.0. The lowest given grade was 1.0 given for the bitterness of initial beer. The lowest given grade for remaining sensory parameters was 2.0. The highest grade was 5.0. It is important that the values for all tasted sensory parameters of both beers were homogeneous (Cv b 30%). The most homogeneous grades were obtained for the liveliness of beer with Ganoderma and initial beer (Cv = 17,704% and Cv = 18,162% respectively), as well as for the bitterness of beer with Ganoderma (Cv = 18,879%). The most heterogeneous were the grades for body and bitterness of initial beer (Cv = 24,123% and Cv = 23,542% respectively) and aroma of beer with Ganoderma (Cv = 23,157%). Statistically a very strong correlation was obtained for all tasted parameters for both beers, and data for overall impression are in very strong correlation with obtained grades for tasted sensory parameters. The coefficient of multiple determination indicates that the variation in grades for overall impression can be explained by simultaneous changes in grades of tasted sensory parameters for initial Pilsner beer 89.39%, and beer with Ganoderma 93.59%. The

Table 2 Basic statistical parameters for sensory properties of analyzed beer samples and obtained overall impression. Beer

Sensorial parameters

Mean

Median

Min.

Max.

Standard deviation

Standard error

Coefficient of variation Cv (%)

Initial Pilsner beer

Aroma Taste Body Bitterness Liveliness Overall impression Aroma Taste Body Bitterness Liveliness Overall impression

3.690 3.648 3.424 3.600 3.962 3.729

4.0 4.0 3.5 4.0 4.0 4.0

2.0 2.0 2.0 1.0 2.0 2.0

5.0 5.0 5.0 5.0 5.0 5.0

0.715 0.703 0.826 0.848 0.720 0.717

0.070 0.069 0.081 0.083 0.070 0.070

19.378 19.285 24.123 23.542 18.162 19.239

3.681 3.790 3.800 3.819 4.057 3.895

4.0 4.0 4.0 4.0 4.0 4.0

2.0 2.0 2.0 2.0 2.0 2.0

5.0 5.0 5.0 5.0 5.0 5.0

0.852 0.849 0.745 0.721 0.718 0.736

0.083 0.083 0.073 0.070 0.070 0.072

23.157 22.387 19.618 18.879 17.704 18.897

Beer with Ganoderma

I. Leskosek-Cukalovic et al. / Food Research International 43 (2010) 2262–2269 Table 3 The results of tested differences of mean grades for sensory properties and overall impression of initial beer and beer with Ganoderma. Sensorial properties and overall impression

Levene's test

t-test

F

p

t

p

Z

p

Aroma Taste Body Bitterness Liveliness Overall impression

6.023⁎ 2.616 1.052 4.252⁎ 0.068 0.072

0.015 0.107 0.306 0.040 0.794 0.788

– 1.579 3.872⁎⁎ – 1.177 1.937

– 0.117 0.000 – 0.242 0.055

0.334 – – 1.975⁎ – –

0.739 – – 0.048 – -

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Table 5 The results of analyzed difference of the main grades of sensory properties and overall impression for tested beers given by male testers.

Wilcoxon test

F, t and Z — sample values applied tests; p — level of significance.; ⁎ – statistically significant differences; ⁎⁎ – statistically very significant differences.

Sensorial properties and overall impression

Mean

Levene's test

t-test

I

G

F

p

t

p

Z

Wilcoxon test p

Aroma Taste Body Bitterness Liveliness Overall impression

3.597 3.661 3.387 3.468 3.952 3.726

4.048 4.145 4.065 4.210 4.242 4.210

0.485 0.001 0.139 7.980 0.456 0.255

0.489 0.975 0.711 0.006⁎⁎ 0.502 0.616

− 3.535⁎⁎ − 5.483⁎⁎ − 5.375⁎⁎ − 4.945⁎⁎ − 2.683⁎ − 5.483⁎⁎

0.001 0.000 0.000 0.000 0.012 0.000

– – – 3.711⁎⁎ – –

– – – 0.000 – –

I — initial beer; G — beer with Ganoderma lucidum extract; F, t and Z — sample values applied tests; p — level of significance; ⁎ – statistically significant differences; ⁎⁎ – statistically very significant differences.

lowest partial influence on overall impression has body for initial beer (r = 0.057, p N 0.05), while aroma for beer with Ganoderma (r = 0.249; p b 0.05). The overall impression for initial beer (r = 0.526; p b 001), and beer with Ganoderma (r = 0.549; p b 0.01) depends mostly on taste. The results of discrimination analysis indicate that tested beer samples statistically differ very significantly in aroma, taste, body, bitterness and liveliness, analyzing together (Wilks' Lambda = 0.883, F = 5.409, p b 0.01). Except for aroma, beer with Ganoderma got higher mean grades for all sensory properties and overall impression (Fig. 3). According to t-test or Wilcoxon Matched Pairs test (when variances of samples were not homogeneous) it was statistically very significantly better in body and significantly better in bitterness than initial Pilsner beer (Table 3 and Fig. 3). It is evident that enrichment of beer with given doses of Ganoderma extract does not impair beer sensory properties and that an even more pleasurable impression can be obtained. The body seems to be considerably better, liveliness, taste and overall impression as well. However, bitterness is the most important fact for the further development of such products. G. lucidum extracts and particularly its terpens have a slightly bitter taste and the question was whether it would be acceptable in combination with hop bitterness in beer. Our preliminary results indicate that these two types of bitterness can be combined and that the obtained bitterness is appreciated as similar as or even more pleasant than bitterness of commercially produced initial beer. Among 105 tasters involved in sensory evaluation participation of female tasters (70.48%) were statistically very significantly higher (χ2 = 17.48; p b 0.01) than male participants (29.52%). Their opinion about sensory acceptability of tested beers was somewhat different from male tasters. Female tasters compared to male gave slightly

higher grades for all sensory properties for initial beer except for its taste (Table 4). For all simultaneously tested sensory properties, statistically female and male mean grades do not differ significantly for initial beer (Wilks' Lambda = 0.963 F = 0.754, p = 0.585), but are very significant for beer with Ganoderma (Wilks' Lambda = 0.855, F = 3.350, p = 0.008). Since the results in all samples were homogenous (Cv b 30%) and variances were homogenous (Levene's test) the significance of difference in average grades for sensory properties between the female and male genders was analyzed by t-test. The obtained results indicate that there were no statistically significant differences in female and male average grades for each sensory property itself as well as for the overall impression for initial beer. Regarding beer with Ganoderma, female and male testers gave the same average grades for liveliness, significantly different for body and very significantly different for aroma, taste, bitterness, and overall impression. The results of discriminant analysis indicate that male testers have found that the tested beers differed very significantly in all sensory properties taken together (Wilks'Lambda = 0.769 F = 3.368, p = 0.010). In their opinion, two beers differ significantly in liveliness and very significantly in all other sensory properties (Table 5). The results of discriminant analysis indicate that female testers gave similar results to the male testers, but according to them, tested beers differ very significantly (Wilks' Lambda = 0.871 F = 4.222, p = 0.001). Regarding their mean grades, initial beer and beer with Ganoderma have the same taste and bitterness. Initial beer has better aroma, while beer with Ganoderma has better body, liveliness, and finely gives a better overall impression. However, according to their

Table 4 Basic statistical parameters and the results of analyzed difference of the main grades of beers sensory properties and overall impression by tasters gender (M — male testers; F — female testers). Beer

Initial Pilsner beer

Beer with Ganoderma

Sensorial properties and overall impression

Mean

Std.Dev.

M

F

M

Aroma Taste Body Bitterness Liveliness Overall impression Aroma Taste Body Bitterness Liveliness Overall impression

3.597 3.661 3.387 3.468 3.952 3.726 4.048 4.145 4.065 4.210 4.242 4.210

3.730 3.642 3.440 3.655 3.966 3.730 3.527 3.642 3.689 3.655 3.980 3.764

0.651 0.688 0.750 0.921 0.663 0.693 0.800 0.721 0.727 0.643 0.669 0.616

Coefficient of variation (%)

Levene's test

t-test

F

M

F

F

p

t

p

0.741 0.714 0.860 0.815 0.746 0.732 0.831 0.858 0.730 0.692 0.728 0.746

18.096 18.787 22.130 26.570 16.772 18.608 19.749 17.395 17.895 15.264 15.776 14.635

19.870 19.617 25.015 22.290 18.818 19.621 23.569 23.564 19.775 18.932 18.300 19.818

1.033 0.089 0.462 2.153 0.488 0.274 1.091 1.516 0.015 0.438 0.403 2.011

0.312 0.765 0.498 0.145 0.486 0.602 0.299 0.221 0.901 0.509 0.527 0.159

0.868 0.128 0.296 − 1035 0.094 0.025 2.964⁎⁎ 2.867⁎⁎ 2.407⁎ 3.821⁎⁎ 1.722 2.935⁎⁎

0.387 0.898 0.768 0.303 0.925 0.980 0.004 0.005 0.018 0.000 0.088 0.004

F and t — sample values applied tests; p — level of significance; ⁎ – statistically significant differences; ⁎⁎ – statistically very significant differences.

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Table 6 The results of analyzed difference of the main grades of sensory properties and overall impression for tested beers given by female testers. Sensorial properties and overall impression

Mean

Levene's test

t-test

I

G

F

p

t

p

Aroma Taste Body Bitterness Liveliness Overall impression

3.730 3.642 3.440 3.655 3.966 3.730

3.527 3.642 3.689 3.655 3.980 3.764

1.841 2.019 1.631 1.445 0.669 0.119

0.177 0.157 0.204 0.231 0.415 0.730

1.781 0.000 − 2.000⁎ 0.000 − 0.129 − 0.299

0.079 1.000 0.049 1.000 0.897 0.766

I — initial beer; G — beer with Ganoderma lucidum extract; F and t — sample values applied tests; p — level of significance; ⁎ – statistically significant differences.

opinion, beer with Ganoderma compared with initial beer differs statistically significantly better only by its body (Table 6). Differences in sensory evaluation of tested beers given by male and female tasters are shown in Fig. 4. Results of the consumer's acceptance test are promising, indicating that Ganoderma alcohol extract can be used as raw material for producing a new type of beer. Their well-known pharmacodynamic properties can be usefully used for producing special beer types with enhanced functionality. Our tasters accepted beer enriched with alcohol extract of Ganoderma in a somewhat different way but generally are very good. Male and female tasters evaluated beer with Ganoderma similarly or even better than initial beer in most important sensory parameters, that define sensorial acceptance of beer. It was somewhat surprising that our male tasters showed greater affinity for new sensory properties than the female tasters did. The female tasters evaluated both beers quite similarly and found no significant differences in quality in comparison to the control initial beer, while

mail tasters estimated the beer with Ganoderma as considerably better. A fact that should be highlighted is the superior bitterness mail tasters detected in beer with Ganoderma in comparison to initial control beer. The quality of the bitterness was placed as somewhat higher and described as more pleasant. It was observed that the addition of Ganoderma influenced the bitterness in beer positively (more harmonic, finer). From the practical point of view, such reaction of the tasters is very important. However, the bitterness in beer is a very complex issue and depends of the psychophysical perception and taste interactions on one side and individual sensory perceptions on the other side. The bitterness evaluation of a beer is influenced by several factors including the fullness/body, degree of sweetness, the content, and type of phenols, content of ethanol, and the temperature of beer tasting. An increase in the degree of fullness can mask the bitterness of a beverage. On the other hand, ethanol increases the intensity of the bitter taste, as well as the duration of the bitter sensation. In our case the ethanol content was the same, but it was noticed that the contribution of the Ganoderma influenced the mouthfeel. Sensory test indicates that perhaps even higher doses of extract can be used to produce beer with a higher content of functionally active Ganoderma terpene. It is worth noting that the sensory properties of such products can be successfully improved in different ways. Medical herbs, which possess biologically active components are just one of the solutions. Another feature that should be highlighted is the fact that sensory evaluation was performed by untrained subjects as the consumer's acceptance test and not by a trained panel. So the results can give just an indication of consumers readiness to accept product like this, regarding their affinity towards specific sensory properties and expected promote functionality. Further development of such product will require the tasting panel, sensory adjustment, and standardization of compounds with healthpromoting properties.

Fig. 4. The principal statistics parameters of analyzed sensorial properties and overall for tested beer samples given by male and female tasters.

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