Olive mill waste is low-quality feed and rarely used in ruminant
nutrition because of its high lignocellulose content, the existence of
anti-nutritional factors such as total polyphenol and condensed tannin, and
low protein contents. This in vitro research was conducted to valorize this waste (crude olive cake, extracted olive cake, and olive leaves) using an exogenous
fibrolytic enzyme produced from Trichoderma longibrachiatum in ruminal nutrition. The enzymatic
activity of this additive was 1161 units of
endoglucanase per millilitre, 113 units of
exoglucanase per millilitre, and 2267 units of xylanases per millilitre. This treatment was applied
by spraying substrates with four doses: 0 (control), 1 (low), 2 (medium),
and 4µLg-1µLg-1 (high) of dry matter olive mill waste in an air-conditioned
room at 26 ∘C for 12 h before in vitro incubation.
For the crude olive cake, this additive at high doses increased degradation
of 14 % of cellulose and 8 % of hemicellulose compared with the control at
12 h before the in vitro incubation. Consequently, it increased dry matter
solubility and reduced sugars at this period compared to the control. Upon
ruminal incubation, the high dose of exogenous fibrolytic enzyme increased
the gas production from the immediately soluble fraction and insoluble fraction,
the rate of gas production for the insoluble fraction, the dry matter
degradability by 26 %, the organic matter degradability by 24 %, the
metabolizable energy value by 28 %, and the microbial crude protein
production by 24 % compared with the control. For olive leaves, an exogenous
fibrolytic enzyme at medium dosage can also hydrolyse the hemicellulose
compound, release fewer sugars, and increase dry matter solubility
compared with the control at 12 h before the in vitro incubation.
Upon in vitro incubation, the medium dose increased the gas production from
immediately soluble and insoluble fractions, the rate of gas production for
the insoluble fraction, the dry matter degradability by 13 %, the organic
matter degradability by 11 %, the metabolizable energy value by 12 %,
and the microbial crude protein production by 12 % compared with the
control. However, the highest dose altered the gas production from insoluble
fractions and decreased microbial crude protein production by 6 % compared
with the control. Under the same conditions, an exogenous fibrolytic
enzyme applied to extracted olive cake did not produce any effect in the chemical
composition and nutritional value. These results showed clearly that
effectiveness of exogenous fibrolytic enzyme varied with incubated waste.
Increasing the nutritional value of crude olive cake and olive leaves using an
exogenous fibrolytic enzyme can encourage breeders to use this waste as
feed at a low cost in animal nutrition. This valorization of waste is a good
solution to reduce pollution of soils and groundwater caused by throwing out
this polluted waste into the environment.
Introduction
The Mediterranean countries contribute 92 % of the world's production of
olive oil (Tufariello et al., 2019). The olive oil extraction generates a
gigantic quantity of solid waste like crude olive cake and olive leaves
(Molina-Alcaide and Yáñez-Ruiz, 2008; Tufariello et al., 2019). The
majority of this waste is thrown out in the nearby environment. This waste decomposes over a long period of time (years) and still presents
economical (i.e. the cost of getting rid of the waste) and environmental problems in soil
and groundwater (Molina-Alcaide and Yáñez-Ruiz, 2008; Dermeche et
al., 2013; Fadel and El-Ghonemy, 2015; Berbel and Posadillo, 2018; Chebaibi
et al., 2019). The fresh or well preserved waste of olive oil production can
be utilized in ruminant feeding with no adverse effects on animal health
(Awawdeh and Obeidat, 2013; Fadel and El-Ghonemy, 2015;
Mannelli et al., 2018; Abid et al., 2020). However, this waste when left
in the open quickly becomes rancid and cannot be eaten by animals
(Molina-Alcaide and Yáñez-Ruiz, 2008). This waste is characterized by
high fibre contents and the existence of anti-nutritional factors,
particularly total polyphenol and condensed tannin (Molina-Alcaide and
Yáñez-Ruiz, 2008). The effect of tannins varies according to their
concentration, ranging from beneficial effects to toxicity and death in high
doses (Makkar, 2003). The fibrous feed can be digested by the rumen
microorganisms, but in most cases only 10 %–35 % of the gross energy ingested
is available as net energy (Varga and Kolver, 1997).
Olive cake is the major solid waste product from the olive oil extraction process.
It represent about 30 % to 40 % of the weight of processed biomass in the
olive mill (Molina-Alcaide and Yáñez-Ruiz, 2008). This solid waste
is a mixture of the olive skin, the crushed pulp residues, and the small
fragments of the olive kernel shell (Berbel and Posadillo, 2018). The use of
olive cake as ruminant feed can reduce the pollution caused by this waste
and improve the quality of ovine and bovine meat and milk in particular.
It increases unsaturated fatty acids, which have positive effects on human
health (Hamdi et al., 2016; Kotsampasi et al., 2017; Mannelli et al., 2018;
Symeou et al., 2019; Chiofalo et al., 2020; Neofytou et al., 2020). Olive
leaves are the solid waste product of separated and clean olives before oil
extraction. It represents around 5 % of the weight of the biomass processed
in the olive mill (Hukerdi et al., 2020). The use of olive leaves as
ruminant feed especially improves the quality of goat and ovine meat and milk. It improves the unsaturated fatty acid profile (Abbeddou et al.,
2011; Hukerdi et al., 2020). To improve the nutritional value of this solid
waste several approaches have been used, such as chemical treatments (Fegeros
et al., 1995; Molina-Alcaide and Yáñez-Ruiz, 2008; Awawdeh and
Obeidat, 2013) and biological treatments with white-rot fungi (Neifer et al.,
2013), Aspergilllus, Trichoderma and Saccharomyces (Fadel and El-Ghonemy, 2015). The biological treatments are more
effective than the chemical treatments due to their greater substrate
specificity and their lower pollution effects (Misra et al., 2007). The
treatment of fibrous feed by biological additives like exogenic fibrolytic
enzymes (EFE) improved the nutritional value, growth performance, milk
production, and economic returns of ruminants (Mohamed et al., 2013; Abid et
al., 2019b, 2020). Therefore, the objective of this research is
to valorize olive mill waste by supplementing ruminant nutrition with EFE.
Materials and methodsSampling and chemical analysis
Crude olive cake, extracted olive cake, and olive leaves samples were
collected from eight olive mills in Sfax (southern Tunisia). Three samples of
each by-product in each oil mill are used. These mills used three phase
centrifugation processes (Tamasi et al., 2016). The dry matter content of the
samples was produced by oven-drying until it reached a constant weight (AOAC 930.15).
Then samples were ground through a mill using a 1 mm sieve and a Cyclotec 1093
Sample Mill (Tecator, Höganäs, Sweden). The crude protein (CP) (AOAC
954.01), ether extract (EE) (AOAC 920.39), and ash (AOAC 942.05) of samples
were determined following the method defined by AOAC (1995). Neutral
detergent fibre (NDF), acid detergent fibre (ADF), and acid detergent lignin
(ADL) were determined by using a fibre analyser (ANKOM220, ANKOM Technology,
Macedon, New York, USA) following the method defined by Van Soest et al. (1991).
Hemicellulose and cellulose were calculated by the difference between NDF
and ADF and between ADF and ADL respectively (Van Soest et al., 1991).
The nitrogen-free extract was calculated by using the formula of NRC (2001)
according to Eq. (1):
Nitrogen free extract (%)=100-(EE(%)+CP(%)+NDF(%)+Ash(%)).
Total phenols, total tannins, condensed tannins, and hydrolysable tannins
compounds were determined following the protocol of Makkar et al. (1993, 2000). Total phenols and total tannins were determined by
using a Folin–Ciocalteu substrate with spectrophotometry at 725 nm of
absorbance. Condensed tannins were determined by using the acid–butanol–HCl–Fe
method with spectrophotometry at 550 nm of absorbance. Hydrolysable tannins
were calculated by the difference between total tannins and condensed
tannins. Reducing sugars was realized following the protocol of Miller
(1959). The soluble dry matter was realized following the protocol of
Elwakeel et al. (2007). Briefly, samples of 1 g dry matter of each substrate
were poured into glass flasks. Then 100 mL of distilled water was added.
This mixture was stored for 12 h in an air-conditioned room at
26 ∘C. After incubation, residues were recovered by filtration
with Whatman 541 filter paper (Whatman Scientific Ltd, Maidstone, Kent,
England). DM losses were determined according to the methods described by
AOAC (1995).
Treatment of olive mill waste using an exogenous fibrolytic enzyme
The exogenous fibrolytic enzyme used in this study was a liquid mixture of two
fibrolytic enzymes, cellulase plus and xylanase plus (Dyadic International
Inc., Jupiter, Florida, USA), used in equal volume. Both enzymes were
obtained by the fermentation of non-recombinant Trichoderma longibrachiatum. The mixture was diluted in
distilled water at three different concentrations, either 1 µL mixture
preparation per 0.2 mL, 2 µL mixture preparation per 0.2 mL, or 4 µL mixture preparation per 0.2 mL. The mixture treatment was applied by
spraying one of the enzyme solutions (or only distilled water in the
control) on the olive mill waste at 0.2 mL of solution per gram of dry matter
olive mill waste in an air-conditioned room at 26 ∘C for 12 h
before in vitro incubations. Thus, the final concentration of this additive was 0
(control), 1 (low), 2 (medium), and 4 µLg-1 (high) dry matter olive
mill waste.
The enzymatic activity of the mixture before dilution in distilled water and
before treatment of substrates was analysed in triplicate at a pH of 6.6 and
a temperature of 39 ∘C, to simulate a normal rumen environment.
The endoglucanase activity and exoglucanase activity were measured according
to the methods of Wood and Bhat (1988) by using carboxymethyl cellulose
sodium salt and cellulose as substrates (Sigma Chemical Co., Saint-Louis,
MO, USA) respectively. The xylanase activity was determined according to the
methods of Biely and Poutanen (1992) by using oat spelt xylan as substrate
(Sigma Chemical Co., Saint-Louis, MO, USA). The enzymatic activity of the
mixture before dilution in distilled water and treatment of substrates used
in this study had 1161 units of endoglucanase per millilitre, 113 units of
exoglucanase per millilitre, and 2267 units of xylanases per millilitre.
Pre-incubation effects
After 12 h of treatment with EFE, solubilize dry matter, crude protein,
cellulose, hemicellulose, lignin, total phenols, and reduced sugars of
treated substrate were analysed in triplicate.
a, b, c Different letters in the same row indicate that the difference is significant (p<0.05). SEM is the standard error of the mean.
Incubation effects
Two rumen-cannulated Holstein cows (650±20 kg body weight) were used
to collect rumen fluid before the morning meal via an electric pump. These
cows received two meals composed by 8 kgd-1 of oat hay and 2 kgd-1 of a
commercially available concentrate. The rumen content of these two cows was
mixed and filtered through four layers of cheesecloth under a continuous
flow of CO2. Filtered ruminal fluid was mixed with the buffer solution of
Menke and Steingass (1988) (1:2v/v) under a continuous flow of CO2 to obtain a fermentation medium. Samples of 200 mg dry matter of substrate, treated with the appropriate EFE dose, were
poured into 120 mL volume serum bottles with 30 mL of the fermentation
medium. Three blank samples (negative controls) were incubated without
substrate to correct the produced gas from the fermentation of residual feed
particles of ruminal fluid. The bottles were instantly closed with rubber
stoppers and incubated in a shaking water bath at 39 ∘C for 96 h. Each treatment was performed in triplicate and repeated 3 times.
Gas pressure was recorded at 2, 4, 6, 8, 12, 24, 48, 72, and 96 h by a
pressure transducer (model PX4200-0100GI, Omega Engineering Inc., Laval, QC,
Canada) related to a visual display transducer (Data Tracker 200, Data Track
Process Instruments Ltd, Christchurch). After each measurement, the produced
gas was released. The net gas was determined by subtracting the gas
production from the blank bottles. The kinetics of gas production indices
were adjusted by the non-linear model procedures of SAS (2011) according to
the model of Ørskov and McDonald (1979) as in to Eq. (2):
GP(t)=A+B(1-e-Ct),
where GP is the cumulative volume of gas produced at the time t in mLg-1 dry
matter, t is the incubation time in h, A is the gas production from
immediately soluble fraction in mLg-1 dry matter, B is the gas production
from insoluble fraction in mLg-1 dry matter, A+B is the potential of gas
production in mLg-1 dry matter, and C is the rate of gas production for the
insoluble fraction in mLh-1.
Metabolizable energy (ME) was estimated by Eq. (3) of Menke and Steingass
(1988). The total volatile fatty acids (VFAs) were estimated according to Eq. (4) of Getachew et al. (1998):
3ME=2.2+0.136×GP+0.0057×CP4VFA=-0.00425+0.0222×GP,
where ME is the metabolizable energy value in MJkg-1 dry matter, VFAs are the
ruminal total volatile fatty acids in mmol per 200 mg dry matter, CP is the
crude protein in percentage of dry matter, and GP is the net gas production in mL
from 200 mg after 24 h of incubation.
At 96 h of incubation, all bottles were put in ice for 5 min and the
fermentation was stopped. The pH was measured immediately. The dry matter
and organic matter degradability at the end of fermentation was measured
according to the protocol of Elghandour et al. (2018). The partitioning
factor of incubation at 24 h (PF 24) and the microbial crude protein (MCP)
was determined with Eqs. (5) and (6) as described by Blümmel et al.
(1997):
PF 24=aDMDGP,
where PF 24 is the partitioning factor of incubation at 24 h, GP is the net
gas production in mL from 1 g dry matter after 24 h of incubation, and
aDMD is the amount of dry matter digestibility in grams at the end of
incubation.
MCP=aDMD-2.2×GP,
where MCP is microbial crude protein in mgg-1 dry matter, GP is the net gas
production (mL) from 200 mg after 24 h of incubation, aDMD is the amount
of dry matter digestibility in grams at the end of incubation, and the 2.2 mgmL-1 is a stoichiometric factor that expresses the C, H, and O (measured in milligrams) required for
the volatile fatty acids associated with the production of gas (measured in millilitres) (Blümmel et al., 1997).
Statistical analysis
Data were analysed by using a factorial model (4×3) to study the
influence of four doses of EFE (control, low, medium, and high) and three
substrates of olive mill waste (crude olive cake, extracted olive cake, and
olive leaves). Data were analysed by using the GLM procedure of SAS (2011)
following Eq. (7):
Yijk=μ+Di+Sj+(D×S)ij+Eijk,
where Yijk is the observation, μ is the overall mean, Di is the effect of the ith dose (i= control, low, medium, and
high), Sj is the effect of jth substrate (j= crude
olive cake, extracted olive cake, and olive leaves), (D×S)ij is the interaction between the dose and the substrate, and Eijk is the random residual error.
The orthogonal contrasts were completed to examine the linear and quadratic
effects of doses for each piece of waste. Duncan's multiple range tests were
performed to separate means (Duncan, 1955) and significance was confirmed at
p<0.05.
ResultsChemical compositions of olive mill waste
Olive mill waste is characterized by low crude protein content (89 to 102 gkg-1 dry matter), reduced sugar (9 to 57 gkg-1 dry matter), and high
fibre content (NDF>400gkg-1 dry matter). The extracted olive
cake caused the largest amount of lignocellulose waste. Olive leaves had high total
polyphenols (77 gkg-1 dry matter) and condensed tannins (58 gkg-1 dry
matter). Crude olive cake had a high fat content (67 gkg-1 dry matter)
(Table 1).
Pre-incubation effects
EFE hydrolysed the fiber compound of olive leaves and olive cake. This
effect was related with the rise in reduced sugars and solubility of dry
matter. For the extracted olive cake, EFE failed to cause any significant
difference in the chemical composition (Table 2).
Incubation effects
The influence of EFE on the gas production from immediately soluble and
insoluble fractions, the rate of gas production for the insoluble fraction,
the dry matter degradability, the organic matter degradability, the
metabolizable energy value, the ruminal total volatile fatty acids, and the
microbial crude protein production of olive mill waste depended on the type
of the substance and the dose of EFE (Tables 3 and 4). Supplementation of
EFE to crude olive cake increased gas production from immediately soluble
and insoluble fractions and the rate of gas production for the insoluble
fraction at all doses. The dry matter degradability, the organic matter
degradability, the metabolizable energy value, the ruminal total volatile
fatty acids, and the microbial crude protein production improved only at the
high dose.
Influence of exogenous fibrolytic enzymes on gas production
kinetics' of olive mill waste.
a, b, c Different letters in the same row indicate that the difference is significant (p<0.05). SEM is the standard error of the mean. A
is the gas production from immediately soluble fraction (mLg-1 DM). B is the
gas production from insoluble fraction (mLg-1 DM). A+B is the potential
of gas production (mLg-1 DM). C is the rate of gas production for the
insoluble fraction (mLh-1).
Effect of exogenous fibrolytic enzymes on fermentation
characteristics of olive mill waste.
a, b, c Different letters in the same row indicate that the difference is significant (p<0.05). SEM is the standard error of the mean. pH
is ruminal pH. DMD is dry matter degradability (%). OMD is organic matter
degradability (%). ME is metabolizable energy value (MJkg-1 DM) =2.2+0.136× net gas production in mL from 200 mg after 24 h of incubation+0.0057×crude protein in % dry matter. VFAs are ruminal total volatile
fatty acids (mmolg-1 DM) =-0.00425+0.0222×net
gas production in mL from 200 mg after 24 h of
incubation. PF24 is partitioning factor at 24 h of incubation
(mg DMD mL-1 gas). MCP is microbial crude protein production
(mgg-1 DM).
For olive leaves, only the medium dose increased the gas production from
immediately soluble and insoluble fractions, the rate of gas production for
the insoluble fraction, the dry matter degradability, the organic matter
degradability, the metabolizable energy value, the ruminal total volatile
fatty acids, and the microbial crude protein production. The lowest dose had
no significant effect. The highest dose had a negative effect in the gas
production from insoluble fraction and microbial crude protein production.
For the extracted olive cake, EFE failed to cause any significant difference
in nutritional value.
DiscussionChemical compositions of olive mill waste
The chemical compositions of solid olive waste were comparable to the
chemical composition of this waste in the review by
Molina-Alcaide and Yáñez-Ruiz (2008). This waste is characterized by
low crude protein (<11gkg-1 dry matter), a high neutral detergent
fibre compound (>400gkg-1 dry matter), and particularly a high lignin
content (>100gkg-1 dry matter). Crude olive cake is also
characterized by high fat content, and the extracted olive cake has low fat
contents. A similar result was found by Álvarez-Rodríguez et al.
(2009). Olive leaves have excessive concentrations of condensed tannins.
Therefore, these by-products cannot be the only components of diet but can constitute a diet supplement.
Pre-incubation effects
The effect of EFE depended on the substrate and the doses of EFE. For the
crude olive cake, the increased dose of EFE linearly hydrolysed
hemicellulose and cellulose, reduced sugars, and increased
dry matter solubility. A similar result was found for fibrous feed such as wheat straw
(Jabri et al., 2019) and Dichanthium aristatum (Díaz et al., 2015). For olive
leaves, the increased dose of EFE linearly decreased the hemicellulose,
increased dry matter solubility, and reduced sugars. A similar result was
found on rich tannin feed like almond hulls (Abid et al., 2019b). This
modification of chemical composition could modify the microorganisms in
the rumen and microbial colonization. However, this additive has no effect
on feeds very rich in lignin fraction like extracted olive cake. Indeed, this
component may prevent the contact of EFE with other commands of substrate
(Hatfield et al., 1999).
The second extraction of crude olive cake particularly increased the
lignin fraction by 102 gkg-1 dry matter. The excessive
concentration of this compound can block the effect of
enzymes.
Incubation effects
The response to EFE depended on the substrate and the doses of EFE. This
additive modified the nutritional value of crude olive cake and olive leaves.
However, it did not affect nutritional value of extracted olive cake. The
absence of an effect on the chemical composition in the pre-incubation period
explained the lack of effect on the nutritional value of this substrate. For the
crude olive cake, the increased dose of EFE linearly increased the gas
production from immediately soluble and insoluble fractions, the gas
production from insoluble fraction, the rate of gas production for the
insoluble fraction, the dry matter degradability, the organic matter
degradability, the metabolizable energy value, the ruminal total volatile
fatty acids, and the microbial crude protein production. Similar results
were found in fibrous tropical foraging material (Sakita et al., 2020). These
improvements in fermentation and degradability cannot be explained
exclusively by the small hydrolysed quantity of cellulose and hemicellulose
(<4 % of dry matter). It was highly probable that these
improvements were due to the synergy between EFE and the ruminal flora. The
increase in microbial production of crude protein following the use of this
additive is mainly due to better accessibility of nutrients for rumen
microorganisms (Getachew et al., 2004). It was very likely that this
improvement was due to the increase in ruminal bacteria. In fact, Newbold et
al. (1992) have shown that EFE produced from Aspergillus oryzae (Amaferm; BioZyme
Enterprises Inc., St Joseph, MO, USA) can stimulate total bacterial numbers
by 34 % and cellulolytic bacterial numbers by 90 % in rumen fluid
(Newbold et al., 1992). Similarly, Salem et al. (2015) showed an increase of
43 % in microbial crude protein production of corn silage and concentrate
supplemented with the same mixture used in this study. Moreover, this
enzymatic additive increased the production of volatile fatty acids. A
similar effect was demonstrated for straws treated with xylanase (Sujani et al., 2015). The increased rate of fermentation of the insoluble fraction
following the use of this additive may be accelerated by the feed transit in the
rumen and consequently increase the energy intake of low-energy feed.
For olive leaves, the increased dose of EFE modified the gas
production from immediately soluble and insoluble fractions, the dry matter
degradability, the organic matter degradability, the metabolizable energy
value, the total volatile fatty acids, and the microbial crude protein
production. Only the medium dose increased the nutritional value of this
waste. The high dose significantly decreased the gas production from the
insoluble fraction by 7 % and microbial crude protein production by 6 %
compared with the control. The negative effect of the highest dose had also
been proven in vivo (Kung et al., 2000) and in vitro (Abid et al., 2019a, b). This
inhibiting effect may be due to the release of anti-nutritional elements of
the olive leaves, like the phenolic compounds, in the ruminal environment.
The latter at high concentrations may inhibit the proliferation of rumen
flora and partially reduce the activity of enzymes (Treacher and Hunt, 1996;
Molina-Alcaide and Yáñez-Ruiz, 2008). The high quantity of the xylan and
xylose released in the pre-incubation period may be causing furfural
(Mosier et al., 2005) and inhibited proliferation of rumen flora (Castro et
al., 1994). The excessive dose of EFE may block the attachment sites of
ruminal microorganisms and the action sites of the endogenous zone (Nsereko et al.,
2000; McAllister et al., 2001; Eun and Beauchemin, 2008).
Conclusions
The EFE hydrolysed cell-wall components, reduced sugars, and increased the
dry matter solubility of crude olive cake and olive leaves. Upon in vitro
incubation, it increased the fermentation, the degradability, the
metabolizable energy value, the volatile fatty acids, and the microbial
crude protein production. Further research work in these areas is needed to
verify the effectiveness of these enzymatic complexes with other fibrous
waste.
Code and data availability
The software code as well as the original data of the paper is available from the corresponding
author upon request.
Author contributions
KA, JJ, and HY performed the experiments. KA analysed the data. KA
wrote this paper. AM, JR, and MK supervised the research project.
Competing interests
The contact author has declared that neither they nor their co-authors have any competing interests.
Disclaimer
Publisher's note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Review statement
This paper was edited by Manfred Mielenz and reviewed by two anonymous referees.
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