Solid substrate growth of white rot fungi on coffee pulp

The presence of several antiphysiological factors limit the use of coffee pulp in monogastric and ruminant feeding. Twenty six white rot fungi were grown under solid substrate conditions in previously ensiled and pressed coffee pulp without adding additional sources of nitrogen. All grew and wholly covered the surface of the substrate. Six of them produced fruiting bodies. The weight loss interval ranged between 6.7–28.0% dry matter before fructification and from 17.0 to 48.7% after fructification. Some fungi biodegraded about 70, 55 and 47% of the total polyphenols, caffeine and permanganate lignin present in the original substrate.     

Biological treatments affect the chemical composition of coffee pulp

Biological treatments were applied to fresh coffee pulp (CoP) to improve its nutritive value for monogastric animals by reducing its content of cellulose and antinutritional factors (ANFs) such as total phenols, tannins and caffeine. Treatments were: (1) ensiling with 0, 50 and 100 gkg(-1) molasses for 2 and 3 months, (2) aerobic decomposition for 0, 7, 14, 21, 28, 35 and 42 days, (3) aerobic bacterial inoculation (Bacillus sp.) for 0, 7, 14, 21 and 28 days. Ensiled CoP (E-CoP) showed higher fat and ash contents than oven-dried-CoP (OD-CoP; P<0.05). Similarly, true protein values tended to increase. The cellulose and total phenols levels of E-CoP were lower than OD-CoP (P<0.05). The E-CoP tannins levels tended to be lower than OD-CoP whereas caffeine levels remained unaffected. Improvement in the nutritional quality of E-CoP was associated with higher fat and protein contents and reduction of cellulose, total phenols and tannins. The aerobic decomposition treatment improved the nutritional quality of CoP by increasing true protein and fat contents. In addition, total phenols, tannins, caffeine and cellulose contents were reduced by an increase in treatment time (P<0.05). Bacterial treatment increased the protein content of CoP after 21 days (from 137 to 392 gkg(-1)) and decreased it after 28 days. Cellulose, total phenols, tannins and caffeine contents reduced with an increase in time of bacterial degradation. Bacterial treatment improved the CoP quality by increasing protein content and reducing cellulose and ANFs, especially after 21 days of treatment. Both the aerobic decomposition (after 21-28 days) and the aerobic bacterial degradation of CoP (after 21 days) appeared more suitable to improve the nutritional quality of CoP than the ensiling.     

Comparative culturing of Pleurotus spp. on coffee pulp and wheat straw: biomass production and substrate biodegradation

The results of the cultivation of six strains of Pleurotus (P. djamor (2), P. ostreatus (2) and P. pulmonarius (2)) on coffee pulp and wheat straw are presented. Metabolic activity associated with biomass of each strain was determined, as well as changes in lignin and polysaccharides (cellulose and hemicellulose), phenolic and caffeine contents in substrate samples colonized for a period of up to 36 days. Analysis were made of changes during the mycelium incubation period (16 days) and throughout different stages of fructification. Greater metabolic activity was observed in the wheat straw samples, with a significant increase between 4 and 12 days of incubation. The degradation of polysaccharide compounds was associated with the fruiting stage, while the reduction in phenolic contents was detected in both substrates samples during the first eight days of incubation. A decrease was observed in caffeine content of the coffee pulp samples during fruiting stage, which could mean that some caffeine accumulates in the fruiting bodies.     

Biotechnological management of coffee pulp — isolation,screening, characterization,selection of caffeine-degrading fungi and natural microflora present in coffee pulp and husk

A total of 272 strains of filamentous fungi were isolated from soil, leaves of coffee plants and coffee cherries collected in coffee-growing areas of Mexico on three semi-synthetic culture media containing coffee extract, coffee extract with sucrose and coffee pulp extract. The isolated strains were purified by conventional techniques and identified by microscopic examination. Strains were selected on the basis of their caffeine-degrading ability in well-defined liquid medium containing caffeine. Most of the isolated microorganisms belong to Aspergillus, Penicillium, Trichoderma, Fusarium, and Humicola genera. Five strains belonging to Aspergillus species and two strains belonging to Penicillium species had the ability to degrade almost 100% of the caffeine in liquid medium. A comparative study on the evaluation of natural microflora present in coffee pulp and coffee husk revealed the presence of a wide variety of microorganisms. The percentage distribution of fungi, bacteria and yeast was almost similar in all the samples, except in coffee husk where the fungal population was slightly higher than in the other two samples. The yeast population was predominant when the coffee pulp was lyophilized immediately after pulping. However, there was a wide diversity in the microbial population with respect to selective media containing functional nutritional groups like cellulose, starch and pectin.     

Biotechnological potential of coffee pulp and coffee husk for bioprocesses

Advances in industrial biotechnology offer potential opportunities for economic utilization of agro-industrial residues such as coffee pulp and coffee husk. Coffee pulp or husk is a fibrous mucilagenous material (sub-product) obtained during the processing of coffee cherries by wet or dry process, respectively. Coffee pulp/husk contains some amount of caffeine and tannins, which makes it toxic in nature, resulting the disposal problem. However, it is rich in organic nature, which makes it an ideal substrate for microbial processes for the production of value-added products. Several solutions and alternative uses of the coffee pulp and husk have been attempted. These include as fertilizers, livestock feed, compost, etc. However, these applications utilize only a fraction of available quantity and are not technically very efficient. Attempts have been made to detoxify it for improved application as feed, and to produce several products such as enzymes, organic acids, flavour and aroma compounds, and mushrooms, etc. from coffee pulp/husk. Solid state fermentation has been mostly employed for bioconversion processes. Factorial design experiments offer useful information for the process optimization. This paper reviews the developments on processes and products developed for the value-addition of coffee pulp/husk through the biotechnological means.     

Temperate forages ensiled with molasses or fresh cheese whey: Effects on conservation quality, effluent losses and ruminal degradation

Effects of ensiling and adding molasses or increasing levels of fresh cheese whey on the conservation and rumen degradability of temperate pasture silages were evaluated. Forage from three paddocks of mixed grass and legume pastures was used to make 45 silages (15 silages per paddock) with 5 treatments, including silage without additives (control), silage with 15 g/kg dehydrated molasses and silage with 20, 50 and 100 g/kg fresh cheese whey. The chemical composition and fermentation quality (i.e., pH, ammonia N, loss of dry matter (DM) and neutral detergent fibre (NDF), effluent production) of the silages were determined. Fresh and ensiled materials were evaluated for in situ rumen degradability. Ensiling reduced DM and NDF rumen degradability (P<0.01). When additives were employed, the reduction of DM degradability of the silages decreased (P≤0.03). Addition of molasses led to the lowest pH (P<0.01) and DM losses (P<0.01), and highest DM degradability (P<0.01). The conservation and DM degradation results of dried molasses silage was superior to those of fresh whey silages. In general, an increase in the level of whey increased DM degradability (P≤0.03), but linearly increased effluent production (P<0.01) and losses (P<0.01).     

Scavenging effect of Indian grape polyphenols on 2,2'-diphenyl-1-picrylhydrazyl(DPPH) radical by electron spin resonance spectrometry

Antioxidant potency of Indian grape cultivars varying in their skin color, seed and polyphenol content (Bangalore blue, Pandhari sahebi, Sharad seedless and Thompson seedless) and their components (whole grapes, pulp with skin and seeds) was examined as 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity using electron spin resonance spectrometry. The total polyphenols in Indian grapes ranged between 3-51%. Extracted polyphenols caused a concentration dependent and significant loss in DPPH radical signal, similar to known antioxidants-Vitamin C, catechin and procyanidin B3 used as references. Among seedless cultivars, polyphenols from Sharad was more potent as antioxidant than Thompson, showing IC50 values of 1250 +/- 30 and 2650 +/- 125 microg/ml, respectively. The inhibitory effect of polyphenols from seedless grape cultivars was as effective as that of seeded variety. The results indicate that polyphenols extracted from Indian grapes/ components (with /without seeds) exhibited free radical scavenging activity and their chemopreventive properties need to be exploited by in vivo model system.     

Effects of pressed beet pulp silage inclusion in maize-based rations on performance of high-yielding dairy cows and parameters of rumen fermentation

Beet pulp contains high amounts of pectins that can reduce the risk of rumen disorders compared to using feedstuffs high in starch. The objective was to study the effects of inclusion of ensiled pressed beet pulp in total mixed rations (TMR) for high-yielding dairy cows. Two TMR containing no or about 20% (on dry matter (DM) basis) beet pulp silage were used. The beet pulp silage mainly replaced maize silage and corn cob silage. The TMR were intentionally equal in the concentrations of energy and utilisable crude protein (CP) at the duodenum. TMR were fed to 39 and 40 dairy cows, respectively, for 118 days. The average daily milk yield was about 43 kg/day. No significant differences in milk yield and milk fat or milk protein content were detected. DM intake of cows was significantly reduced by the inclusion of beet pulp silage (23.0 v. 24.5 kg/day). However, a digestibility study, separately conducted with sheep, showed a significantly higher organic matter digestibility and metabolisable energy concentration for the TMR that contained beet pulp silage. In vitro gas production kinetics indicated that the intensity of fermentation was lower in the TMR that contained beet pulp silage. In vitro production of short-chain fatty acids, studied using a Rusitec, did not differ between the TMR. However, the inclusion of beet pulp silage in the ration caused a significant reduction in the efficiency of microbial CP synthesis in vitro. The amino acid profile of microbial protein remained unchanged. It was concluded that beet pulp silage has specific effects on ruminal fermentation that may depress feed intake of cows but improve digestibility. An inclusion of beet pulp silage of up to 20% of DM in rations for high-yielding dairy cows is possible without significant effects on milk yield and milk protein or milk fat.     

Effect of wilting and the additives,straw, molasses and urea on the fermentation pattern of maize silage

The process of ensiling was studied in fresh maize (15% dry matter (DM)), wilted maize (18 and 24% DM) and maize mixed with 5–20% of wheat straw (18, 25 and 29% DM). Silages with 24% DM were preserved better than those with lower dry matter content. There was a significant change, with time, in pH, titrable acidity, volatile fatty acids, lactic acid, number of lactic acid bacteria, volatile nitrogen and soluble sugars in all the treatments. There was a significant decline in volatile fatty acids (P<0.05) and ammonia (P<0.01) production, and a significant increase in soluble sugar (P<0.01) in silages made after wilting. A significant decline in titrable acidity (P<0.01), volatile fatty acid production (p<0.05) and ammonia nitrogen (P<0.01), and a significant increase in pH (P<0.01) were found in silages of maize mixed with wheat straw. The overall rate of fermentation decreased during the first few days of fermentation in wilted and wheat straw silages, but the final products had characteristics of a good silage. In the second experiment the effect of urea and molasses was studied on wheat straw plus maize (15:85) silage with an initial DM content of 31–34%. Three levels of molasses (0, 3 and 6% of fresh weight) and two levels of urea (0 and 0.5% of fresh weight) were studied. Urea treatment with 3% molasses was found to be the best on the basis of silage characteristics.     

Biogenic amine production in grass, maize and total mixed ration silages inoculated with Lactobacillus casei or Lactobacillus buchneri

AIMS: To investigate the effects of inoculating Lactobacillus casei or Lacobacillus buchneri on the production of biogenic amines (BA) in silage. METHODS AND RESULTS: Wilted festulolium (Lolium perenne x Festuca pratensis), whole crop maize or a total mixed ration, consisting of wet brewer grains, lucerne hay, cracked maize, sugarbeet pulp, soyabean meal and molasses, was ensiled with or without the inoculation of either L. casei (>10(6) CFU g(-1)) or L. buchneri (>10(6) CFU g(-1)). Silages were opened after 60 days of storage, and the concentrations of histamine, tyramine, putrescine and cadaverine were determined. The inoculation of L. casei decreased all the BA regardless of the silage type. The effects of L. buchneri varied between the three silages; the tyramine and putrescine were increased in maize but were lowered in festulolium. Histamine was reduced in festulolium and the by-products, whereas no change was found in the maize silage. None of the inoculant strains produced the four BA in a synthetic medium, accounting for the actual ensiling except for tyramine and putrescine in maize. CONCLUSIONS: Wide variation would be found in the production of BA owing to the ensiling materials. The inoculation of L. casei can lower the BA concentration, while the effects of L. buchneri may vary considerably. The screening of BA-producing activity may help to reduce the risk of BA contamination in inoculated silage. SIGNIFICANCE AND IMPACT OF THE STUDY: Strains of decarboxylase-negative L. buchneri can enhance the aerobic stability of silage without a concern regarding the large production of putrefactive BA.     

Studies on fermentation of sorghum silage during storage — Effect of urea and molasses

Eighteen silages involving various treatments were prepared from Jowar (Sorghum vulgare) at the dough stage in 500 ml flasks. The treatment combinations were urea at 0% (U₀), 0.5% (U₁) and 1% (U₂), and molasses at 0% (M₀) and 1% (M₁). The flasks were opened for analysis at 30- (I₁), 60- (I₂) and 120- (I₃) day intervals. Urea levels increased the pH values irrespective of the presence of molasses. However, with increasing urea levels, the lactic acid increased in the absence of molasses while butyric acid increased in its presence. Fermentation quality was better for the silage prepared without additives and with molasses alone but was very poor when urea was added with molasses. Quality of silage with M₀U₀, M₀U₁ and M₁U₀ was maintained to the end of the experiment, whereas M₀U₂ silage deteriorated during the later part of the storage period.     

Packed bed column fermenter and kinetic modeling for upgrading the nutritional quality of coffee husk in solid-state fermentation.

Studies were carried out to evaluate solid-state fermentation (SSF) for the upgradation of the nutritional quality of coffee husk by degrading the caffeine and tannins present in it. SSF was carried out by Aspergillus niger LPBx in a glass column fermenter using factorial design experiments and surface response methodology to optimize bioprocess parameters such as the substrate pH and moisture content and aeration rate. The first factorial design showed that the moisture content of the substrate and aeration rate were significant factors for the degradation of toxic compounds, which was confirmed by the second factorial design too. The kinetic study showed that the degradation of toxic compounds was related to the development of the mold and its respiration and also to the consumption of the reducing sugars present in coffee husk. From the values obtained experimentally for the oxygen uptake rate and CO(2) evolved, the system determined a biomass yield (Y(x/o)) of 3.811 (g of biomass).(g of consumed O(2))(-1) and a maintenance coefficient (m) of 0.0031 (g of consumed O(2)).(g biomass of biomass)(-1).h(-1). The best results on the degradation of caffeine (90%) and tannins (57%) were achieved when SSF was carried out with a 30 mL.min(-1) aeration rate using coffee husk having a 55% initial moisture content. The inoculation rate did not affect the metabolization of the toxic compounds by the fungal culture. After SSF, the protein content of the husk was increased to 10.6%, which was more than double that of the unfermented husk (5.2%).     

Coffee, caffeine, and coronary heart disease

PURPOSE OF REVIEW: This review summarizes and highlights recent advances in current knowledge of the relationship between coffee and caffeine consumption and risk of coronary heart disease. Potential mechanisms and genetic modifiers of this relationship are also discussed. RECENT FINDINGS: Studies examining the association between coffee consumption and coronary heart disease have been inconclusive. Coffee is a complex mixture of compounds that may have either beneficial or harmful effects on the cardiovascular system. Randomized controlled trials have confirmed the cholesterol-raising effect of diterpenes present in boiled coffee, which may contribute to the risk of coronary heart disease associated with unfiltered coffee consumption. A recent study examining the relationship between coffee and risk of myocardial infarction incorporated a genetic polymorphism associated with a slower rate of caffeine metabolism and provides strong evidence that caffeine also affects risk of coronary heart disease. Several studies have reported a protective effect of moderate coffee consumption, which suggests that coffee contains other compounds that may be beneficial. SUMMARY: Diterpenes present in unfiltered coffee and caffeine each appear to increase risk of coronary heart disease. A lower risk of coronary heart disease among moderate coffee drinkers might be due to antioxidants found in coffee.     

Caffeine restriction: effect on mild hypertension.

OBJECTIVE--To determine the effects on blood pressure of modifying dietary caffeine intake in patients with mild and borderline hypertension by monitoring ambulatory and clinic blood pressure. DESIGN--Four way, randomised, crossover trial of four consecutive two week dietary regimens: normal diet, caffeine free diet alone, caffeine free diet with decaffeinated instant coffee, caffeine free diet with caffeinated instant coffee (instant coffee phases conducted double blind). SETTING--Hospital hypertension clinic, Scotland. PATIENTS--52 patients (23 men; aged 26-67 years) with untreated borderline or mild hypertension (diastolic blood pressure 90-105 mm Hg) who normally drank a minimum of three cups of coffee daily. MAIN OUTCOME MEASURES--Mean ambulatory blood pressure over 24 hours; mean morning, daytime, and night time ambulatory blood pressure; sitting clinic blood pressure at 1700; plasma caffeine concentration at 1700 on the last day of each regimen. RESULTS--Mean 24 hour ambulatory blood pressure was not different between regimens. There was no difference in blood pressure variability between regimens. During the caffeine free diet alone morning ambulatory diastolic blood pressure was higher (2.8 mm Hg) than during the caffeine free diet with caffeinated coffee. Mean sitting clinic systolic blood pressure was higher at 1700 (4.7 mm Hg) with a caffeine free diet than with the caffeine free diet with caffeinated coffee (p less than 0.05). Dietary compliance as assessed by plasma caffeine concentration was excellent. There was no significant correlation between plasma caffeine concentration and blood pressure. CONCLUSIONS--Drinking caffeinated instant coffee over a two week period does not adversely influence blood pressure in patients with borderline or mild hypertension; abstinence is of no benefit.     

Allantoin has a limited role as nitrogen source in cultured coffee cells

In plants the ureides allantoin (ALN) and allantoic acid (ALA) are formed in purine metabolism, and in some legumes both compounds play an important role as nitrogen (N) sources. In coffee plants, ALN and ALA are catabolites of caffeine degradation. Caffeine is found throughout the coffee plant and in some parts this alkaloid can accumulate up to 4% dry basis. Therefore, caffeine degradation via ureides may make an important contribution to N metabolism of the plant. Using coffee cell suspension as a model we investigated the contribution of ALN as a source of N in coffee. ALN was incorporated in the liquid medium and after 20 d of cultivation, cell mass, NO(3), NH(4), amino acids, soluble proteins, ALN and caffeine were determined in the cells. The activity of glutamine synthetase was also studied. The results showed that despite being taken up by cells ALN does not contribute significantly as a source of N in coffee cells. Compared with mineral N sources, cells grown with ALN-N accumulated much less mass. The inclusion of ALN in the medium caused significant alterations in the content of some N compounds indicating a stress condition.     

The effects of barley, unmolassed sugar-beet pulp and molasses supplements on organic matter, nitrogen and fibre digestion in the rumen of cattle given a silage diet

In a 4 × 4 Latin-square experiment with a 2 × 2 factorial arrangement of treatments, 4 cattle fitted with a rumen and duodenal cannula were given four grass-containing diets [480 g kg⁻¹ of the total dry matter (DM) intake] and barley (BU), barley + molasses (2:1) (BM), sugar-beet pulp (SU) or sugar-beet pulp + molasses (SM). Duodenal flow was estimated using Cr-mordanted straw and CoEDTA as markers, and microbial nitrogen entering the small intestine using purine bases of nucleic acids.

Molasses-containing diets had a higher (P < 0.01) organic matter (OM) digestibility. The proportion of digestible OM apparently disappearing in the rumen averaged 0.72 and was not significantly affected by the diet. When cattle received molasses, the quantity of microbial N entering the small intestine was higher (P < 0.05) and there was a trend towards a higher efficiency of microbial N synthesis (28.8 vs. 25.6 g N kg⁻¹ OM apparently digested in the rumen). When S diets were consumed, total non-ammonia N flow at the duodenum exceeded N intake by 7.0 g day⁻¹ and when B diets were consumed, it was 0.7 g day⁻¹ less than N intake. Feed N degradability in the rumen and apparent N digestibility of S diets were lower (P < 0.05; P < 0.001) than those of B diets.

Rumen (P < 0.05) and total (P < 0.001) digestibility of neutral detergent fibre (NDF) and acid detergent fibre (ADF) was higher when S diets were given. The proportion of digestible fibre disappearing in the rumen was not affected by the diet. The rate and extent of silage and hay DM degradation were not significantly affected by the diet. However, dietary inclusion of molasses decreased (P < 0.05) the lag time of both hay and silage DM degradation.

The rumen dilution rate of liquid averaged 0.097 and that of particles, 0.049; neither was significantly different for either B and S diets or U and M diets. Duodenal liquid flow was higher (P < 0.05) for M diets.

Average rumen pH was not affected by the diet, but the molasses diets increased (P < 0.05) the range in rumen pH. The BM diet was associated with higher (P < 0.01) rumen ammonia concentration than the other diets. Low rumen ammonia concentrations (< 2 mM) were observed for long periods between feeds. The molar proportion of butyrate was higher on B diets and there was a trend towards a higher proportion of acetate and propionate on S diets. Molasses tended to increase the molar proportion of propionate and butyrate.     

Robusta coffee beans post-harvest microflora: Lactobacillus plantarum sp. as potential antagonist of Aspergillus carbonarius

Coffee contamination by ochratoxigenic fungi affects both coffee quality as well as coffee price with harmful consequences on the economy of the coffee exporting countries for whom which is their main source of income. Fungal strains were isolated from coffee beans and identified as black Aspergilli. Ochratoxigenic moulds like Aspergillus carbonarius were screened and selected for detailed studies. Also lactic acid bacteria (LAB) were isolated from silage coffee pulp and their antifungal activity was tested on dual-culture agar plate. Ten of the isolated LAB demonstrated antifungal effect against A. carbonarius. API 50 CH and APIZYM were used to perform phenotypic identification. 16S rDNA sequencing was made to confirm the results.     

Effect of combined ensiling of sorghum and soybean with or without molasses and lactobacilli on silage quality and in vitro rumen fermentation

Two sorghum (Sorghum bicolor (L.) Moench) varieties (SG₁ & SG₂), with the former showing higher grain and total DM yield, but also increased tannin contents compared to the latter, and one soybean (Glycine max (L.) Merr.) variety (INCASOY-35) were sown, harvested (at pasty grain state), chopped and ensiled (CIAP, Cuba). Silages were made in following combinations: either SG₁ or SG₂ combined with soybean in two proportions (0.4 and 0.6). All silages were prepared with or without molasses (3.5% of fresh material) and Lactobacillus sp. as inoculant (3 × 10⁵ colony forming units/g). Silage quality parameters included pH, ammonia, lactate, acetate, butyrate and water soluble carbohydrates content. Further, both fresh and ensiled materials were incubated in vitro with buffered rumen fluid to study the fermentation characteristics. Silage of a good quality could be produced with both sorghum varieties alone, but combined silages showed improved quality compared to soybean silage (p<0.05 for all quality characteristics). Addition of molasses and bacterial inoculant further improved silage quality (p<0.05 for all quality characteristics). In vitro incubation (24 h) of ensiled material resulted in lower acetate and higher propionate proportion compared to fresh forages. However, ensiling without molasses and inoculant reduced in vitro short chain fatty acid production and hence the apparent rumen degradability of organic mater as well as the fermentation rate. As expected, a higher proportion of sorghum increased the molar propionate proportion and the fractional fermentation rate, whereas ammonia (mmol/L) concentrations were reduced. SG₁ silages produced higher molar propionate proportions, lower acetate proportions and ammonia concentrations.     

Reversed-phase liquid chromatographic investigation of UV-absorbing low-molecular-weight compounds in saliva

The reversed-phase mode of high-performance liquid chromatography was used to determine the intra- and inter-individual levels of UV-absorbing low-molecular-weight compounds in saliva. Many of the compounds known to occur in serum were also found in saliva; however, concentrations in saliva are lower. Both the intra- and inter-individual levels of these compounds vary significantly; in most cases, the inter-individual variance is 2–3 times the intra-individual variance.

Caffeine and its metabolites in saliva are also reported. A greater number of metabolites were found in the saliva of habitual coffee drinkers. After caffeine was administered orally, paraxanthine, theobromine, theophylline, 1-methylxanthine, and 1-methyluric acid were found in the saliva of an individual who did not drink coffee regularly. In this subject, the serum half-life for caffeine was 3.49 h and the saliva half-life was 3.27 h. The half-life of caffeine in an habitual coffee drinker who had refrained from caffeine products for four days was 4.39 h.     

Incorporation of caffeine into the macromicete fungus Pleurotus sajor-caju growing on coffee pulp

TWhen the chemical composition of secondary metabolites from the Pleurotus sajor-caju growing on coffee pulp were study, it was found that the fungus has the faculty of incorporating caffeine inside its fructiferous body. Component of the substrate (around 1.3% on dry basis) did not show a structural change over the alkaloid; this constitutes an unexpected outcome for a species belonging to realm of the fungi.