Nova Biotechnol Chim (2021) 20(2): e898 DOI: 10.36547/nbc.898 1 Nova Biotechnologica et Chimica Molecular identification and technological properties of yeasts isolated from spontaneously fermented cassava waste pulp Adelodun L. Kolapo1,, Raoofat O. Salami2, Gbemisola O. Onipede1 1Department of Biological Sciences, Faculty of Science, Augustine University, Ilara-Epe, Lagos State, Nigeria 2Department of Microbiology, Faculty of Science, Olabisi Onabanjo University, Ago Iwoye, Ogun State, Nigeria  Corresponding author: adelodun.kolapo@augustineuniversity.edu.org Article info Article history: Received: 15th March 2021 Accepted: 10th September 2021 Keywords: Cassava waste pulp Livestock feed Molecular identification Technological properties Yeasts Abstract The aim of this work was to report on molecular identification and technological properties of the yeast flora isolated from spontaneously fermented cassava waste pulp. This was done with a view of obtaining yeast strains that could be used as a starter culture for the fermentation of cassava waste pulp. Molecular identification was based on the nucleotide sequence of the ITS region of the genomic DNA of the yeast isolates while the technological properties evaluated include linamarase (UmL- 1), gelatinase, and haemolytic activity, growth at pH 2.5, and tolerance to 2 % bile salt. All the representative five isolated yeasts were identified as Geotrichum silvicola KLP04, KLP06, KLP07, KLP08 and Geotrichum candidum KLP05. The isolates exhibited linamarase activity ranging between 3.3 and 4.2 with strain KLP04 having the highest value and strain KLP05 the least. None of the isolates demonstrated gelatinase and haemolytic activity except strain Geotrichum silvicola KLP08 which was partially haemolytic. All the examined yeasts exhibited good growth at pH 2.5, with strain KLP08 having the highest viable counts of 4.1 log10cfumL -1 and strain KLP04 the least value of 3.5 log10cfumL -1 after 72 h of growth. All the identified yeasts showed strain-specific tolerance to 2 % bile salt with strain KLP04 having the highest viable count of 4.3 log10cfumL -1 and strain KLP08 the least value of 2.2 log10cfumL -1 at the end of 72 h of incubation. Based on all the examined technological properties, Geotrichum silvicola KLP04 strain had the highest potential to be considered for starter culture for the fermentation of cassava waste pulp. © University of SS. Cyril and Methodius in Trnava Introduction The maize grain is a major feed grain and a standard component of livestock diets where it serves as a source of energy (Heuzé et al. 2017). For instance, its proportion in monogastric tropical diets could range between 50 and 70 % (PAN 1995). However, the high consumption of maize both by the human population and the livestock industries coupled with its low level of production have continuously generated a demand-supply gap with a concomitant price increase. The attendant effect of this scenario on the increased cost of animal feeds has been an age-long one for which cheaper alternative feedstuffs have been developed to replace the expensive conventional ones (Salami and Odunsi 2003). Due to their cheapness, agricultural wastes and by-products of food processing have become the first line of choice as unconventional feed materials in livestock mailto:adelodun.kolapo@augustineuniversity.edu.org Nova Biotechnol Chim (2021) 20(2): e898 2 industries. Examples of these materials include sorghum spent grains, wheat offals as well as cassava by-products. According to Lukuyu et al. (2014), cassava by-products that have found application in feeding livestock include cassava leaf, cassava leaf meal, cassava leaf protein concentrate, cassava peels, cassava stumps, cassava sievate and cassava pomace/pulp/bagasse/starch residue. Cassava meal provides dietary energy to over 500 million people in the world (ARC 2014). FAO (2001) reported that the global production of cassava in the year 2000 was 172 million tons with Africa accounting for 45 %, Asia 28 %, and Latin America and the Caribbean 19 %. Nigeria, Brazil, Thailand, Congo (DRC), and Indonesia are the five top producing countries. Current statistics show that Nigeria still accounts for 20 % of cassava global production (FAO 2020). In contrast to Latin America and Southeast Asia, where the majority of cassava is exported for industrial purposes or animal feed; about 70 to 80 % of cassava produced in Nigeria is utilized for human consumption (Dada et al. 2010) and only a reported 5 % of cassava was used as livestock feed (Apata and Babalola 2012). However, in recent times, the industrial potential of cassava to produce starches for textiles, pharmaceutical, food, alcohol, acetone, and dextrin industries are largely being exploited. This current trend is adding another stream of cassava waste to those that have been known to be generated from the processing of cassava tuber for human food. It has been estimated that the supply-demand gap for cassava starch and high-quality cassava flour in Nigeria are 290,000 and 485,000 metric tons (MT) per year, respectively (PWC 2020). This corresponds to the respective estimated generation of 870,000 and 485,000 MT of cassava pulp as waste annually. The high cyanogenic glycosides contents as well as low protein content of cassava wastes constitute a restraint to their full exploitation as livestock feed. Different processes found to be effective in reducing cyanogenic glycosides include sun- drying, ensiling and soaking plus drying (Tewe 1992; Salami and Odunsi 2003). In addition, fermentation of cassava products and by-product with starter microorganisms such as Saccharomyces cerevisiae and Lactobacillus spp. (Oboh 2006; Ubalua 2007), Trichoderma viride (Ezekiel et al. 2010), Aspergillus niger and Saccharomyces cerevisiae (Iyayi and Losel 2001), Rhizopus oligosporus and Aspergillus niger (Kolapo et al. 2021), and Rhizopus oryzae (Vlavonou 1988) resulted in a product with higher protein content, lower cyanogenic glycosides and phytate content. In addition, the inclusion of microorganisms with probiotic potentials to the animal diet is known to promote growth and enhance the performance of livestock (Nagpal et al. 2015; Arowolo and He 2018). In this regard, some yeasts, such as Saccharomyces cerevisiae (Alizadeh et al. 2016), selenium yeast and Phaffia rhodozyma yeast (Shurson 2018) have shown remarkable usefulness. Utilization of cassava and its wastes for livestock feeding has long been realized as various reports on their use for feeding poultry (Ravindran 1991; Salami and Odunsi 2003; Adeyemo et al. 2014; Omede et al. 2017), pigs (Iyayi and Tewe 1988; Unigwe et al. 2014) aquaculture (Solomon et al. 1999; Okoli 2020) and ruminants (Smith 1988; Lukuyu et al. 2014; Oloruntola et al. 2019) have been documented. In addition, with the increasing demand for cassava starch and high-quality cassava flour in Nigeria, many medium and large-scale industries are being attracted to the cassava starch and flour processing sector. In Nigeria, the cassava flour industries have been estimated to generate 870,000 and 485,000 MT of cassava waste pulp annually. Like previous cassava wastes generated from processing of cassava for human food, appropriate technologies might be deployed to ensure that this new stream of waste (cassava waste pulp) is incorporated into livestock feeding programs. In order to achieve this, the understanding of the yeasts profile and their technological properties of spontaneously fermented cassava waste pulp is necessary. To the best of our knowledge, investigation which focused on molecular identification of the yeast profile of spontaneously fermented cassava waste pulp has not been reported. Thus, this study aimed to report on molecular identification and technological properties of the yeast flora isolated from spontaneously fermented cassava waste pulp. This was done with a view of obtaining yeast strains that Nova Biotechnol Chim (2021) 20(2): e898 3 could be used as starter culture for the fermentation of cassava waste pulp. Experimental Preparation and spontaneous fermentation of cassava waste pulp Freshly harvested cassava tubers of the TMS 92/0067 variety were obtained from the Root and Tuber Expansion Programme of the International Institute of Tropical Agriculture, located in Ogere, Ogun State, Nigeria. The cassava tubers were harvested after 12 months of planting. The tubers were peeled by removing both the bark (outermost thin brown layer) and the cortex of cassava tuber. After peeling, 1 kg of cassava tubers was washed and grated using a mechanical grater to obtain cassava pulp. The pulp was mixed with 0.5 L of clean tap water and stirred vigorously. The resulting suspension was screened using a double layer of cheese cloth to extract the cassava starch. The residual mass was rinsed with excess water for about three times to extract as much starch as possible. The residue left thereafter was cassava waste pulp (CWP). The CWP was divided into three portions. Each portion was packed into a salt bag and the mouth tied up and left for 7 days for spontaneous fermentation to take place at the ambient temperature (30 ºC). Isolation of microorganisms Ten grams (10 g) of fermented CWP from each of the triplicate setups were homogenized in 90 mL of sterile buffered peptone water. The three homogenates were ten-fold serially diluted using the same diluent. Aliquots of serially diluted samples were pour-plated on Yeast and Mould Agar supplemented with 100 mg.L-1 chloramphenicol (Oxoid, Basingstoke Hampshire, UK). Distinct colonies were streaked on the medium of isolation twice to obtain pure cultures (Schwan et al. 2007). The ability of all the yeast isolates to produce linamarase enzyme was evaluated (as described in the subsequent section). Pure cultures of yeast isolates with significant linamarase activities were selected and maintained on agar slants at 4 ºC for further molecular characterization studies. Preliminary characterization of yeast isolates Yeast isolates were assessed macroscopically for colour and appearance. A microscopic examination was done to assess the presence/absence of hyphae and arthrospores. Molecular identification of isolates Genomic DNA extraction from overnight cultures of the isolates grown in Luria-Bertani broth (Acumedia, Michigan, USA) was performed by using the ZR Fungal/Bacterial DNA MiniPrep™ Kit (Zymo Research, California, USA) as described by Adedeji et al. (2017). Fresh cultures were centrifuged at 10,000 × g for 1 min. The microbial cells were lysed by bead beating in a lysis buffer, and the lysate was subsequently centrifuged. The supernatant was passed through a column matrix to allow for DNA binding. The bound DNA was purified and eluted from the column matrix. Agarose gel electrophoresis technique was used to verify the integrity of the eluted DNA, while quantification was carried out using Qubit 2.0 fluorometer (Thermo Fisher Scientific, Massachusetts, USA). PCR amplification of the internal transcribed spacer (ITS) region of genomic DNA of the yeast isolates was done using primers ITS1: 5′ TCCGTAGGTGAACCTGCGG 3′ and ITS4: 5′ TCCTCCGCTTATTGATATGC 3′ (White et al. 1990). PCR was performed in a total volume of 50 μL containing 30 – 50 ng DNA, 100 mM of each primer, 0.05 U.μL-1 Taq DNA polymerase, 4 mM MgCl2, and 0.4 mM of each dNTP. The amplification reaction was performed with a C1000 Touch thermal cycler (Bio-Rad Laboratories, California, USA). The thermal cycling condition used was an initial denaturation at 96 °C for 5 min, followed by 30 cycles of denaturation at 96 °C for 45 s, annealing at 56 °C for 30 s and extension at 72 °C for 2 min, followed by a final extension at 72 °C for 5 min and a holding period at 4 °C for infinite time. The PCR amplicons were analysed by electrophoresis in 1 % (w/v) agarose gel with ethidium bromide, 1 kb DNA ladders were loaded Nova Biotechnol Chim (2021) 20(2): e898 2 in 5 μL volumes, while 7 μL of the sample was loaded with 2 μL of loading dye. The gel was allowed to run for 2 h at 60 V. Gel results were visualized with a ChemiDoc™ MP System (Bio- Rad Laboratories, California, USA) to confirm the expected size of the product. The remaining PCR products were purified using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Germany). The sequencing of the purified PCR products was done with PRISM™ Ready Reaction Dye Terminator Cycle Sequencing Kit using the dideoxy chain termination method and electrophoresed with the model ABI PRISM® 3500XL DNA Sequencer (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. ChromasLite version 2.33 software was used for the analysis of electropherograms, (sense and antisense) resulting from sequencing reaction for good quality sequence assurance. The resulting electropherograms were edited using BioEdit Sequence Alignment Editor. After this, the resulting consensus sequences obtained were Blast in the NCBI (www.ncbi.nlm.nih.gov) database with the Basic Alignment Search Tool (BLASTn) for homology in order to identify the probable organism in question (Dabassa et al. 2019). These sequences were deposited in the GenBank (https://www.ncbi.nlm.nih.gov/genbank/) under accession numbers MW232914, MW233017, MW233034, MW233050, and OK070746. Phylogenetic analysis The phylogenetic analysis was based on the sequence of each species in order to establish relationships among them. The evolutionary history was inferred by using the Maximum Likelihood method and the Hasegawa-Kishino-Yano model (Hasegawa et al. 1985). The tree with the highest log likelihood (-2931.96) was shown. The percentage of trees in which the associated taxa clustered together was shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree was drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 6 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. There was a total of 485 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (Kumar et al. 2018). Evaluation of technological properties of isolated yeasts Linamarase activity The method described by Oyewole (2001) and O’Brien et al. (1991) was used to evaluate the ability of the yeast isolates to produce linamarase (EC 3.2.1.21). Linamarase activity of the isolates was quantitatively determined as β-glucosidase glycohydrolase. The estimation was carried out by measuring the release of p-nitrophenol from p- nitrophenyl-β-D-glucoside using a spectrophotometer. A loop full of 18h culture was added to 6 mL of 10g.L-1 of p-nitrophenyl-β-D- glucoside in 50 g.L-1 sodium citrate (pH 6.0) and incubated at 37 ºC for 15 min. Three milliliters of 0.1 M sodium carbonate (Na2CO3) were subsequently added to terminate the reaction. The absorbance was then measured at 420 nm using the Jenway model 6305 UV/VIS spectrophotometer. A standard curve enabled the conversion of the absorbance obtained to the quantity of p- nitrophenol released from p-nitrophenyl-β-D- glucoside. One unit of activity was defined as the amount releasing 1 μmol of p-nitrophenol from p- nitrophenyl-β-D-glucoside in 1 min under the assay condition. Haemolytic and gelatinase activity The yeast isolates were cultured in Malt Extract Broth at 37 °C for 12 – 18 h and then transferred onto blood agar (Difco, Michigan, USA) plates supplemented with 5 % defibrinated whole sheep blood as described by Yoon et al. (2008). After 24 – 48 h, the plates were examined for haemolytic reaction. A partial lysis of red blood cells and greening zone was recorded as α-haemolysis while a clear zone around yeast growth was taken as 4 http://www.ncbi.nlm.nih.gov/ https://www.ncbi.nlm.nih.gov/genbank/ Nova Biotechnol Chim (2021) 20(2): e898 3 β-haemolysis and no lysis was recorded as γ- haemolysis. Nutrient gelatin medium, consisting of gelatin, peptone, and beef extract was used for the determination of gelatinase activity. 3 mL of gelatin medium that had been previously adjusted to pH 6.8 using 0.1 M NaOH was dispensed into test tubes which were then autoclaved at 121 ºC for 15 min. The tubes were allowed to cool in an upright position. For 24 h, yeast cultures were inoculated into the tubes and incubated at 35 ºC for 48 h. A positive result was indicated by partial or complete liquefaction of the inoculated tube at 4 ºC. In a tube with a negative result, the content of the tube remained completely solidified at the end of refrigeration. Growth at low pH The ability of the yeast isolates to grow at low pH was evaluated using the method described by Conway et al. (1987). Fresh culture of yeast strains was inoculated into Malt Extract Broth (1 % v/v) with pH adjusted to 2.5 using 3N HCl. The inoculated broth was then incubated at 37 oC for 72 h. Viable cell counts of samples that were aseptically taken at 0, 24, 48, and 72 h were determined. Each determination was carried out in triplicates. Tolerance to bile salts The ability of the strains to grow in the presence of bile was determined according to the method of Conway et al. (1987). The procedure was carried out by inoculating 1% (v/v) fresh culture of selected yeast strains into Malt Extract Broth supplemented with 2% bile salts. This was followed by incubation at 37oC for 72 h. Viable cell counts were determined at 0, 24, 48, and 72 h. Each determination was carried out in triplicates. Statistical Analysis Data obtained was expressed as means ± standard deviation. Analysis of variance was carried out on the data obtained to determine the significance of differences. A two-tailed P-value of less than 0.05 was considered as statistically significant. Values that were significantly different were separated using the Duncan Multiple Range test using SPSS for windows Verson 17.0 statistical package. Results and Discussion A total of nineteen yeast isolates were obtained from the three samples of the investigated naturally fermented cassava waste pulp. Out of these, five isolates that demonstrated significant linamarase activity were further studied. The macroscopic appearance of the yeast isolates was creamy white with a glossy surface. Microscopic examination of the isolates revealed the presence of true hyphae and arthrospores. The blast of the sequences of yeasts isolated from naturally fermented Cassava Waste Pulp is shown in Table 1. The four isolates from this study were identified as Geotrichum silvicola while the fifth isolate was identified as Geotrichum candidum. Fig.1 shows the maximum likelihood phylogenetic tree. It describes the evolutionary relationship between and among strains of organisms based on their character. The marked strains denoted with, KLP04, KLP05, KLP06, KLP07 and KLP08, are those from the present study while the others are the related/similar strains retrieved from the GenBank. As shown in the tree, the strains from this study are not closely related to the strains already deposited in GenBank as the degree of relatedness (homology) they shared with the reference strains is less than 70 %, even though they share relatively high similarity (Stackebrandt and Goebel 1994). A previous report on phenotypic characterization of yeasts presented in some Nigerian traditional fermented foods such as burukutu, ogi, and pito confirmed the presence of Candida glabrata, Debaryomyces hansenii, Candida krusei, Candida colliculosa, Pichia anomala, Pichia farinosa, and Pichia membranefaciens (Alakeji et al. 2015). In a related development, Omemu et al. (2007) have isolated Saccharomyces cerevisiae, Candida krusei, Candida tropicalis, Geotrichum candidum, Geotrichum fermentans, and Rhodotorula graminis during the fermentation of maize for ogi production. Taxonomic characterization based on morphological, physiological, and biochemical data indicated the presence of Rhodotorula glutinis 5 Nova Biotechnol Chim (2021) 20(2): e898 2 (Fresenius) F. C. Harrison var. glutinis – a linamarin degrading yeast in cassava wastewater treatment lagoons (Vasconcellos et al. 2009). Previous characterization of yeast ecology of fermented cassava products has documented the presence of different species of the following genera: Saccharomyces, Candida, Hansenula, Penicillium, Geotrichum, Rhodotorula, Pichia, and Zygosaccharomyces (Amoa-Awua et al. 1997; Oyewole 2001; Coulin et al. 2006; Schwan et al. 2007). In another development, Galactomyces spp was reported to be one of the yeasts involved in the fermentation of sour cassava starch (Lacerda et al. 2005). However, Geotrichum silvicola sp. nov. has been described as a novel asexual arthroconidial yeast species related to the genus Galactomyces (Pimenta et al. 2005). Recently, Geotrichum silvicola has been isolated from spontaneously fermenting motoho, a southern African non- alcoholic sorghum beverage (Moodley et al. 2019). The present study is the first to report the presence of Geotrichum silvicola in cassava fermented products. Table 1. Molecular identification of yeasts isolated from spontaneously fermented cassava waste pulp based on the ITS region of the genomic DNA. Isolate Code Identity Organisms in GenBank with significant alignment with isolate Max. score Total score Query cover Identity [%] Accession number KLP04 Geotrichum silvicola Geotrichum silvicola CBS 9194 500 500 95 96.43 MW232914 KLP05 Geotrichum candidum Geotrichum candidum AUMC10284 472 472 98 93.48 OK070746 KLP06 Geotrichum silvicola Geotrichum silvicola CBS 9194 478 478 95 93.75 MW233017 KLP07 Geotrichum silvicola Geotrichum silvicola CBS 9194 461 461 95 92.90 MW233034 KLP08 Geotrichum silvicola Geotrichum silvicola CBS 9194 572 572 90 97.36 MW233050 Fig. 1. The maximum likelihood phylogenetic tree of the sequenced isolates and other reference strains retrieved from GenBank (*strains ending with KLP 04, KLP05, KLP06, KLP07, and KLP08 are isolates from the present study). 6 Nova Biotechnol Chim (2021) 20(2): e898 3 The linamarase activity (UmL-1) of the yeast isolated from fermented cassava waste pulp is shown in Table 2. The isolates exhibited activity ranging between 3.3 and 4.2 with strain KLP04 having the highest value and strain KLP05 the least. There is no significance difference among KLP06, KLP07, and KLP08. The value obtained in this report is within the range (0.5 – 5.8) reported by Nwokoro and Anya (2011) for different fractions of linamarase enzyme of Lactobacillus delbrueckii NRRL B-763. Various workers have reported that yeasts and lactic acid bacteria are the major microorganisms responsible for the fermentation of cassava (Essers et al. 1995; Amoa- Awua et al. 1996; Kobawila et al. 2005). The exhibited high linamarase activity of the isolated yeasts in the present study is indicating their capabilities to degrade the cyanogenic glycosides in cassava waste pulp; hence they could be used as starter cultures to produce fermented cassava waste pulp. Table 2. Linamarase (U.mL-1), gelatinase and haemolytic activity of yeasts isolated from fermented cassava waste pulp. Isolate code Linamarase activity Gelatinase activity Haemolytic activity KLP04 4.2 ± 0.2a Negative γ-haemolysis KLP05 3.3 ± 0.1c Negative γ-haemolysis KLP06 3.7 ± 0.1b Negative γ-haemolysis KLP07 3.6 ± 0.0b Negative γ-haemolysis KLP08 3.6 ± 0.1b Negative α-haemolysis Values are means ± standard deviation (n = 3). Within each column, values with different superscripts are significantly different. The gelatinase and haemolytic activities of the isolated yeasts are shown in Table 2. None of the isolates demonstrated the two activities except strain KLP08, which was partially haemolytic. The results obtained in this study, except for strain KLP08, are similar to the findings of Franz et al. (2001), Mannu et al. (2003) and Banwo et al. (2012) on enterococci of food origin. Gelatinase genes (Gel gene) may be silent, and phenotypes may be negative, even in the presence of Gel gene (Franz et al. 2001; Yoon et al. 2008). Most yeast present in food satisfies the important criteria of safety due to long history of safe human consumption in traditional fermented food products. Hence, yeasts have the Generally Regarded as Safe (GRAS) status (FDA 2001). All yeast isolates (except strain KLP08) in the present study could be tentatively assigned GRAS status as they exhibited safety potentials. The result of the present study underscores the absolute necessity to establish the safety status of every strain that will be used for food fermentation as intra-species variation was observed for haemolytic activity of the isolated yeasts. The quantitative determination of the effect of different conditions such as pH 2.5 and 2 % bile salt on the growth pattern of the isolated yeasts revealed strain-specific responses. All the examined yeasts exhibited good growth at pH 2.5 after the intervals of 24, 48, and 72 h (Fig. 2). At the end of 72 h of growth, strain KLP08 showed the highest viable counts of 4.1 log10cfu.mL -1 and strain KLP04 the least value of 3.5 log10cfu.mL -1. These results are comparable to the findings of Alakeji et al. (2015), who reported highest (6.78 log10cfu.mL -1) and least (2.18 log10cfu.mL -1) viable counts for Candida colliculosa PII and Pichia membranefaciens BA2 respectively after grew at pH 2.5 for 72 h. In a related development, Psomas et al. (2001) and Kourelis et al. (2010) reported that strains of Saccharomyces cerevisiae, Candida albicans and Debaryomyces hansenii have survived at pH 3.0. From the results obtained for the influence of 2 % bile salt on the growth pattern of the examined yeast (Fig. 3), all the yeasts showed strain-specific tolerance to 2 % bile salt. In this regard, strain KLP04 had the highest viable count 4.3 log10cfu.mL -1 and strain KLP08 the least value 2.2 log10cfu.mL -1 at the end of 72 h of incubation. Debaryomyces hansenii OA3 and Candida glabrata SPY3 were reported to have the highest (6.25 log10cfu.mL -1) and least (1.88 log10cfu.mL -1) viable counts respectively after grew under 2 % bile salts for 72 h (Alakeji et al. 2015). Similarly, in-vitro studies have shown that Wickerhamomyces anomalus survived excellently in >0.6 % bile salts (Garcia-Hernadenz et al. 2012; Bonatsou et al. 2015). Klaenhammer and Kullen (1999) had stated that important criteria for the selection of probiotic strains are the maintenance of their cell integrity and retaining of their beneficial metabolic functions during gastrointestinal passage. 7 Nova Biotechnol Chim (2021) 20(2): e898 2 Fig. 2. Effect of pH 2.5 on the growth of selected yeast strains. Fig. 3. Effect of 2 % bile salts on the growth of selected yeast strains. In this connection, there is the need for the strains to survive the natural barriers in the intestine, including body temperature, low pH and elevated bile concentration, as Czerucka et al. (2007) had stated that the stomach pH at fed state could be as low as 2.5 for 3 h. All the isolated yeasts examined in the present study had impressive growth at the body temperature of 37 ºC, low pH of 2.5 and under 2 % bile salt condition. This may confirm that they are eminently qualified as probable probiotic that could be used for the fermentation of cassava waste pulp for the production of animal feed. However, further consideration of their safety potentials may disqualify strain KLP08 on the ground of being partially haemolytic. Conclusion Based on all the technological properties examined in this study, Geotrichum silvicola KLP04 strain had the highest potential to be considered for starter culture for the fermentation of cassava waste pulp as it demonstrated the highest linamarase activity and tolerance to 2 % bile salt in addition to having good growth at pH 2.5 and demonstration of GRAS status. Conflict of Interest The authors declare that they have no conflict of interest. References Adedeji BS, Ezeokoli OT, Ezekiel CN, Obadinaa AO, Somorin YM, Sulyokd M, Adeleke RA, Warth B, Nwangburukah CC, Omemu AM, Oyewole OB, Krska R (2017) Bacterial species and mycotoxin contamination associated with locust bean, melon and their fermented products in south-western Nigeria. Int. J. Food Microbiol. 258: 73-80. Adeyemo AI, Sani A, Aderibigbe TA, Abdurrasheed MO, Agbolade JO (2014) A study of Aspergillus niger- hydrolyzed cassava peel meal as a carbohydrate source on the histology of broiler chickens. SpringerPlus 3: 31. Alakeji TP, Banwo K, Ogunremi OR, Sanni AI (2015) Functional properties of yeasts isolated from some Nigerian traditional fermented foods. J. Microbiol. Biotechnol. Food Sci. 4: 437-441. Alizadeh M, Rodriguez-Lecompte JC, Rogiewicz A, Patterson R, Slominski BA (2016) Effect of yeast-derived products and distillers dried grains with solubles (DDGS) on growth performance, gut morphology, and gene expression of pattern recognition receptors and cytokines in broiler chickens. Polut. Sci. 95: 507-517. Amoa-Awua WK, Frisvad JC, Sefa-Dedeh S, Jakobsen M (1997) The contribution of moulds and yeasts to the fermentation of 'agbelima' cassava dough. J. Appl. Microbiol. 83: 288-296. Amoa-Awua WKA, Appoh FE, Jakobsen M (1996) Lactic acid fermentation of cassava dough into agbelima. Int. J. Food Microbiol. 31: 87-98. Apata DF, Babalola TO (2012) The use of cassava, sweet potato and cocoyam, and their products by non-ruminants. Int. J. Food Sci. Nutr. 2: 54-62. Arowolo MA, He J (2018) Use of probiotics and botanical extracts to improve ruminant production in the tropics: a review. Animal Nutrit. 4: 241-249. ARC (2014) Cassava. Agricultural Research Council, Accessed 24/08/2021, https://www.arc.agric.za/arc- iic/Pages/Cassava.aspx. Banwo K, Sanni A, Tan H, (2012) Technological properties and probiotic potential of Enterococcus faecium strains isolated from cow milk. J. Appl. Microbiol. 114: 229-241. Bonatsou S, Benítez A, Rodríguez-Gomez F, Panagou EZ, Arroyo-Lopez FN (2015) Selection of yeasts with multifunctional features for application as starters in natural black table olive processing. Food Microbiol. 46: 66-73. 8 https://www.arc.agric.za/arc-iic/Pages/Cassava.aspx https://www.arc.agric.za/arc-iic/Pages/Cassava.aspx Nova Biotechnol Chim (2021) 20(2): e898 3 Conway PL (1987) Selection criteria for probiotic microorganisms. Asia Pacific J. Clin. Nutrit. 5: 10-14. Coulin P, Farah Z, Assanvo J, Spillmann H, Puhan Z (2006) Characterisation of the microflora of ‘attieke’, a fermented ´cassava product, during traditional small-scale preparation. Int. J. Food Microbiol. 106: 131-136. Czerucka D, Piche T, Rampal P, (2007) Review article: yeast as probiotics – Saccharomyces boulardii. Aliment. Pharmacol. Therapeut. 26: 767-778. Dabassa KA, Han DY, Bacha K, Bai FY (2019) Occurrence and molecular identification of wild yeasts from Jimma Zone, SouthWest Ethiopia. Microorganisms. 7: 633 Dada AD, Ali GA, Afolabi OO, Siyanbola WO (2010) Innovative approaches to industrial utilisation of cassava in a developing economy. Afri. J. Sci. Technol. Innov. Develop. 2: 154-174. Essers AJA, Bennik MHJ, Nout MJR (1995) Mechanisms of increased linamarin degradation during solid substrate fermentation of cassava. World J. Microbiol. Biotechnol. 11: 118-128. Ezekiel OO, Aworh OC, Blaschek HP, Ezeji TC (2010) Protein enrichment of cassava peel by submerged fermentation with Trichoderma viride ATCC 36316. Afr. J. Biotechnol. 9: 187- 194 FAO (2001) FAO Bulletin of Statistics. 2: 47-248. FAO (2020) FAO in Nigeria: Nigeria at a glance. FDA (2001) Partial list of microorganisms and microbial- derived ingredients that are used in foods. Franz CMAP, Muscholl-Silberhorn AB, Yousif NMK, Vancanneyt M, Swings J, Holzapfel WH (2001) Incidence of virulence factors and antibiotic resistance among enterococci isolated from food. Appl. Environ. Microbiol. 6: 4385-4389. García-Hernández Y, Rodríguez Z, Brandão LR, Rosa CA, Nicoli JR, Iglesias AE, Peréz-Sanchez T, Salabarría RB, Halaihel N (2012) Identification and in-vitro screening of avian yeasts for use as probiotic. Res. Vet. Sci. 93: 798- 802. Hasegawa M, Kishino H, Yano T (1985) Dating the human- ape split by a molecular clock of mitochondrial DNA. J. Mol. Evol. 22: 160-174. Heuze V, Tran G, Lebas F (2017) Maize grain Feedipedia, a programme by INRA, CIRAD, AFZ and FAO. https://www.feedipedia.org/node/556. Iyayi EA, Losel DM (2001) Protein enrichment of cassava by-product through solid state fermentation by fungi. J. Food Technol. Afr. 6: 116-118. Iyayi EA, Tewe OO (1988) Effect of protein deficiency on utilization of cassava peel by growing pigs. In Proceedings of the IITA/ILCA/University of Ibadan Workshop. 14-18 November, pp. 54-59. Klaenhammer TR, Kullen MJ (1999) Selection and design of probiotics. Int. J. Food Microbiol. 50: 45-57. Kolapo AL, Salami RA, Onajobi I, Oluwafemi F, Fawole AO, Adejumo OE (2021) Detoxification and nutritional enrichment of cassava waste pulp using Rhizopus oligosporos and Aspergillus niger The Annal. Universit. Dunarea de Jos of Galati Fascicle VI – Food Technol. 45: 52-68. Kourelis A, Kotzamanidis C, Litopoulou-Tzanetakis E, Scourasi ZG, Tzanetakis N, Yiangou M (2010) Preliminary probiotic selection of dairy and human yeast strains. J. Biol. Res.-Thessalon. 13: 93-104. Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Mol. Biol. Evol. 35: 1547- 1549. Lacerda ICA, Miranda RL, Borelli BM, Nunes AC, Nardi RMD, Lachance MA, Rosa CA (2005) Lactic acid bacteria and yeasts associated with spontaneous fermentations during the production of sour cassava starch in Brazil. Int. J. Food Microbiol. 105: 213-219. Lukuyu B, Okike I, Duncan A, Beveridge M, Blümmel M (2014) Use of cassava in livestock and aquaculture feeding programs. ILRI Discussion Paper 25, International Livestock Research Institute, Nairobi, Kenya, 83 p. Mannu L, Paba A, Daga E, Comunian R, Zanetti S, Dupre I, Sechi LA (2003) Comparison of the incidence of virulence determinants and antibiotic resistance between Enterococcus faecium strains of dairy, animal and clinical origin. Int. J. Food Microbiol. 88: 291-304. Moodley SS, Dlamini NR, Steenkamp L, Buys EM (2019) Bacteria and yeast isolation and characterisation from a South African fermented beverage. S. Afr. J. Sci. 115: 1-6. Nagpal R, Shrivastava B, Kumar N, Dhewa T, Sahay H (2015) Microbial feed additives. In Puniya AK (Eds.), Rumen microbiology: from evolution to revolution, Springer, India., pp. 161- 17. Nwokoro O, Anya FO (2011) Linamarase enzyme from Lactobacillus delbrueeckii NRRL B-763: purification and some properties of a β-glucosidase. J. Mex. Chem. Soc. 55: 246-250. O’Brien GM, Taylor AJ, Poulter NH (1991) Improved enzymic assay for cyanogens in fresh and processed cassava. J. Sci. Food Agric. 56: 277-289. Oboh G (2006) Nutrient enrichment of cassava peels using a mixed culture of Saccharomyces cerevisae and Lactobacillus sp. solid media fermentation techniques. Electronic J. Biotechnol. 9: 46-49. Okoli IC (2020) Cassava Waste as Feedstuff 1: Cassava peel production, processing, and nutrient composition. Research Tropica, Accessed 12/03/2021, https://researchtropica.com/cassava-waste-as-feedstuff-1/. Oloruntola OD, Ayodele SO, Jimoh OA, Agbede JO (2019) Dietary cassava peel meal, methionine, and multi- enzyme supplementation in rabbits’ nutrition: effect on growth, digestibility, and carcass traits. J. Basic Appl. Zool. 80: 46. Omede AK, Ahiwe EU, Zhu ZY, Fru-Nji F, Ilji PA (2017) Improving cassava quality for poultry feeding through application of biotechnology. In Waisundara VY (Eds.), Cassava, Intechopen, London. Omemu AM, Oyewole OB, Bankole MO (2007) Significance of yeasts in the fermentation of maize for ogi production. Food Microbiol. 24: 571-576. Oyewole OB (2001) Characteristics and significance of yeasts involvement in cassava fermentation for ‘fufu’ production. Int. J. Food Microbiol. 65: 213-218. 9 https://www.feedipedia.org/node/556 https://researchtropica.com/cassava-waste-as-feedstuff-1/ Nova Biotechnol Chim (2021) 20(2): e898 2 PAN (1995) Annual reports. Poultry Association of Nigeria, Lagos Nigeria. Pimenta RS, Alves PDD, Correa A, Lachance M, Prasad GS, Rajaram Sinha BR, Rosa CA (2005) Geotrichum silvicola sp. nov., a novel asexual arthroconidial yeast species related to the genus Galactomyces. Int. J. System. Evol. Microbiol. 55: 497-501. Psomas E, Andrighetto C, Litopoulou-Tzanetaki E, Lombardi A, Tzanetakis N (2001) Some probiotic properties of yeast isolates from infant faeces and Feta cheese. Int. J. Food Microbiol. 69 :125-133. PWC (2020) Harnessing the economic potential of cassava production in Nigeria. Accessed 06/10/2020. https://www.pwc.com/ng/en/publications/economic- potential-of-cassava-production-in-nigeria.html. Ravindran V (1991) Preparation of cassava leaf products and their use as animal feed. Roots, tubers, plantain and bananas in animal feeding. 111-125. Salami RI. Odunsi AA. (2003) Evaluation of processed cassava peel meals as substitutes for maize in the diets of layers. Int. J. Poult. Sci. 2: 112-116. Shurson G.C. (2018) Yeast and yeast derivatives in feed additives and ingredients: Sources, characteristics, animal responses, and quantification methods. Anim. Feed Sci. Technol. 235: 60-76. Schwan RF, Almeida EG, Souza-Dias MAG, Jespersen L (2007) Yeast diversity in rice-cassava fermentations produced by the indigenous Tapirape people of Brazil. FEMS Yeast Res. 7: 966-972. Smith OB (1988) A review of ruminant responses to cassava- based diets. In Proceeding of the IITA/ILCA/University of Ibadan Workshop. 14-18 November, pp. 39-53. Solomon SG, Lamai SL, Tiamiyu LO (1999) Studies on the nutritional value of cassava (Manihot utillisima) peels as energy source in the diet of Oreochromis niloticus fry fed in indoor glass aquaria. In 13th Annual Conference of the Fisheries Society of Nigeria (FISON), 3-8 November 1996 New Bussa, Nigeria, pp. 76-83. Stackebrandt E, Goebel BM (1994) A place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int. J. System. Bacteriol. 44: 846-849. Tewe OO (1992) Detoxification of cassava products and effects of residual toxins on consuming animals. In Machin D, Nyvold S (Eds.), Roots, tubers, plantains and bananas in animal feeding. Proceedings of the FAO Expert Consultation held in CIAT, Cali, Colombia 21-25 January 1991; FAO Animal Production and Health Paper, pp. 95. Ubalua AO (2007) Cassava wastes: treatment options and value addition alternatives. Afr. J. Biotechnol. 6: 2065- 2073. Unigwe CR, Fasanmi OG, Okorafor UP, Nwufoh OC, Oladele-Bukola MO (2014) Replacement value of sundried cassava peels meal for maize on growth performance and haematology of grower pigs. Schol. Academ. J. Biosci. 2: 27-32. Vasconcellos SP, Cereda MP, Cagnon JR, Foglio MA, Rodrigues RA, Manfio GP, Oliveira VM (2009) In vitro degradation of linamarin by microorganisms isolated from cassava wastewater treatment Lagoons. Braz. J. Microbiol. 40: 879-883. Vlavonou BM (1998) Cassava processing technologies in Africa. In Natalie DH (Eds.), Praise of cassava. Int. Inst. Trop. Agric., pp. 25-32. White TJ, Burns T, Lee S, Taylor J (1990) Amplification and sequencing of fungal ribosomal RNA genes for phylogenetics. In Innis MA, Gelfand DH, Sninsky JJ, White TJ (Eds.), PCR Protocols: A Guide to Methods and Applications, Academic Press Inc., New York, pp. 315-322. Yoon MY, Kim YJ, Hwang HJ (2008) Properties and safety aspects of Enterococcus faecium strains isolated from Chungkukjang, a fermented soy product. LWT-Food Sci. Technol. 41: 925-933. 10 https://www.pwc.com/ng/en/publications/economic-potential-of-cassava-production-in-nigeria.html https://www.pwc.com/ng/en/publications/economic-potential-of-cassava-production-in-nigeria.html http://www.feedipedia.org/node/8146 http://www.feedipedia.org/node/8146 http://www.feedipedia.org/node/8146 http://www.feedipedia.org/node/8142 http://www.feedipedia.org/node/8142 http://www.feedipedia.org/node/8142