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Article

Biocatalyst Potential of Cellulose-Degrading Microorganisms Isolated from Orange Juice Processing Waste

by
Ioanna Zerva
,
Nikolaos Remmas
and
Spyridon Ntougias
*
Laboratory of Wastewater Management and Treatment Technologies, Department of Environmental Engineering, Democritus University of Thrace, Vas. Sofias 12, 67132 Xanthi, Greece
*
Author to whom correspondence should be addressed.
Beverages 2019, 5(1), 21; https://0-doi-org.brum.beds.ac.uk/10.3390/beverages5010021
Submission received: 29 December 2018 / Revised: 3 February 2019 / Accepted: 11 February 2019 / Published: 2 March 2019
(This article belongs to the Special Issue Novel Beverages and Novel Technologies for Their Production)
Browse Figure
Versions Notes

Abstract

:
Cellulases can be applied as macerating and peeling enzymes in the orange juice processing industry. In this work, indigenous cellulose-degrading microorganisms were isolated from orange juice processing waste through successive enrichment procedures using carboxymethyl cellulose (CMC) as the sole carbon source. A total of 24 microbial isolates were screened for their ability to grow in CMC liquid medium, resulting in the selection of seven isolates. The latter were further assessed by determining their endo-1,4-β-d-glucanase, exo-1,4-β-d-glucanase, and β-1,4-d-glucosidase activities, of which their respective activities were as high as 3.89, 10.67, and 10.69 U/mg protein. All cellulose-degraders selected belonged to the genus Paenibacillus, although to distinct operational taxonomic units related to P. xylanexedens, P. tundrae, and P. pabuli (operational taxonomic unit—OTU#1) and to P. wynnii, P. odorifer, and P. donghaensis (OTU#2) spectrum. Regarding the cellulase activities of the orange juice processing waste, endo-1,4-β-d-glucanase activity (4.00 ± 0.11 U/g) was exerted only extracellularly, whereas exo-1,4-β-d-glucanase (2.60 ± 0.19 U/g) and β-1,4-d-glucosidase (5.69 ± 0.23 U/g) activities were exhibited both extracellularly and intracellularly. In conclusion, orange juice processing waste can be considered as a valuable source for the isolation of cellulose-degrading microbiota with potential uses in beverage industry, solid state fermentation and energy production.

1. Introduction

Oranges are one of the most cultivated and processed citrus fruits, with 23.89 million tons of orange juice being annually consumed worldwide [1]. Due to the high moisture content, which exceeds 80% of the fresh fruit biomass, oranges are considered to be easily spoiled crops. Thus, preservation approaches should be applied to expand their storage life. By valorizing orange fruits through conversion into various crop-processing products, mainly juice and sweet commodities, food lifetime duration is expanded, creating new business challenges [2].
In the recent years, the fruit juice industry has become one of the largest agro-industrial sectors worldwide. Indeed, an increased global interest has been sighted on juice production and commercialization [2,3]. Fruit juices mainly consist of long polymeric saccharides like cellulose, hemicellulose, pectin, and starch, which are considered the major constituents of orange dietary fibers [4]. Cellulose, a β-1,4-linked glucose-based biopolymeric carbohydrate, is the major plant structural polysaccharide and the most abundant biomolecule in the biosphere [5].
In beverage industries, orange crops are subjected to washing and thereafter to peeling, followed by chopping into pieces. At present, enzymatic methods for peeling are considered superior to chemical, thermal, and mechanical methods, due to the reduced flesh damage of the fruits and the lowered peeling losses, which increase the quantity and improve the quality of the produced juice [6]. Apart from the peeling of the fruits, cellulolytic enzymes can contribute to juice clarification and viscosity, which are crucial organoleptic characteristics of consumers’ preference, reducing crop processing cost and increasing fruit juice yield. In addition, such enzymes significantly meliorate the consistency and the turbidity of cloudy juices and concentrates [7].
The applicability of enzymes depends upon the polysaccharides composition of the various fruit juices. For instance, Navel orange pulp contains a total dietary fiber of 35.4–36.9% dry weight (d.w.), with cellulose, hemicellulose, pectin, and lignin accounting for 25.3%, 5.4%, 15.7–16.3%, and 2.2–3.0% d.w., respectively [2]. Moreover, the total protein and fat content of orange dietary fiber concentrate is estimated to be 8.75% and 1.48% d.w. respectively, whereas the inorganic content is equal to 2.8% d.w. [8]. Regarding orange peels, the pectin, hemicellulose, cellulose, lignin, protein, fat, and ash content accounts for 42.5%, 10.5%, 9.21%, 0.84%, 6.5%, 3.5%, and 2.0% d.w., respectively [9]. Mixtures of enzymes, such as cellulases, hemicellulases, and pectinases, have been widely applied as macerating enzymes in fruit juice industries. During juice processing, maceration of fruits with such enzymes is performed after both crushing and juice extraction, hydrolyzing polysaccharides like cellulose and pectins into mono- and di-saccharides [6]. Apparently, macerating enzymes are key agents to ameliorate juice quality from a broad variety of fruits treated in juice processing agro-industries [7,10]. Apart from maceration, commercial mixtures of such enzymes, for example, Peelzyms series (NOVO Nordisk Ferment), are known as peeling agents that have been widely used in processing citrus fruits, including oranges [11].
The hydrolytic cleavage of β-1,4-glycosidic bonds in cellulose is achieved by means of enzymes known as cellulase complexes. The latter is comprised of endo-1,4-β-d-glucanase (EC 3.2.1.4), which cleaves the internal bonds of cellulose moiety, exo-1,4-β-d-glucanase (EC 3.2.1.91), which cleaves the ends of the cellulose chain releasing cellobiose, and β-1,4-d-glucosidase (EC 3.2.1.21), which hydrolyzes cellobiose and cellulose-derived oligosaccharides from the non-reducing ends to glucose [10,12].
Considering orange peel microbiota, the dominant fungal taxa have been recently reported to be members of the genera Penicillium, Meyerozyma, Paraphaeosphaeria, and Candida [13]. In addition, Pichia terricola and Hanseniaspora opuntiae have been found to be the prevailing cultured yeast species during spontaneous fermentation of orange peel [14]. Penicillium sp. OP1 isolated from Japanese mandarin peels has been reported to be capable of degrading cellulose by 63% in a 2-week incubation period [15].
Paenibacillus spp. have also been reported to produce highly active and stable cellulases capable of degrading both soluble and microcrystalline cellulose at relatively low cost [16,17,18]. In addition, P. xylanexedens and P. tundrae are considered to be effective xylan- and cellulose-degrading bacteria [19]. Recent reports have also stated the biotechnological potential of cellulolytic enzymes from Paenibacillus spp. in juice clarification [20]. Despite the use of cellulases in fruit juice clarification [6], no indigenous cellulolytic bacteria, including Paenibacillus strains, have been isolated from orange juice processing waste until now. Only pathogenic to orange tree bacteria, (e.g., Xanthomonas axonopodis pv. citri, which causes citrus canker) have been found to exert low cellulase activities to penetrate their host (e.g., Citrus sinensis). Moreover, almost all the cellulose-degrading microorganisms used in the solid-state fermentation of orange peels/wastes are allochthonous fungi.
Thus, this work aims to uncover the cellulolytic potential of microbiota obtained from orange juice processing waste, by estimating their endo-1,4-β-d-glucanase, exo-1,4-β-d-glucanase, and β-1,4-d-glucosidase activities in order to find applications in food enzyme technology, in particular in the beverage industry as improvers of orange juice organoleptic properties, thus resulting in the valorization of the cellulose content of wastes generated by the orange juice processing industry.

2. Materials and Methods

2.1. Enrichment and Isolation of Cellulose-Degrading Microorganisms

An enrichment procedure was followed to isolate cellulolytic microorganisms from orange juice processing waste. This was obtained from an orange juice factory processing Navel variety fruits, where three independent bulk samples were combined. In particular, orange juice processing waste was subjected to ten-fold dilution (10 g to 100 mL) under aerobic conditions in a defined medium comprising of 2 g/L sodium carboxymethyl cellulose (CMC) (Sigma-Aldrich, St. Louis, MO, USA), 1.84 g/L Na2HPO4, 11.8 g/L KH2PO4, 1.4 g/L (NH4)2SO4, and 0.1 g/L MgSO4 and placed in an incubation chamber at 28 °C. After 1 week of incubation, 1 mL of the exhausted medium was inoculated to 99 mL of the above described minimal medium and incubated under the same incubation conditions. The enrichment procedure was finalized by further inoculating 1 mL of the second exhausted medium to 99 mL of the above-mentioned fresh cellulose-containing medium, followed by 1 week of incubation at 28 °C. The third exhausted medium obtained was subjected to ten-fold dilution series in order to isolate cellulolytic microorganisms, where cellulose-based medium was solidified with 1.7% w/v agar. Determination of physicochemical composition was performed as previously described by Ntougias et al. [21].

2.2. Cellulase Activities of Orange Juice Processing Waste and Cellulose-Degrading Microorganisms Isolated from this Orange Juice Waste

The conducted enrichment procedure resulted in the isolation of 24 cellulose-growing microorganisms, which were further evaluated in terms of their cellulolytic potential by assessing their cellulases activities induced in defined growth medium consisting of carboxymethyl cellulose (CMC) as the sole carbon and energy source. Despite that the isolated strains grew on CMC agar, 17 out of 24 isolated strains failed to sustain growth on liquid cultures, probably due to their ability to catabolize agar without cellulose assimilation. The remaining seven microbial isolates were further assessed through estimation of their extracellular and intracellular cellulolytic activities. To estimate such potential, endo-1,4-β-d-glucanase, exo-1,4-β-d-glucanase, and β-1,4-d-glucosidase activities were determined in their lysate and exhausted growth medium.
All cellulose-degrading microbiota obtained from orange juice processing waste were incubated in the aforementioned cellulose-based liquid medium for a period of 1 week. The biomass from each cellulolytic isolate was centrifuged at 12,000× g for 5 min and the exhausted broth was obtained in order to measure endo- and exo-cellulase activities. On the other hand, the centrifuged biomass was rinsed twice in 20 mM sodium acetate and further resuspended in the same buffer. The resuspended cells were sonicated for 15 min under 0.6 s pulse/0.4 s intervals, at 50% power level of a UP200S device (24 kHz frequency/200 W input power, Hielscher, Germany), as previously described in Remmas et al. [22]. Disrupted cells were removed by centrifugation at 15,000× g for 15 min (4 °C) and the cell-free lysate was collected for estimating intracellular cellulolytic activities. The protein content of both exhausted broth and cell-free lysate was determined according to the methods of Bradford [23]. To estimate the cellulolytic activities of orange juice processing waste, 10 g of this waste was mixed with 20 mL of 20 mM sodium acetate for 20 min at 200 rpm and then subjected to sonication under the previously mentioned conditions. Following centrifugation at 15,000× g for 15 min (4 °C), the biomass residues were removed and the lysate was used for downstream analysis. The same mixture was also prepared (although was not sonicated) in order to assess the extracellular cellulolytic activities of orange juice processing waste.
An aliquot of 1.6 mL 4 mM p-nitrophenyl-β-d-cellobioside (Apollo Scientific, Stockport, UK) dissolved in 0.02 M acetate buffer was mixed with 0.4 mL cell-free lysate or exhausted broth (orange juice processing waste extract, as appropriate) in order to assess the intracellular and extracellular exo-1,4-β-d-glucanase activities of the cellulolytic isolates (or orange juice processing waste), respectively. The enzymatic reaction was carried out at 40 °C by determining the absorbance change at 410 nm against blank [24]. The β-1,4-d-glucosidase activities were determined by following the same protocol as for the exo-1,4-β-glucanase activities, but using 4-nitrophenyl β-d-glucopyranoside as the cleavage substrate. A standard curve was constructed by measuring the color change of reference standards derived from 1 g/L p-nitrophenol solution, which was dissolved in 20 mM acetate buffer.
Endo-1,4-β-d-glucanase activity was assessed by adding 0.4 mL lysate or culture broth (orange juice processing waste extract), as appropriate, in 1.6 mL 0.5% w/v CMC (dissolved in 20 mM acetate buffer) and placed at 50 °C for 12 h [25]. A color reaction was achieved by boiling the mixture for a period of 15 min in the presence of 2 mL dinitrosalicylic acid (DNS) reagent, which was prepared by adding 10 g/L DNS, 2 g/L phenol, 0.5 g/L w/v Na2SO3, and 10 g/L NaOH [26]. The color developed was stabilized by adding 0.67 mL 1.42 M potassium sodium tartrate solution. The color change against blank was assessed at 540 nm and the standard curve was made by performing serial dilutions from 0.4% w/v glucose, which were further reacted and stabilized with DNS and potassium sodium tartrate solution, respectively.
Endo-1,4-β-d-glucanase as well as exo-1,4-β-d-glucanase and β-1,4-d-glucosidase activities are expressed as U/mg protein or U/g d.w., where 1 enzyme unit represents the amount of cellulase required for the release of 1 µmole/min of glucose or p-nitrophenol, respectively. In all enzyme assays, blanks containing cell-free lysate/exhausted broth (as appropriate) in the absence of substrate and substrate in the absence of cell-free lysate/exhausted broth (as appropriate) were included and samples’ estimated values were corrected for any background activity determined.

2.3. Phylogenetic Identification of Cellulose-Degrading Bacteria

Extraction of genomic DNA from cellulose-degrading microorganisms was carried out by employing the Macherey-Nagel’s “NucleoSpin Tissue” (Düren, Germany). PCR products were amplified in a TP600 thermocycler (TaKaRa, Shiga, Japan) through the use of the forward primer 341f (5′-CCT ACG GGA GGC AGC AG-3′) and the reverse primer 907r (5′-CCG TCA ATT CMT TTG AGT TT-3′) [27]. A reaction mixture of 50 μL was made by adding 1 μL of 40 ng genomic DNA, 10× buffer, 1.5 mM MgCl2, 0.2 mM dNTP, 0.5 μM each of the forward and reverse primer and 5 U Taq polymerase (Minotech Biotechnology, Heraklion, Greece). PCR amplification was achieved by placing the PCR mixture for 3 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 58 °C, and 30 s at 72 °C. The PCR reaction was completed by keeping the temperature at 72 °C for 5 min. The amplified PCR sequences were inserted into pMD20 vector (TaKaRa, Shiga, Japan) by TA-ligation and the ligated products were cloned into Escherichia coli DH5a cells provided by TaKaRa (Japan). Plasmid DNA was extracted from the transformed E. coli cells by employing the Macherey-Nagel’s “NucleoSpin Plasmid kit” (Germany). All sequencing reactions were carried out at Eurofins Genomics (Ebersberg, Germany). Amplicons were merged by the “CAP3 Sequence Assembly Program” of Huang and Madan [28] and assembled sequencing reads were deposited in GenBank (accession numbers MK332505-MK332511). The amplified 16S rRNA gene sequences were aligned with their related amplicons by using the “Clustal Omega” platform [29]. The phylogenetic tree was constructed by using the software MEGA 7 through the application of the Jukes-Cantor model and the inference of the phylogram topology by the “neighbor-joining” approach based on bootstrapping of 1000 trees [30,31,32].

2.4. Statistical Analysis of Enzyme Data

Analysis of variance (ANOVA) conducted by Duncan’s multiple comparison test was employed to evaluate cellulolytic activities among the cellulose-degrading bacteria isolated, whereas Student’s t-test was performed to assess significant differences between the bulk/surface and lysed orange juice processing waste.

3. Results and Discussion

A cultivation approach, which was based on the performance of an enrichment procedure, was employed in order to isolate cellulose-degrading microbiota from orange juice processing waste, whereas a range of enzymatic assays were performed to assess their cellulolytic potential through estimation of their endo-1,4-β-d-glucanase, exo-1,4-β-glucanase, and β-1,4-d-glucosidase activities. The cellulose-degrading microorganisms were obtained from orange juice processing waste, which was characterized by low pH of 3.86 ± 0.02, extremely high organic matter (e.g., 99.05 ± 0.02% w/w), and high total soluble and anthrone-reactive carbohydrate content of 51.97 ± 2.00 and 4.12 ± 0.04% w/w, respectively (Table 1). The nitrogen content of the orange juice processing waste was equal to 0.68 ± 0.05% w/w, resulting in an extremely high C/N ratio of 84.5. The total phosphorus and phenolics concentrations were estimated to be 12.68 ± 0.14 and 12.48 ± 0.14 mg/g d.w., respectively (Table 1).
Similar endo-1,4-β-d-glucanase activities were detected in the bulk/surface (3.71 U/g d.w.) and in the lysed (4.00 U/g d.w.) orange juice processing waste, indicating that internal cleavage of cellulose occurred only extracellularly. On the other hand, exo-1,4-β-d-glucanase, which unchains cellobiose units from cellulose and cellulose oligomers ends, and β-1,4-d-glucosidase, which cleaves cellobiose and other β-1,4-linked oligomers to release glucose, exhibited significantly higher activities in the lysed biomass than in the non-sonicated bulk (for p < 0.05 and p < 0.01, respectively) (Table 2). This intimates that such enzymes acted both extracellularly and intracellularly, due to the fact that small molecules like cellulose oligomers and cellobiose can insert into the cytoplasm, inducing exo-1,4-β-glucanases and β-1,4-glucosidases.
In comparison to previous studies, orange juice processing waste exerted among the highest cellulolytic activities reported in the literature for orange peel and pulp (Table 3). Dong et al. [33] estimated cellulase activity of 16.7 and 36.1 U/g f.w. in orange peels and pulp, respectively, which is approximately 38% and 34% lower and higher than the detected activity in the present research work (i.e., 26.9 U/g f.w.), respectively. In addition, the cellulase activities determined by Marín et al. [34] and Lei et al. [35] in orange peels and pulp respectively were 40-fold and 100-fold lower than the cellulase activity determined in the current study (Table 3).
None of the microbial strains growing in CMC liquid medium exhibited intracellularly any of the cellulolytic activities examined. The selected cellulolytic isolates showed a narrow range of extracellular endo-1,4-β-d-glucanase activities, fluctuating from 2.82 to 3.89 U/mg protein and from 1.52 to 3.03 U/L (Table 4). The highest endo-1,4-β-d-glucanase activity was expressed by the isolate CEL-11 (3.89 U/mg protein), whereas the respective lowest activities were exerted by the isolates CEL-3 and CEL-8 (2.87 and 2.82 U/mg protein, respectively), when the activity values were estimated as U/mg protein.
The seven selected microbial isolates exerted extracellular exo-1,4-β-d-glucanase activities, which ranged from 0.98 to 10.67 U/mg protein and from 0.29 to 17.70 U/L (Table 4). The highest exo-1,4-β-d-glucanase activity was exhibited by the isolate CEL-9 (10.67 U/mg protein whereas the lowest exo-1,4-β-d-glucanase activities were exhibited by the isolates CEL-7 and CEL-8 (0.98 and 1.96 U/mg protein, respectively).
The β-1,4-d-glucosidase activities of the selected microbial isolates ranged from 0.07 to 10.69 U/mg protein and from 0.10 to 12.55 U/L (Table 4). Among them, microbial strains CEL-7 and CEL-11 exhibited the highest β-1,4-d-glucosidase activities (9.52 and 10.69 U/mg protein, respectively), whereas CEL-1 and CEL-6 expressed the lowest activities (0.13 and 0.07 U/mg protein, respectively).
The selected microbial isolates exhibiting cellulolytic activity were phylogenetically characterized through partial small subunit ribosomal RNA gene sequencing. DNA from these isolates was amplified with primers targeting the 16S rRNA gene, indicating that the selected strains belong to bacteria. All selected isolates were placed within the spectrum of the genus Paenibacillus, although they were divided into two distinct operational taxonomic units (OTUs) (Figure 1). The most abundant out consisted of the isolates CEL-1, CEL-6, CEL-7, CEL-9, and CEL-11 (OTU#1), whereas OTU#2 was comprised of the isolates CEL-3 and CEL-8. Members of OTU#1 were identical to 16S rRNA gene (100% similarity) with the species Paenibacillus xylanexedens, P. tundrae, and P. pabuli, whereas OTU#2 isolates were associated with the species Paenibacillus wynnii, P. odorifer, and P. donghaensis, showing equal phylogenetic similarities of 99.8% (Figure 1).
The phylogenetic distinctness among the two OTUs is also reflected in the expression of endo-1,4-β-d-glucanase, where the respective activities of the isolates of OTU#2 were statistically lower than those of the members of OTU#1 (Table 4). However, intra-strain variation in the expression of endo-1,4-β-d-glucanase and β-1,4-d-glucosidase activities appeared to occur among members of each OTU (Table 4).
The cellulase activities determined in the current study were greater than those measured in allochthonous fungi, when expressed as U/mg. In particular, the endo-1,4-β-d-glucanase activities of the selected Paenibacillus species ranged within 2.82 to 3.89 U/mg, whereas the respective activities of Emericella variecolor, Aspergillus niger, A. fumigatus, A. awamori, Fusarium moniliforme, and Rhizopus oryzae strains fluctuated within 7 × 10−4 to 39.2 × 10−3 U/mg (Table 5). As expected, endo-1,4-β-d-glucanase activities were greater in allochthonous fungi when expressed as U/L (Table 5), since the Paenibacillus species of the present study were grown in CMC as the sole carbon and energy source in contrast to the fungal strains reported in Table 5, which were grown on the various carbonaceous substrates (apart from cellulose) of the orange peels and pulp.
Even though orange waste is considered to be a preferable substrate for solid state fermentation applications, attempts at isolating indigenous cellulolytic microbiota have been limited, probably due to the important fraction of sugar monomers and easily hydrolyzed oligomers (e.g., anthrone-reactive carbohydrates, Table 1), which are available to microorganisms, favoring the growth of non-cellulolytic microbial communities and restricting the isolation of the slow cellulase-producing microbiota. The implementation of the successive enrichment steps overcame this limitation, permitting the isolation of the most effective cellulose-degraders.
Thus, we can conclude that the high cellulase activities of Paenibacillus isolates selected in the current study demonstrate their biotechnological potential to serve as biocatalysts, whose application is not limited to juice clarification, but also in their valorization in solid state fermentation and energy recovery from cellulosic residues.

Author Contributions

I.Z. performed the physicochemical analysis of orange juice processing waste and the isolation of cellulose-degrading microorganisms; I.Z. and N.R. performed the enzyme assays and the phylogenetic analysis of cellulose-degrading microorganisms; S.N. designed and supervised the work; S.N., N.R., and I.Z. drafted and edited the paper, and read and approved the final manuscript.

Funding

This research was implemented through the Operational Program “Human Resources Development, Education and Lifelong Learning”, code MIS (OPS) 5006203, and was co-financed by the European Union (European Social Fund) and Greek national funds.

Acknowledgments

This work was carried out in the frame of the research project “Optimizing the energy recovery of waste from oranges juice industries using specialized native microorganisms as a starting culture”, code MIS (OPS) 5006203, which was implemented through the Operational Program “Human Resources Development, Education and Lifelong Learning” and was co-financed by the European Union (European Social Fund) and Greek national funds.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Food and Agriculture Organization of the United Nations. Citrus Fruit—Fresh and Processed, Statistical Bulletin, 2016; Food and Agriculture Organization of the United Nations: Rome, Italy, 2017. [Google Scholar]
  2. Toushik, S.H.; Lee, K.-T.; Lee, J.-S.; Kim, K.-S. Functional applications of lignocellulolytic enzymes in the fruit and vegetable processing industries. J. Food Sci. 2017, 82, 585–593. [Google Scholar] [CrossRef] [PubMed]
  3. Arsad, P.; Sukor, R.; Wan Ibadullah, W.Z.; Mustapha, N.A.; Meor Hussin, A.S. Effects of enzymatic treatment on physicochemical properties of sugar palm fruit juice. Int. J. Adv. Sci. Eng. Inf. Technol. 2015, 5, 308–312. [Google Scholar] [CrossRef]
  4. Vaillant, F.; Millan, A.; Dornier, M.; Decloux, M.; Reynes, M. Strategy for economical optimization of the clarification of pulpy fruit juices using crossflow microfiltration. J. Food Eng. 2001, 48, 83–90. [Google Scholar] [CrossRef]
  5. Huang, J.; Xia, T.; Li, G.; Li, X.; Li, Y.; Wang, Y.; Wang, Y.; Chen, Y.; Xie, G.; Bai, F.W.; et al. Overproduction of native endo-β-1,4-glucanases leads to largely enhanced biomass saccharification and bioethanol production by specific modification of cellulose features in transgenic rice. Biotechnol. Biofuels 2019, 12, 11. [Google Scholar] [CrossRef] [PubMed]
  6. Sharma, H.P.; Patel, H.; Sugandha. Enzymatic added extraction and clarification of fruit juices–A review. Crit. Rev. Food Sci. Nutr. 2017, 57, 1215–1227. [Google Scholar] [CrossRef] [PubMed]
  7. Avendaño, K.A.; Anguiano, M.; López, C.E.; Montañez, L.E.; Sifuentes, L.; Balagurusamy, N. Microbial enzymes: Applications in food processing. Agro Food Ind. Tech. 2016, 27, 63–67. [Google Scholar]
  8. Grigelmo-Miguel, N.; Martín-Belloso, O. Characterization of dietary fiber from orange juice extraction. Food Res. Int. 1998, 31, 355–361. [Google Scholar] [CrossRef]
  9. Cypriano, D.Z.; da Silva, L.L.; Tasic, L. High value-added products from the orange juice industry waste. Waste Manag. 2018, 79, 71–78. [Google Scholar] [CrossRef] [PubMed]
  10. Singh, R.; Singh, R.K. Role of enzymes in fruit juices clarification during processing: A review. Int. J. Biol. Technol. 2015, 6, 1–12. [Google Scholar]
  11. Sanchez-Bel, P.; Egea, I.; Serrano, M.; Romojaro, A.; Pretel, M.T. Obtaining and storage of ready-to-use segments from traditional orange obtained by enzymatic peeling. Food Sci. Technol. Int. 2012, 18, 63–72. [Google Scholar] [CrossRef] [PubMed]
  12. Mamma, D.; Christakopoulos, P. Biotransformation of citrus by-products into value added products. Waste Biomass Valorization 2014, 5, 529–549. [Google Scholar] [CrossRef]
  13. Wang, F.; Chen, L.; Liu, S.; Li, F.; Zhang, X.; Chen, H.; Liu, Y. Studying safe storage time of orange peel (Citrus reticulata) using high-throughput sequencing and conventional pure culture. Food Sci. Nutr. 2018, 6, 2545–2552. [Google Scholar] [CrossRef] [PubMed]
  14. Liu, R.; Zhang, Q.; Chen, F.; Zhang, X. Analysis of culturable yeast diversity in spontaneously fermented orange wine, orange peel and orangery soil of a Ponkan plantation in China. Ann. Microbiol. 2015, 65, 2387–2391. [Google Scholar] [CrossRef]
  15. Fujii, K.; Shintoh, Y. Degradation of mikan (Japanese mandarin orange) peel by a novel Penicillium species with cellulolytic and pectinolytic activity. J. Appl. Microbiol. 2006, 101, 1169–1176. [Google Scholar] [CrossRef] [PubMed]
  16. Maki, M.; Leung, K.T.; Qin, W. The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass. Int. J. Biol. Sci. 2009, 5, 500–516. [Google Scholar] [CrossRef] [PubMed]
  17. Grady, E.N.; MacDonald, J.; Liu, L.; Richman, A.; Yuan, Z.C. Current knowledge and perspectives of Paenibacillus: A review. Microb. Cell Fact. 2016, 15, 203. [Google Scholar] [CrossRef] [PubMed]
  18. Xu, J.; Xiong, P.; He, B. Advances in improving the performance of cellulase in ionic liquids for lignocellulose biorefinery. Bioresour. Technol. 2016, 200, 961–970. [Google Scholar] [CrossRef] [PubMed]
  19. Nelson, D.M.; Glawe, A.J.; Labeda, D.P.; Cann, I.K.O.; Mackie, R.I. Paenibacillus tundrae sp. nov. and Paenibacillus xylanexedens sp. nov., psychrotolerant, xylan-degrading bacteria from Alaskan tundra. Int. J. Syst. Evol. Microbiol. 2009, 59, 1708–1714. [Google Scholar] [CrossRef] [PubMed]
  20. Lee, J.P.; Seo, G.-W.; An, S.-D.; Kim, H. A cold-active acidophilic endoglucanase of Paenibacillus sp. Y2 isolated from soil in an alpine region. J. Appl. Biol. Chem. 2017, 60, 257–263. [Google Scholar] [CrossRef]
  21. Ntougias, S.; Papadopoulou, K.K.; Zervakis, G.I.; Kavroulakis, N.; Ehaliotis, C. Suppression of soil-borne pathogens of tomato by composts derived from agro-industrial wastes abundant in Mediterranean regions. Biol. Fertil. Soils 2008, 44, 1081–1090. [Google Scholar] [CrossRef]
  22. Remmas, N.; Melidis, P.; Voltsi, C.; Athanasiou, D.; Ntougias, S. Novel hydrolytic extremely halotolerant alkaliphiles from mature landfill leachate with key involvement in maturation process. J. Environ. Sci. Health A Tox. Hazard. Subst. Environ. Eng. 2017, 52, 64–73. [Google Scholar] [CrossRef] [PubMed]
  23. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254. [Google Scholar] [CrossRef]
  24. Baldrian, P.; Valášková, V.; Merhautová, V.; Gabriel, J. Degradation of lignocellulose by Pleurotus ostreatus in the presence of copper, manganese, lead and zinc. Res. Microbiol. 2005, 156, 670–676. [Google Scholar] [CrossRef] [PubMed]
  25. Ghose, T.K. Measurement of cellulase activities. Pure Appl. Chem. 1987, 59, 257–268. [Google Scholar] [CrossRef] [Green Version]
  26. Miller, G.L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 1959, 31, 426–428. [Google Scholar] [CrossRef]
  27. Muyzer, G.; Brinkhoff, T.; Nübel, U.; Santegoeds, C.; Schäfer, H.; Waver, C. Denaturing gradient gel electrophoresis (DGGE) in microbial ecology. In Molecular Microbial Ecology Manual; Akkermans, A., van Elsas, J., de Bruijn, F., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1998; pp. 1–27. [Google Scholar]
  28. Huang, X.; Madan, A. CAP3: A DNA sequence assembly program. Genome Res. 1999, 9, 868–877. [Google Scholar] [CrossRef] [PubMed]
  29. Sievers, F.; Wilm, A.; Dineen, D.; Gibson, T.J.; Karplus, K.; Li, W.; Lopez, R.; McWilliam, H.; Remmert, M.; Söding, J.; et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 2011, 7, 539. [Google Scholar] [CrossRef] [PubMed]
  30. Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol. Biol. Evol. 2016, 33, 1870–1874. [Google Scholar] [CrossRef] [PubMed]
  31. Jukes, T.H.; Cantor, C.R. Evolution of protein molecules. In Mammalian Protein Metabolism; Munro, H.N., Ed.; Academic Press: New York, NY, USA, 1969; pp. 21–132. ISBN 0125106017. [Google Scholar]
  32. Saitou, N.; Nei, M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1987, 4, 406–425. [Google Scholar] [CrossRef] [PubMed]
  33. Dong, T.; Xia, R.; Wang, M.; Xiao, Z.; Liu, P. Changes in dietary fibre, polygalacturonase, cellulase of navel orange (Citrus sinensis (L.) Osbeck ‘Cara Cara’) fruits under different storage conditions. Sci. Hortic. 2008, 116, 414–420. [Google Scholar] [CrossRef]
  34. Marín, M.; Sánchez, A.; Artola, A. Production and recovery of cellulases through solid-state fermentation of selected lignocellulosic wastes. J. Clean. Prod. 2019, 209, 937–946. [Google Scholar] [CrossRef]
  35. Lei, Y.; Liu, Y.; Zeng, W.; Deng, X. Physicochemical and molecular analysis of cell wall metabolism between two navel oranges (Citrus sinensis) with different mastication traits. J. Sci. Food Agric. 2010, 90, 1479–1484. [Google Scholar] [CrossRef] [PubMed]
  36. Srivastava, N.; Srivastava, M.; Manikanta, A.; Singh, P.; Ramteke, P.W.; Mishra, P.K.; Malhotra, B.D. Production and optimization of physicochemical parameters of cellulase using untreated orange waste by newly isolated Emericella variecolor NS3. Appl. Biochem. Biotechnol. 2017, 183, 601–612. [Google Scholar] [CrossRef] [PubMed]
  37. Gomes, H.A.R.; Silva, A.J.D.; Gómez-Mendoza, D.P.; Santos Júnior, A.C.M.D.; Cologna, N.D.M.D.; Almeida, R.M.; Miller, R.N.G.; Fontes, W.; Sousa, M.V.; Ricart, C.A.O.; et al. Identification of multienzymatic complexes in the Clonostachys byssicola secretomes produced in response to different lignocellulosic carbon sources. J. Biotechnol. 2017, 254, 51–58. [Google Scholar] [CrossRef] [PubMed]
  38. Attri, I.; Gurumayum, S.; Kaur, S.; Nagal, S.; Tuli, M. Utilization of orange peel and wheat straw for cellulase enzyme production from Aspergillus terreus MTCC 7600. Ecol. Environ. Conserv. 2016, 22, S297–S302. [Google Scholar]
  39. Li, P.-J.; Xia, J.-L.; Shan, Y.; Nie, Z.-Y.; Wang, F.-R. Effects of surfactants and microwave-assisted pretreatment of orange peel on extracellular enzymes production by Aspergillus japonicus PJ01. Appl. Biochem. Biotechnol. 2015, 176, 758–771. [Google Scholar] [CrossRef] [PubMed]
  40. Kannahi, M.; Elangeswari, S. Enhanced production of cellulase on different fruit peel under submerged fermentation. Int. J. Pharm. Sci. Res. 2015, 32, 161–165. [Google Scholar]
  41. Gutiérrez-Soto, G.; Medina-González, G.E.; García-Zambrano, E.A.; Treviño-Ramírez, J.E.; Hernández-Luna, C.E. Selection and characterization of a native Pycnoporus sanguineus strain as a lignocellulolytic extract producer from submerged cultures of various agroindustrial wastes. BioResources 2015, 10, 3564–3576. [Google Scholar] [CrossRef]
  42. Tsiklauri, N.; Khvedelidze, R.; Zakariashvili, N.; Aleksidze, T.; Bakradze-Guruli, M.; Kvesitadze, E. Higher basidial fungi isolated from different zones of Georgia—Producers of lignocellulosic enzymes. Bull. Georg. Natl. Acad. Sci. 2014, 8, 102–109. [Google Scholar]
  43. Delabona, P.D.S.; Pirota, R.D.P.B.; Codima, C.A.; Tremacoldi, C.R.; Rodrigues, A.; Farinas, C.S. Effect of initial moisture content on two Amazon rainforest Aspergillus strains cultivated on agro-industrial residues: Biomass-degrading enzymes production and characterization. Ind. Crops Prod. 2013, 42, 236–242. [Google Scholar] [CrossRef]
  44. Bansal, N.; Tewari, R.; Soni, R.; Soni, S.K. Production of cellulases from Aspergillus niger NS-2 in solid state fermentation on agricultural and kitchen waste residues. Waste Manag. 2012, 32, 1341–1346. [Google Scholar] [CrossRef] [PubMed]
  45. Díaz, A.B.; De Ory, I.; Caro, I.; Blandino, A. Enhance hydrolytic enzymes production by Aspergillus awamori on supplemented grape pomace. Food Bioprod. Process. 2012, 90, 72–78. [Google Scholar] [CrossRef]
  46. Omojasola, P.F.; Jilani, O.P. Cellulase production by Trichoderma longi, Aspergillus niger and Saccharomyces cerevisae cultured on waste materials from orange. Pak. J. Biol. Sci. 2008, 11, 2382–2388. [Google Scholar] [CrossRef] [PubMed]
  47. Niture, S.K.; Pant, A. Production of cell wall-degrading enzymes by a pH tolerant estuarine fungal isolate Fusarium moniliforme NCIM1276 in different culture conditions. World J. Microbiol. Biotechnol. 2007, 23, 1169–1177. [Google Scholar] [CrossRef]
  48. Hamdy, H.S. Purification and characterization of the pectin lyase secreted within the macerating fluid of Rhizopus oryzae (went & prinsen geerligs) grown on orange peel. Ind. J. Biotechnol. 2006, 5, 284–291. [Google Scholar]
  49. Saito, K.; Kawamura, Y.; Oda, Y. Role of the pectinolytic enzyme in the lactic acid fermentation of potato pulp by Rhizopus oryzae. J. Ind. Microbiol. Biotechnol. 2003, 30, 440–444. [Google Scholar] [CrossRef] [PubMed]
  50. Ismail, A.-M.S. Utilization of orange peels for the production of multienzyme complexes by some fungal strains. Process Biochem. 1996, 31, 645–650. [Google Scholar] [CrossRef]
Beverages 05 00021 g001 550
Figure 1. Phylogenetic position of cellulose-degrading bacteria isolated from orange juice processing waste.
Figure 1. Phylogenetic position of cellulose-degrading bacteria isolated from orange juice processing waste.
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Table
Table 1. Physicochemical composition of orange juice processing waste.
Table 1. Physicochemical composition of orange juice processing waste.
ParameterMean ± SE
pH *3.86 ± 0.02
Electrical Conductivity (μS/cm) *511 ± 0.33
Dry weight content (%)14.89 ± 0.05
Organic matter (% w/w)99.05 ± 0.02
Carbon (% w/w)57.45 ± 0.01
Ash content (% w/w)0.95 ± 0.03
Nitrogen (% w/w)0.68 ± 0.05
Soluble carbohydrates (% w/w)51.97 ± 2.00
Anthrone-reactive C (% w/w)4.12 ± 0.04
Phosphorus (mg/g d.w.)12.68 ± 0.14
Soluble phenolics (mg/g d.w.)12.48 ± 0.14
* 1/10 w/v, waste-to-water ratio.
Table
Table 2. Cellulolytic activities of orange juice processing waste prior and after sonication.
Table 2. Cellulolytic activities of orange juice processing waste prior and after sonication.
Endo-1,4-β-d-Glucanase Activity (U/g d.w.)Exo-1,4-β-d-Glucanase Activity (U/g d.w.)β-1,4-glucosidase Activity (U/g d.w.)
Sonicated waste4.00 ± 0.112.60 ± 0.195.69 ± 0.23
Non-sonicated waste3.71 ± 0.161.69 ± 0.163.93 ± 0.14
p-value0.2000.022 *0.003 **
Values are expressed as Mean ± SE; * and ** statistically significant for p < 0.05 and p < 0.01, respectively.
Table
Table 3. Cellulase activities exerted by orange peel and pulp.
Table 3. Cellulase activities exerted by orange peel and pulp.
SubstrateCellulase ActivityReference
Orange pulp6.5 mg/(g.min) or 36.1 U/g f.w.[33]
Orange peel3 mg/(g.min) or 16.7 U/g f.w.
Orange peel
(mixed with compost and bulking agent)		7.8 mg/(g.h) or 0.1 U/g d.w.[34]
Orange pulp0.29 U/g f.w.[35]
Orange juice processing waste
(orange peel)		4.0 U/g d.w. or 26.9 U/g f.w.Present study
U: μmoles glucose released/min.
Table
Table 4. Cellulolytic activities of cellulose-degrading microbial isolates obtained from orange juice processing waste.
Table 4. Cellulolytic activities of cellulose-degrading microbial isolates obtained from orange juice processing waste.
Endo-1,4-β-d-Glucanase ActivityExo-1,4-β-d-Glucanase Activityβ-1,4-Glucosidase Activity
IsolateU/mg proteinU/L brothU/mg proteinU/L brothU/mg proteinU/L broth
CEL-13.52 ± 0.05 (b)1.52 ± 0.02 (a)4.42 ± 0.11 (d)5.58 ± 0.14 (d)0.13 ± 0.02 (a)0.17 ± 0.02 (a)
CEL-32.87 ± 0.06 (a)2.02 ± 0.04 (b)5.55 ± 0.09 (e)8.38 ± 0.14 (f)6.30 ± 0.20 (c)9.51 ± 0.30 (b)
CEL-63.41 ± 0.10 (b)1.65 ± 0.05 (a)3.61 ± 0.07 (c)4.77 ± 0.09 (c)0.07 ± 0.03 (a)0.10 ± 0.04 (a)
CEL-73.64 ± 0.07 (b)2.84 ± 0.05 (c)0.98 ± 0.04 (a)1.30 ± 0.05 (b)9.52 ± 2.40 (d)12.55 ± 3.16 (b)
CEL-82.82 ± 0.10 (a)2.09 ± 0.07 (b)1.96 ± 0.26 (b)0.29 ± 0.04 (a)3.29 ± 0.08 (b)0.49 ± 0.01 (a)
CEL-93.40 ± 0.10 (b)3.03 ± 0.09 (d)10.67 ± 0.14 (f)17.70 ± 0.23 (g)7.26 ± 0.24 (c)12.04 ± 0.40 (b)
CEL-113.89 ± 0.08 (c)2.84 ± 0.06 (c)5.59 ± 0.08 (e)6.20 ± 0.09 (e)10.69 ± 0.04 (d)11.86 ± 0.04 (b)
Values are expressed as Mean ± SE; Lack of letter in common within the same column denotes statistically significant differences at p < 0.05.
Table
Table 5. Cellulase activities of microorganisms growing on orange peels, pulp and residues.
Table 5. Cellulase activities of microorganisms growing on orange peels, pulp and residues.
Cellulase-Producing MicroorganismFermentative SubstrateCellulase ActivityIsolation SourceReference
Xanthomonas axonopodis pv. citri Xac 306citrus pulp0.12 U/mLhost: sweet orange[9]
Emericella variecolor NS3orange peel31 U/grotten wood[36]
Clonostachys byssicola RCFS6orange peel0.27 U/mLsoil[37]
Aspergillus terreus MTCC 7600orange peel15.8 U/mLsoil[38]
Aspergillus japonicus PJ01orange peel1.6 U/mLsoil[39]
Aspergillus niger strain-Trichoderma viridae strainorange peel0.30 U/mL-0.62 U/mLsoil[40]
Pycnoporus sanguineus CS2orange peel0.16 U/mLoak forest[41]
Pleurotus ostreatus GK10-Fomes sp. KA20-Ganoderma sp. GM 04orange peel22.1 U/mL-17.3 U/mL-5.9 U/mLsoil[42]
Aspergillus niger P47C3-Aspergilus fumigatus P40M2orange bagasse39.2 U/g 39.0 U/gsoil[43]
Aspergillus niger NS2-2orange peel1.0 U/gagricultural residues[44]
Aspergillus awamori 2B.361 U2/1orange peel2.8 U/gnot reported[45]
Trichoderma longibrachiatum strain-Aspergillus niger strain-Saccharomyces cerevisiae strainorange albedo, orange pulp & orange peel1.8, 1.3 & 1.1 U/mL-1.9, 1.6 & 1.2 U/mL-1.6, 1.4 & 1.1 U/mLrotten wood and palm wine[46]
Fusarium moniliforme NCIM1276wheat bran & orange pulp27.5 U/gsoil[47]
Rhizopus oryzae strainorange peel2.4 U/gsoil[48]
Rhizopus oryzae NBRC 4707orange peel0.7 U/gnot reported[49]
A. niger A-20-A. oryzae 1911-Memnoniella sp. 6-P. chrysogenum 3486-P. oxalicum 7dried orange peels1.3 U/mL-1.1 U/mL-3.4 U/mL-0.9 U/mL-2.4 U/mLnot reported[50]

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Zerva, I.; Remmas, N.; Ntougias, S. Biocatalyst Potential of Cellulose-Degrading Microorganisms Isolated from Orange Juice Processing Waste. Beverages 2019, 5, 21. https://0-doi-org.brum.beds.ac.uk/10.3390/beverages5010021

AMA Style

Zerva I, Remmas N, Ntougias S. Biocatalyst Potential of Cellulose-Degrading Microorganisms Isolated from Orange Juice Processing Waste. Beverages. 2019; 5(1):21. https://0-doi-org.brum.beds.ac.uk/10.3390/beverages5010021

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Zerva, Ioanna, Nikolaos Remmas, and Spyridon Ntougias. 2019. "Biocatalyst Potential of Cellulose-Degrading Microorganisms Isolated from Orange Juice Processing Waste" Beverages 5, no. 1: 21. https://0-doi-org.brum.beds.ac.uk/10.3390/beverages5010021

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