International Journal of Food Science and Technology 2018 1

Original article
Use of coffee by-products for the cultivation of Pleurotus
citrinopileatus and Pleurotus salmoneo-stramineus and its impact
on biological properties of extracts thereof

Ana C. Freitas,1* Mariana B. Antunes,1,2 Dina Rodrigues,1 S
ergio Sousa,1 Manuela Amorim,1
Maria F. Barroso, Ana Carvalho,3 Sandra M. Ferrador2 & Ana M. Gomes1
3

1 CBQF - Centro de Biotecnologia e Qu
ımica Fina – Laborat
orio Associado, Escola Superior de Biotecnologia, Universidade Cat

olica
Portuguesa, Rua Arquiteto Lob~ao Vital, 172, 4200-374 Porto, Portugal


2 Bioinvitro, Biotecnologia Lda, Rua Eng.° Jos
e Rodrigo Carvalho, 95, 4480-484, Arvore, Vila do Conde, Portugal
3 REQUIMTE/LAQV, Instituto Superior de Engenharia do Porto, Instituto Polit
ecnico do Porto, Rua Dr. Ant
onio Bernardino de Almeida,
431, 4200-072 Porto, Portugal

(Received 14 December 2017; Accepted in revised form 12 March 2018)

Summary Incorporating spent coffee grounds (SCGs), a by-product from coffee brewing, in growth substrate of ben-
eficial edible mushrooms is an approach that has to be further studied due to its potential positive out-
comes: environmental impact mitigation, production costs reduction and beneficial impact on consumer
health. Hence, cultivation of Pleurotus citrinopileatus and Pleurotus salmoneo-stramineus was tested using
SCG which enabled maximum production yield of P. citrinopileatus which was of 25.1% (w/w). Variable
antidiabetic potential was observed between aqueous and enzymatic extracts (3.8%–29% inhibition)
regardless species and substrates, whereas aqueous extract of P. citrinopileatus grown in substrate without
SCG stood out presenting the highest antioxidant activity and inhibition activity of angiotensin I-convert-
ing enzyme (IC50 = 123 lg mL
1). Ethanolic and aqueous extracts of both Pleurotus species grown in the
presence or absence of SGC proved to be an interesting prebiotic source for growth of Bifidobacterium
animalis Bo in comparison with fructooligosaccharides (FOS).
Keywords Antioxidant and prebiotic, caffeine, extraction, mushrooms, Pleurotus spp., spent coffee grounds, yield.

commercial and biological potential and were selected

Introduction
as target species in this study. Saprophyte mushrooms
Mushrooms are not only added-value foods because of as Pleurotus spp. are known to be easily cultivable in a
their sensorial and nutritional characteristics but also diversity of organic substrates including different cereal
due to their biological properties (Carrasco-Gonz
alez straws or other agricultural by-products (Petre et al.,
et al., 2017). They are a good source of protein, fibre, 2016). Coffee by-products, in particular, spent coffee
vitamins and minerals and of bioactive compounds grounds (SCGs) which consist of residues of used cof-
such as phenolic compounds, polysaccharides, terpenes fee grounds resulting from soluble coffee production,
and steroids (Moon & Lo, 2013), which have been have also begun to be tested as an important alterna-
used as a medicinal or nutraceutical application tive. This by-product is important because almost 50%
(Sabaratnam et al., 2011). of the world coffee production is processed for soluble
Solid-state fermentation as bioconversion processes coffee (Ramalakshmi et al., 2009; Esquivel & Jim
enez,
where food protein is recovered from lignocellulosic 2012) resulting in over ten million tons of solid resi-
materials has been used successfully for the cultivation dues generated yearly from coffee agro-industry world-
of mushrooms (Philippoussis, 2009) namely several wide (Echeverria & Nuti, 2017). Spent coffee grounds
species of Pleurotus genus such as P. ostreatus, possess high content of water (80%–85%), organic
P. sajor caju or P. eryngii. Other less explored species load with 8.6% cellulose, 36.7% hemicellulose and
such as P. citrinopileatus (golden oyster) and P. salmo- 13.6% of protein being an acidic residue (Mussato
neo-stramineus (pink oyster) also have tremendous et al., 2011a).
Added-value uses of SCGs are environmentally
*Correspondent: E-mail: afreitas@porto.ucp.pt sound and fundamental for circular economy; disposal

doi:10.1111/ijfs.13778
© 2018 Institute of Food Science and Technology
2 Coffee by-products for mushrooms cultivation A. C. Freitas et al.

or utilisation of SCG has included sewer discharge, coffee shop and sterilised at 121 °C through 120 min.
sanitary land fill, incineration or cattle feed (Ramalak- Once cooled, it was mixed, under aseptic conditions,
shmi et al., 2009). However, the chemical composition with sterilised substrate I. Filter bags containing
of SCGs makes this by-product a substrate of high 3.5 Kg of substrate were inoculated with 10%(w/w)
biotechnological value able to be used as a substrate spawn from one of the two precultured Pleurotus
for cultivation (Mussato et al., 2011a). Some studies strains. Four replicates were used for each production
have been published on culturing of Pleurotus spp. making a total of sixteen bags. Inoculated bags were
using this agro-industrial by-product (Mart
ınez-Car- incubated in darkness at 22–24 °C until mycelia had
rera et al., 1985; Thielke, 1989; Vel
azquez-Cede~
no completely covered the substrate and appearance of
et al., 2002; Mata et al., 2005; Salmones et al., 2005). primordia which took 20–24 days for substrate I and
Moreover, research is warranted to perceive its impact 25–30 days for substrate II. Mushroom fructification
on growth and bioactives composition of different occurred after 5–10 days at 18–20 °C under photoperi-
Pleurotus spp. which composition is easily affected by ods of 12 h depending of the substrate and cultured
the growth substrate and conditions. Furthermore, to Pleurotus strain. After fructification and mushroom
test for such bioactivity, extracts are normally pre- growth to an average size of 5 cm, entire clean mush-
pared. Water-based extraction is not only food com- rooms from each producing bag were cut off and dried
patible, nonexpensive and environment-friendly but in a ventilated drier over 24 h between 40 and 60 °C
also could have lower selectivity and extraction effi- and subsequently milled to less than 1.0 mm using a
ciency (Herrero et al., 2006). Enzyme-assisted extrac- grinder. Production yield, resulting from the mixture
tion (EAE) has potential because hydrolytic action of three fructifications, was obtained by determining
enzymes on natural-derived products could weaken or the ratio of mushroom wet weight in comparison with
disrupt cell wall structure as well as breakdown com- original substrate wet weight. The content of caffeine
plex interior storage compounds releasing compounds in the organic substrates and in the mushrooms was
such as polysaccharides, peptides or amino acids determined according to ISO 20481:2008.
(Wang et al., 2010). Based on the above rationale, this
study endeavoured to pursue several objectives: (i) to
Proximate composition of mushrooms and extraction
cultivate P. citrinopileatus and P. salmoneo-stramineus,
testing the effect of incorporating SCG in commercial Moisture and organic matter were determined accord-
organic substrate; (ii) to evaluate the impact of differ- ing to AOAC methods (1990). Protein content was
ent extraction modes on the yield and biological determined by the Kjeldahl method adapted from US
potential of the extracts. To our best knowledge, this ISO 5983-1 using 4.38 as converting factor to protein
is the first four-factor study combining the effects of (Kulshreshtha et al., 2013). Total fat was determined
species-substrate-extraction-bioactivity, where different by gravimetric Soxhlet method using petroleum ether
extraction modes are applied to the selected Pleurotus for extraction, whereas total sugar content was deter-
species grown on different substrates reporting their mined indirectly by subtracting protein and fat content
consequent biological potential (antioxidant, antidia- from total organic content.
betic, antihypertensive and prebiotic activities). Seven replicated hot water extraction (HWE) and
enzymatic-assisted extraction (EAE) with Alcalase
(Sigma-Aldrich, USA) an extracellular alkaline pro-
Material and methods
tease (Subtilisin, EC 3.4.21.62) were performed accord-
ing to Rodrigues et al. (2017).
Strains and culture conditions
Three replicated ethanolic extractions (EtOH_Ext)
Strains of Pleurotus citrinopileatus (synonym of Pleuro- were performed with 5 g of dried and milled mush-
tus cornucopiae var. citrinopileatus; M2502) and Pleuro- room dispersed in 45 mL of ethanol (70% v/v; Pan-
tus salmoneo-stramineus (synonym of P. ostreatoroseus reac, Spain) and incubated in an agitated water bath
or P. djamor; PD2708) were obtained from Mycelia at 25 °C for 14 h. The ethanolic solution was then
(Nevele, Belgium). centrifuged at 5000 g for 10 min at 4 °C (centrifuge
Cultivation was performed using sterile filter bags Medifriger BL-S; JP Selecta, Spain), and the super-
with two different sterilised organic substrates. Sub- natant brought almost to dryness with a rotary evapo-
strate I, consisted in 38%(w/w) sawdust, 5%(w/w) rator (B€uchi, R-210, Switzerland). The residue was
wood shavings, 20%(w/w) wood straw, 18%(w/w) redissolved in 5.0 mL deionised water, and the extract
crushed grain corn, 13%(w/w) wheat bran, 6%(w/w) frozen at
80 °C until lyophilisation.
crushed oil seed cake with 68%(w/w) water content, All extracts were frozen (
80 °C) and lyophilised
was obtained from Mycelia. Substrate II consisted on and then weighed and stored in desiccators in the
50%(w/w) of substrate I and 50%(w/w) sterilised cold dark, at room temperature until further analysis. The
SCG. Spent coffee grounds were obtained from local extraction yield was calculated based on the ratio of

International Journal of Food Science and Technology 2018 © 2018 Institute of Food Science and Technology
 Coffee by-products for mushrooms cultivation A. C. Freitas et al. 3

weight of lyophilised extract to the weight of extracted each probiotic strain grown in different carbon sources,
dried mushroom. Content of total sugars in the differ- specific growth rates (h
1) were calculated.
ent extracts was determined by the phenol-H2SO4 The analysis of sugars and organic acids by HPLC
method (Dubois et al., 1956) using glucose (0– was done according to Rodrigues et al. (2011) in sam-
200 lg mL
1) as a standard. ples of 500 lL collected from each batch culture at
different sampling points (0, 6, 12, 24 and 48 h).
Evaluation of biological properties of the extracts
Statistical analysis
Antidiabetic activity
The a-glucosidase inhibitory activity was determined Data are presented as average plus standard deviation.
in 96-well plates according to the method described in One-way analysis of variance was carried out using
Rodrigues et al. (2017), and antidiabetic activity was SPSS (v 23.0, SPSS Chicago, IL, USA) associated to
expressed as percentage inhibition of a-glucosidase. Tukey test to assess the different significant sources of
variation (P = 0.05).
Antioxidant activity
Total phenolic content (TPC) in all different extracts
Results and discussion
was determined according to Mendes et al. (2016). For
antioxidant capacity assays, ferric reduction activity
Fructification and proximate composition of cultivated
power (FRAP) was determined according to Paz et al.
mushrooms
(2015), whereas scavenging of radical ABTS•+ (ABTS)
and oxygen radical absorption capacity (ORAC) Production yield and proximate composition of
assays were in turn performed according to Mendes P. citrinopileatus and of P. salmoneo-stramineus mush-
et al. (2016). All assays were performed in triplicate, rooms are displayed in Table 1. No negative effect
and quantifications were based on calibration curves from the incorporation of SGC in organic substrate
using gallic acid for TPC, ascorbic acid for FRAP and was observed for the production of P. citrinopileatus
ABTS as well as Trolox for ORAC, respectively. both at yield level and mushroom composition. The
maximum expected yield, which according to substrate
Antihypertensive activity supplier Mycelia would be of 25%(w/w), was observed
All extracts were evaluated by the angiotensin I-con- in both substrates including substrate II which incor-
verting enzyme (ACE) assay according to Tavares porated 50%(w/w) sterilised SCG. No statistical differ-
et al. (2011). Inhibition activity of ACE was deter- ences were observed in terms of organic content and
mined as the amount of inhibitor concentration able total protein for P. citrinopileatus grown in both
to lower the ACE activity to 50% of the initial value organic substrates which were similar to those pub-
(IC50; lg mL
1). lished by Rodrigues et al. (2015). Significant differ-
ences were observed for total fat, total sugar and
Prebiotic activity and sugars and organic acid analysis content of caffeine. Pleurotus citrinopileatus grown in
Potential prebiotic activity of mushroom extracts was substrate II, incorporating SCG, had slightly higher
evaluated in vitro by measuring their impact on the content in total fat, less total sugar content and had
growth of two different probiotic strains, namely Lac- 0.08 g/100 gdry mushroom of caffeine. Higher values of
tobacillus acidophilus Ki (DSM, Australia) and Bifi- total fat (3.5 g/100 gdry mushroom) but lower total sugar
dobacterium animalis Bo (CSK, Netherlands). Prebiotic content (54.7% g/100 gdry mushroom) were reported by
activity was assessed in triplicate by enumeration of Rodrigues et al. (2015) for P. citrinopileatus. Accord-
viable cell numbers of probiotic strains grown in MRS ing to our knowledge, no reports with cultivation of
broth without glucose but supplemented with each of P. citrinopileatus using SCG were found in the litera-
the extracts (2%) throughout 48 h. At each sampling ture. However, according to Salmones et al. (2005),
point (0, 4, 6, 8, 10, 12, 24 and 48 h), inoculated MRS four of the Pleurotus spp. studied (P. ostreatus, P. pul-
broth was plated, in duplicate, on MRS agar and the monarius and P. djamor) were able to increase their
viable cells were enumerated according to procedures productivities when cultured in sterilised coffee pulp.
described in Rodrigues et al. (2015). Strains growth in No significant differences in terms of biological effi-
MRS broth with 2% of glucose or 2% of fruc- ciency among strains of P. ostreatus and P. pulmonar-
tooligosaccharides (FOS¸ OraftiÒP95) (positive con- ius cultivated in pasteurised coffee pulp were also
trols) as well as without glucose (negative control) was reported by Vel
azquez-Cede~ no et al. (2002).
included. The selection of the two probiotic strains and Negative impact of SCG was however observed for
of the extracts (ethanolic and hot water extracts) was production of P. salmoneo-stramineus. Statistically sig-
based on previous growth curves obtained by absor- nificant lower production was obtained using substrate
bance measurements at 660 nm (data not shown). For II representing 85.2% of losses (Table 1) with

© 2018 Institute of Food Science and Technology International Journal of Food Science and Technology 2018
4 Coffee by-products for mushrooms cultivation A. C. Freitas et al.

Table 1 Production yield and proximate composition of Pleurotus citrinopileatus and of Pleurotus salmoneo-stramineus mushrooms cultivated
in the different organic substrates as well as total sugar content in the different extracts from both species cultivated in different organic
substrates

Total
Yield Total solids Organic matter Total Protein Total Fat Sugar1 Caffeine
Organic (gmushroom/ (g/100 (g/100 (g/100 (g/100 (g/100 (g/100
Mushroom substrate 100 gsubstrate) gmushroom) gdry mushroom) gdry mushroom) gdry mushroom) gdry mushroom) gdry mushroom)

P. citrinopileatus Substrate I 25.1  0.5a 9.7  0.1a 93.4  0.1ª 23.5  1.1a 2.0  0.0a 67.9 <LOD2
Substrate II 25.1  0.6a 9.6  0.1ª 92.5  0.2ª 24.1  1.6a 2.5  0.3b 65.9 0.08  0.01
P. salmoneo- Substrate I 25.0  0.3ª 17.7  0.1b 90.5  0.1b 32.8  0.4b 2.1  0.6a 55.6 <LOD2
stramineus Substrate II 3.7  0.2b 17.4  0.2b 91.5  0.1ab 27.4  0.5ab 2.1  0.4a 62.1 0.06  0.01

Ethanolic
Aqueous extracts (HWE) Enzymatic extracts (EA_Alcalase) extracts (EtOH_Ext)

Total Total Total

Organic sugar a-Glucosidase sugar a-Glucosidase sugar a-Glucosidase
Mushroom substrate Yield3 content4 inhibition (%) Yield content inhibition (%) Yield content inhibition (%)

P. citrinopileatus Substrate I 67  2a 117  5a 20  3a 58  5a 89  3a 29  2a 25.4  0.4a 299  2b -

Substrate II 62  3a 109  3a 15  3ab 62  4ab 75  4b 7  1c 30  4bc 365  9a 1.2  0.2a
P. salmoneo- Substrate I 64  4a 79  1b 3.8  0.7c 62  4ab 57  2c 16.2  0.5b 31.8  0.3c 233  12c -
stramineus Substrate II 60  3a 87  5b 11.1  0.8b 59  3a 51  3b 20  6ab 26  1ab 191  9d 7.1  0.2b
1
Total sugar (g/100 gdry mushroom) = Organic matter - total protein - total fat.
2
LOD = 0.005.
3
Extraction yield defined as glyoph extract/100 gdry mushroom.
4
Total sugar in extracts defined as mg/glyoph extract.
Caffeine content in Spawn II was of 0.11  0.02 and 0.07  0.01 g/100 gdry substrate for P. citrinopileatus and P. salmoneo, respectively. Different
letters in a column indicate significant differences (P < 0.05) between mushrooms produced in the two different organic substrates.

mushrooms being smaller and without characteristic by aqueous and enzymatic extraction than by ethano-
oyster cap (data not shown) presenting also differences lic extraction. Lower yields values were however
in terms of proximate composition: lower total pro- reported by Lee et al. (2007) for hot and cold water
tein, higher total sugar and 0.06 g/100 gdry mushroom of extraction (41–52 g/100 g) or ethanolic extraction
caffeine. Variable values of biological efficiency [(- (20 g/100 g) on P. citrinopileatus mushrooms.
mushroom wet weight/substrate dry weight) 9 100%] No significant effects between species and substrates
and strain-dependent have been reported for P. djamor were observed (P > 0.05) for aqueous extracts. Some
mushrooms grown in coffee pulp (Salmones & Mata, variations were however observed between enzymatic
2002; Salmones et al., 2005). extracts and ethanolic extracts (Table 1). For these two
Mushrooms, independent of the species grown in types of extracts, statistically significant higher values
substrate II, had similar caffeine contents to those (P < 0.05) were recorded for P. citrinopileatus grown in
observed in the dry substrate (0.07–0.11 g/100 gdry sub- substrate II incorporating SCG, whereas for P. salmo-
strate) before inoculation suggesting that fungal myce- neo-stramineus, higher values were observed for
lium was not able to degrade caffeine during growth extracts resulting from fruit bodies grown in substrate
but alternatively accumulated it thus justifying, at least I, without SCG. These values evidence some probable
in part, its content a posteriori in the fruiting bodies. synergistic effects between the impact of SCG on
This is in agreement with Salmones et al. (2005) obser- structural and compositional characteristics of myce-
vations who reported decreased content in the coffee lium cells in the fruiting bodies decreasing the extrac-
pulp substrate precisely during fruiting stages and tion yield for P. salmoneo-stramineus but not for
0.17%–0.22% of caffeine in the respective mushrooms. P. citrinopileatus. Due to sample constraints, only total
sugar content was determined in the different extracts
(Table 1). As expected, higher content of total sugar
Different extraction methods and biological potential of
was observed in the ethanolic extracts with highest
the extracts
value of all recorded in the extract from P. citrinopilea-
Extraction yields obtained by different extraction tus grown in substrate II. Lower values of total sugar
modes on both species grown in the different substrate content were observed generally in the extracts
are displayed in Table 1. Higher yields were obtained obtained from P. salmoneo-stramineus independently of

International Journal of Food Science and Technology 2018 © 2018 Institute of Food Science and Technology
 Coffee by-products for mushrooms cultivation A. C. Freitas et al. 5

organic substrate used for mushroom cultivation but particular the extract from P. citrinopileatus grown in
dependent of extraction mode: EtOH Ext>HWE> substrate I, were shown to have the highest antidiabetic
EAE_Alc (Table 1). potential (Table 1). In terms of species, higher values
were also obtained with P. citrinopileatus, especially in
the aqueous and enzymatic extracts obtained from
Antidiabetic activity
mushrooms grown in substrate I. The general lower
In general, a-glucosidase inhibitory activity was lower values of antidiabetic potential compared to acarbose
in all extracts (Table 1) in comparison with acarbose (a contradict those published by Khan & Tania (2012)
drug able to reduce serum glucose levels and used as in vivo trials with aqueous extracts of Pleurotus spp.
positive control) which was approximately of 89%. mushrooms which were promising by promoting the
Variable antidiabetic potential was observed in the reduction in blood glucose levels (16%–23%) in rats.
aqueous and enzymatic extracts regardless the species
or organic substrates. The extracts that showed less
Antioxidant activity
potential were the ethanolic extracts evidencing that a
high content of polysaccharides seems not be the main The antioxidant potential of the different extracts was
factor in the inhibitory activity of the a-glucosidase but evaluated by different methods (Fig. 1). Some correla-
in turn, SCG in the organic substrate appears as a posi- tion is observed between the higher values of TPC
tive factor in particular for extracts of P. salmoneo- observed and higher yield values (Table 1); higher con-
stramineus. In general, enzymatic extracts, and in tent of TPC was recorded in aqueous and enzymatic

Figure 1 (a) Total phenolic content (TPC), (b) ferric reduction activity power (FRAP), (c) scavenging of radical ABTS•+ (ABTS) and (d) oxy-
gen radical absorption capacity (ORAC) of the extracts of Pleurotus citrinopileatus grown in substrate I ( ) or substrate II ( ) and of Pleurotus
salmoneo-stramineus grown in substrate I ( ) or substrate II ( ), respectively. For each type of extraction, different lowercase letters indicate
significant differences (P < 0.05) between extracts obtained from both species grown in the different substrates.

© 2018 Institute of Food Science and Technology International Journal of Food Science and Technology 2018
6 Coffee by-products for mushrooms cultivation A. C. Freitas et al.

extracts in comparison with ethanolic extracts (Fig. 1a) (39  0.8 g ET/glyoph extract), the recorded values were
which are particularly rich in total sugars (Table 1), among those with higher antioxidant activity. The
suggesting that HWE and EAE_Alc are probably ethanolic extracts, independently of the species and
more favourable to extract phenolic compounds or organic substrate, only present some antioxidant
other compounds that could have antioxidant activity. potential regarding FRAP (Fig. 1b). This tendency
Edible mushrooms contain many antioxidant com- contrasts with main observation reported by Lee et al.
pounds such as carotenoids, flavonoids, phenolic com- (2007) where ethanolic extracts had consistently higher
pounds, polysaccharides and antioxidant enzymes (Vaz antioxidant activities than water extracts. In terms of
et al., 2010). Good antioxidant capacities have been enzymatic extracts resultant from Alcalase, some
reported for extracts of mushrooms including those of potential against radicals ABTS•+ and oxygen was
P. citrinopileatus which have been attributed to the observed for those resultant from P. salmoneo-strami-
presence of ascorbic acid, a- and c-tocopherols and neus grown on substrate I or II (Fig. 1c–d).
total phenols (Lee et al., 2007). On the other hand, To evaluate the relationship between TPC and
and independently of the extraction mode, the content antioxidant activity determined by each different
in TPC changed according to mushroom species as method, the respective correlation coefficients were
well as organic substrate without a generalised trend. determined. A correlation coefficient of 0.728 was
For example, in aqueous extracts, higher values were obtained between TPC and ORAC, which suggests
recorded in extracts from P. citrinopileatus grown on that the phenolic compounds present in the extracts
substrate I (11.9  0.6 mg GAE/glyoph extract) and from appear to have a good absorption capacity for oxy-
P. salmoneo-stramineus grown in substrate II (11.2  genated radicals, in a positive relation to the species,
0.6 mg GAE//glyoph extract), respectively. These values organic substrate and extraction mode.
are included in the range values of those published by
Mishra et al. (2013) which varied between 3.94 and
Antihypertensive activity
21.67 mg tannic acid equivalents per g of mycelium
for several species of Pleurotus including P. djamor Antihypertensive potential of the extracts, determined as
and P. citrinopileatus. In turn, by EAE with alcalase, IC50, revealed that ethanolic extracts of both species were
the highest value was recorded in extracts from not able to exert any activity against ACE. In fact, a rele-
P. salmoneo-stramineus grown in substrate I (11.8  vant antihypertensive activity was only observed with
0.8 mg GAE/glyoph extract). In ethanolic extracts, slightly aqueous and enzymatic extracts from P. citrinopileatus
higher values were recorded for both species grown in grown in both organic substrates. These oscillated
substrate II (6.8–7.0 mg GAE/glyoph extract). Flavonoids, between good inhibition activity (50<IC50<150) observed
chlorogenic acid and protocatechuic acid were with aqueous extract of P. citrinopileatus grown in sub-
extracted from SCG by Mussato et al. (2011b) where strate I (IC50=123 lg mL
1) and intermediate inhibition
extraction using 60% methanol in a solvent/solid ratio activity (150<IC50<400) observed in aqueous extract of
of 40 mL g
1 SCG during 90 min was able to produce P. citrinopileatus grown in substrate II (IC50 = 276
extracts with high content of phenolic compounds and lg mL
1) as well in enzymatic extracts of P. citrinopilea-
high antioxidant activity. Chlorogenic acid has been tus grown in substrate I (IC50 = 178 lg mL
1) and II
reported as one of the most abundant phenolic com- (IC50 = 243 lg mL
1), respectively. According to these
pounds in SGC (Lee et al., 2007). According to results, apparently neither Alcalase activity nor organic
Ramalakshmi et al. (2009), the presence of phenolic substrate containing SCG were factors of added value in
compounds including chlorogenic acid in appreciable terms of antihypertensive activity. Surprisingly, no anti-
amounts along with brown pigments in SCG extracts hypertensive activity was recorded with any of the
makes this coffee by-product a good source of antioxi- extracts from P. salmoneo-stramineus despite the higher
dants which could also possibly have a role in antitu- content of protein in their fruiting bodies, even in those
mour activity. extracts resulting from Alcalase extraction. Abdullah
In terms of antioxidant activity observed in the dif- et al. (2012) evaluated the ACE inhibitory activity of
ferent extracts (Fig. 1a–d), no general tendency was some edible culinary-medicinal mushrooms extracts,
observed considering extraction mode, mushroom spe- including some species of Pleurotus, extracted by boiling
cies or organic substrate and was not in agreement in water for 30 min reporting values of IC50 between 50
with TPC contents. The aqueous extracts of and 67 lg mL
1.
P. citrinopileatus grown in substrate I stood out in
relation to the others presenting the highest values of
Prebiotic activity
TPC (11.9  0.6 mg EAG/glyoph extract), and of FRAP
(2.4  0.2 mg EAA/glyoph extract); for scavenging activ- The selection of tested extracts and probiotic strains
ity of radical ABTS•+ (7.1  0.8 mg EAA/glyoph was based on absorbance growth curves through 24 h
extract) and oxygen radical absorption capacity of incubation at 37 °C of four probiotic bacteria

International Journal of Food Science and Technology 2018 © 2018 Institute of Food Science and Technology
 Coffee by-products for mushrooms cultivation A. C. Freitas et al. 7

(L. casei L26, L. acidophilus Ki, B. animalis Bo e and source was observed. The decrease in pH values was
B. lactis Bb12) in microplate using MRS broth supple- much steeper in MRS supplemented with glucose and
mented with controls (2%) or with all of the different FOS shortly after 4 h (Fig. 2d–f). Intermediate reduc-
extracts (2%) as carbon sources (data not shown). tion was observed in MRS supplemented with extracts
From this study, two probiotic bacteria were selected of P. citrinopileatus while the lowest pH reduction
as well as two types of extracts for confirmation of occurred in MRS supplemented with extracts of
prebiotic activity over 48 h of incubation at 37 °C: (i) P. salmoneo-stramineus. This trend was more pro-
the aqueous and ethanolic extracts were selected nounced in MRS with ethanolic extracts where lower
because of the higher exponential growth; (ii) B. ani- pH values were verified over time (Fig. 2c and f) com-
malis Bo was chosen because it was the most promis- pared to those obtained in MRS with aqueous extracts
ing in comparison with B. lactis Bb12, and (iii) (Fig. 2e). This fact is certainly related to the higher
L. acidophilus Ki was selected mainly because this is a availability of fermentable sugars present in the
more fastidious and challenging probiotic bacteria in ethanolic extracts. There was no particular effect by
comparison with L. casei L26. SCG in the organic substrate on pH profile over 48 h
Probiotic viable cells and pH variation (Fig. 2) as of incubation.
well as sugars consumption and organic acids produc- Concerning the number of viable cells of L. aci-
tion (Table 2) were determined over 48 h of incubation dophilus Ki in MRS supplemented with the ethanolic
at 37 °C. The higher prebiotic potential was observed extracts and their respective pH variation over 48 h of
using ethanolic extracts regardless the mushroom spe- incubation (Fig. 2c–f), a similar scenario is observed in
cies or organic substrate, which is certainly related with general to that obtained with B. animalis Bo. How-
its higher total sugar contents being 2.2–3.3 higher than ever, some aspects can be highlighted: (i) after 12 h, a
HWE extracts (Table 1). The evolution of the number more marked decrease in the number of viable cells of
of viable cells of B. animalis Bo in MRS supplemented L. acidophilus Ki in MRS supplemented with ethanolic
with the ethanolic (Fig. 2a) or aqueous extracts extract of P. salmoneo-stramineus produced with sub-
(Fig. 2b) of both species of mushrooms grown on the strate I (without SCG) was observed (Fig. 2c); (ii) a
two organic substrates was very similar to those greater difference was observed between the number of
observed in MRS supplemented with the positive con- viable cells of L. acidophilus Ki in MRS supplemented
trols glucose or FOS controls during the exponential with the other three ethanolic extracts in comparison
phase (0–8 h), and beginning of the stationary phase with MRS supplemented with glucose or with FOS,
(8–12 h). After 10 h of incubation, slightly higher val- particularly after 24 and 48 h; (iii) the pH variation in
ues of viable cells of B. animalis Bo were recorded in MRS with each of the four ethanolic extracts was sim-
MRS supplemented with the ethanolic extracts, partic- ilar to that obtained with B. animalis Bo. The pH vari-
ularly after 24 and 48 h with extracts of P. salmoneo- ation was not affected by the decrease in viable cells of
stramineus, compared to MRS supplemented positive L. acidophilus Ki in MRS supplemented with ethanolic
controls (Fig. 2a). It was also noted that the numbers extract of P. salmoneo-stramineus produced on sub-
of viable cells of B. animalis Bo in MRS supplemented strate I (Fig. 2c–f). Specific growth rates of L. aci-
with the aqueous extracts (Fig. 2b), regardless of the dophilus Ki obtained in MRS with ethanolic extracts
mushroom species and the organic substrate, remained were statistically significant higher than those obtained
more stable after 24 and 48 h of incubation, than with in MRS with FOS, in particular with extracts resultant
the ethanolic extracts for which it was registered a from P. salmoneo-stramineus grown in substrate II
decline after 24 h (Fig. 2a,b). Thus, the ethanolic and incorporating SCG (Fig. 2c); statistically significant
aqueous extracts of both species of mushrooms shown lower specific rates were obtained in MRS with glu-
to be a very efficient alternative source for growth of cose.
B. animalis Bo demonstrating a good prebiotic poten- In general, all the selected ethanolic and aqueous
tial. Specific growth rates of B. animalis Bo obtained in extracts were evidenced as good alternative carbon
MRS with ethanolic extracts are slightly higher than sources showing good prebiotic potential in compar-
those obtained in MRS with FOS, in particular with ison with the recognised prebiotic FOS. Mushrooms
extracts resultant from P. citrinopileatus grown in both have been referred as potential candidates for alterna-
of the organic substrates (Fig. 2a); statistical significant tive prebiotic sources because they contain polysaccha-
lower specific rates were obtained in MRS with or rides such as chitin, hemicellulose, b- and a-glucans,
without glucose. With aqueous extracts, a similar sce- mannans, xylans and galactans (Aida et al., 2009);
nario was observed but specific growth rates in MRS Pleurotus is an excellent source of crude fibre (10%)
with these extracts were slightly lower than those and b-glucans (25%) (Carrasco-Gonz
alez et al., 2017).
obtained in MRS with FOS. According to Synytsya et al. (2008), the variable use
Regarding the variation in pH over 48 h of incuba- of different extracts from P. ostreatus and P. eryngii
tion, in general, a greater effect of the type of carbon by probiotics evidences different chemical structure of

© 2018 Institute of Food Science and Technology International Journal of Food Science and Technology 2018
8 Coffee by-products for mushrooms cultivation A. C. Freitas et al.

Figure 2 Number of viable cells (a, b and c) and respective variation in pH (d, e and f) of Bifidobacterium animalis Bo or Lactobacillus aci-
dophilus Ki throughout 48 h of incubation at 37 °C in the presence (s) or absence (d) of glucose, with FOS ( ) or with ethanolic extracts (a,
c, d and f) or hot water extracts (b and e) of Pleurotus citrinopileatus grown in substrate I (&) or II (h) and of Pleurotus salmoneo-stramineus
grown in substrate I (▲) or II (4), respectively. For each growth curve, specific rate growth (h
1) is presented.

the polysaccharides behind the prebiotic potential; used for synbiotic effect. In another example, Rodri-
according to the authors, at least two types of glucans gues et al. (2016) reported that digested Pholiota
and proteoglucan complexes from Pleurotus could be nameko enzymatic extract obtained with Flavourzyme

International Journal of Food Science and Technology 2018 © 2018 Institute of Food Science and Technology
 Table 2 Content of glucose and of FOS in the respective positive controls (MRS with glucose or MRS with FOS) and of organic acids over 48 h of incubation at 37 °C in
controls or in MRS added with different extracts and inoculated with Bifidobacterium animalis Bo or Lactobacillus acidophilus Ki, respectively

Glucose/FOS (g L
1) Lactic acid (g L
1) Acetic acid (g L
1)

B. animalis B. animalis B. animalis L. acidophilus B. animalis B. animalis L. acidophilus Ki

Time (hr) Bo L. acidophilus Ki Bo EtOH_Ext Bo HWE Ki EtOH_Ext Time (hr) Bo EtOH_Ext Bo HWE EtOH_Ext

Con. Neg. 0 - - 0.591  0.008 0.36  0.02 0.47  0.02 0 2.125  0.005 2.25  0.03 2.265  0.004
6 - - 0.951  0.006 0.914  0.004 0.92  0.020 6 2.714  0.002 2.97  0.04 2.61  0.02
12 - - 0.981  0.007 0.99  0.02 0.997  0.004 12 2.96  0.02 3.117  0.007 3.01  0.02

© 2018 Institute of Food Science and Technology

24 - - 0.97  0.02 0.96  0.06 0.95  0.02 24 2.98  0.01 3.41  0.08 3.12  0.04
48 - - 0.87  0.06 0.80  0.03 0.88  0.03 48 3.18  0.02 3.39  0.01 3.39  0.01
Con. Pos. 0 14.3  0.2 14.6  0.3 0.41  0.03 0.420  0.001 0.46  0.02 0 2.13  0.04 2.236  0.001 2.30  0.02
Glucose 6 10.6  0.9 13.4  0.4 4.46  0.04 3.24  0.01 4.7  0.1 6 2.18  0.02 2.47  0.02 2.12  0.02
12 5.8  0.3 6.23  0.03 10.6  0.1 10.4  0.2 10.33  0.04 12 2.20  0.03 2.764  0.007 2.42  0.02
24 0.75  0.05 1.02  0.03 14.8  0.2 14.71  0.03 14.19  0.05 24 2.32  0.03 2.668  0.008 2.3  0.2
48 0.66  0.04 0.68  0.01 14.5  0.4 13.76  0.03 14.58  0.03 48 2.47  0.03 2.61  0.05 2.001  0.003
Con. Pos. FOS 0 24.8  2.1 24.3  0.6 0.62  0.02 0.379  0.007 0.390  0.006 0 2.181  0.001 2.18  0.06 2.24  0.03
6 12.2  1.2 12.8  0.1 4.7  0.1 3.8  0.2 4.880  0.005 6 2.271  0.008 2.4  0.1 2.301  0.006
12 8.0  0.3 7.8  0.1 11.14  0.02 10.46  0.07 10.83  0.04 12 2.261  0.009 2.764  0.007 2.230  0.009
24 2.0  0.2 2.03  0.01 15.3  0.2 15.0  0.1 15.1  0.1 24 2.11  0.02 2.668  0.008 2.17  0.02
48 1.9  0.3 1.65  0.01 15.6  0.2 16.6  0.6 14.67  0.06 48 2.15  0.02 2.61  0.05 1.840  0.007
P. citrinopileatus 0 - - 3.1  0.2 1.2  0.2 0.50  0.02 0 2.57  0.03 2.45  0.06 2.650  0.009
Substrate I 6 - - 5.2  0.1 3.81  0.04 5.091  0.005 6 2.59  0.03 2.77  0.06 2.51  0.05
12 - - 8.42  0.07 4.12  0.04 7.92  0.02 12 2.28  0.01 3.272  0.004 2.46  0.01
24 - - 9.4  0.1 4.02  0.04 9.12  0.08 24 2.19  0.01 3.337  0.009 2.01  0.02
48 - - 9.2  0.2 3.78  0.04 8.7  0.1 48 2.26  0.03 3.368  0.007 1.93  0.01
P. citrinopileatus 0 - - 4.3  0.2 1.6  0.3 0.54  0.01 0 2.58  0.01 2.3  0.1 2.80  0.02
Substratre II 6 - - 4.96  0.03 3.8  0.2 4.8  0.3 6 2.65  0.03 2.68  0.02 2.63  0.02
12 - - 8.44  0.06 4.24  0.05 7.7  0.2 12 2.31  0.03 3.14  0.02 2.60  0.02
24 - - 8.6  0.2 4.11  0.06 8.4  0.2 24 2.27  0.03 3.15  0.04 2.13  0.04
48 - - 8.3  0.2 3.94  0.03 7.9  0.2 48 2.50  0.01 3.27  0.01 2.10  0.02
P. salmoneo-stramineus 0 - - 0.46  0.03 1.2  0.1 0.44  0.03 0 2.31  0.06 2.09  0.04 2.37  0.01
Substrate I 6 - - 3.5  0.5 3.3  0.3 3.83  0.06 6 2.2  0.1 2.72  0.05 2.26  0.03
12 - - 6.36  0.04 3.66  0.04 6.40  0.02 12 2.35  0.04 3.06  0.01 2.41  0.03
24 - - 6.24  0.06 3.51  0.03 6.2  0.1 24 2.44  0.02 3.07  0.01 2.26  0.01
48 - - 6.25  0.03 3.45  0.04 6.01  0.08 48 2.55  0.02 3.20  0.03 2.20  0.02
P. salmoneo-stramineus 0 - - 0.72  0.03 1.023  0.004 0.57  0.03 0 2.29  0.02 2.29  0.02 2.43  0.02
Substrate II 6 - - 3.83  0.02 2.41  0.01 3.86  0.08 6 2.41  0.03 1.3  0.1 2.471  0.005
12 - - 6.71  0.01 3.20  0.09 6.6  0.2 12 2.20  0.02 3.201  0.005 2.30  0.02
24 - - 6.62  0.03 3.12  0.08 6.4  0.2 24 2.38  0.03 3.23  0.01 2.16  0.01
48 - - 6.43  0.02 3.07  0.09 6.15  0.06 48 2.60  0.02 3.32  0.05 2.221  0.009
Coffee by-products for mushrooms cultivation A. C. Freitas et al.

International Journal of Food Science and Technology 2018

9
10 Coffee by-products for mushrooms cultivation A. C. Freitas et al.

selectively increases bifidobacteria, hindering growth of extract) or prebiotic activity in comparison with FOS.
Clostridium histolyticum as well as member of the This very much opens doors to a more efficient use of
Clostridium coccoides/Eubacterium rectale group, which SCG where sustainability and cost-effectiveness are
associated to consistent increase in short-chain fatty needed within a circular economy perspective (zero
acids and lactic acid production suggests its potential waste).
prebiotic character. These results confirmed the poten-
tial prebiotic activity observed for this extract on
L. acidophilus La5 and B. animalis BB12 (Rodrigues Acknowledgments
et al., 2017). The authors are grateful to ORAFTI, CSK and DSM
Consumption of glucose and FOS was similar for providing the FOS and the probiotic strains,
among both probiotic strains: 57%–59% consumption respectively. This work was supported by national
of glucose after 12 h, reaching values of 95% after funds through FCT/MEC (PIDDAC), project refer-
48 h, whereas consumption of FOS was of 68% after ence IF/00588/2015 with scientific collaboration of
12 h reaching values of 92%–93% after 48 h CBQF under the FCT project UID/Multi/50016/2013.
(Table 2). As expected, the consumption of glucose
and FOS during fermentation was accompanied by the
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© 2018 Institute of Food Science and Technology International Journal of Food Science and Technology 2018