INTRODUCTIONNapa cabbage (Brassica pekinesis L.) is one of themost cultivated agricultural products in Indonesia. In2014 its production reached 602 468 t. The local leaderin this field was the West Java province that yields14.92 tons of napa cabbage per ha [1]. Since over 20% ofnapa cabbage cannot be utilised [2], this waste amountmakes napa cabbage production inefficient.Napa cabbage waste contains the same essentialcomponent, namely, polysaccharides in the form ofcellulose, as napa cabbage itself. Cellulose is knownto be a constituent component of plant cell walls, and itaccount for as much as 30–50% of total lignocellulose [3].Currently, napa cabbage waste is used as animalfeed, while its value could be increased, e.g., throughproduction of cellulose-degrading enzymes.Cellulose-degrading enzymes can be producedfrom napa cabbage waste, which is high in cellulosecontent, by yeast. Enzyme production by indigenouscellulolytic yeast requires optimal conditions, however, itis influenced by external factors, especially, temperature.Thus, too low temperatures can inhibit enzymeproduction because of the plasma membrane fluiditydecrease which leads to disturbed metabolic activity [4].On the other hand, too high temperature can damagecells and the structure of proteins, which are constituentsof enzymes. The fact that temperature is an easilycontrolled parameter makes it possible to support yeastgrowth during the fermentation of napa cabbage wastefor cellulose-degrading enzyme production. Therefore,the aim of this research was to characterise and identifyindigenous yeast isolated from napa cabbage waste.STUDY OBJECTS AND METHODSThe object of the research was napa cabbage wastefrom the Gedebage Central Market in Bandung City,Copyright © 2019, Utama et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 InternationalLicense (http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix,transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license.Foods and Raw Materials, 2019, vol. 7, no. 2E-ISSN 2310-9599ISSN 2308-4057Research Article DOI: http://doi.org/10.21603/2308-4057-2019-2-321-328Open Access Available online at http:jfrm.ruIndigenous yeast with cellulose-degrading activityin napa cabbage (Brassica pekinensis L.) waste:Сharacterisation and species identificationGemilang L. Utama* , Widia D. Lestari, Indira L. Kayaputri,Roostita L. BaliaUniversitas Padjadjaran, Bandung, Indonesia* e-mail: g.l.utama@unpad.ac.idReceived July 31, 2019; Accepted in revised form August 27, 2019; Published October 21, 2019Abstract: Napa cabbage waste contains an organic component, cellulose, which can be utilised as an ingredient for cellulose-degradingenzyme production with the help of indigenous yeast. The aim of the research was to identify and characterise potential indigenousyeast isolated from napa cabbage waste, which has cellulose-degrading activity. Indigenous yeast were isolated and characterisedusing the RapID Yeast Plus System, then turbidity was used to determine the yeast total population. Indigenous yeast was grownat napa cabbage waste at 27, 37, and 40°C for three days, and cellulose-degrading activity was determined by the DinitrosalicylicAcid (DNS) method. The potential yeast isolate with the highest cellulose-degrading activity was identified by a sequence analysisof the rRNA gene internal transcribed spacer (ITS) region with using primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4(5′- TCCTCCGCTTATTGATATGC-3′). The results were compared to the GenBank database using the Basic Local Alignment SearchTools/BLAST algorithm. Three species of indigenous yeast were isolated from napa cabbage waste (S2, S6, and S8). S8, incubated at 37ºCfor three days, demonstrated the highest cellulose-degrading enzyme activity (1.188 U/mL), with the average activity of 0.684U/mL.Species identification results indicated that the S8 isolate had a 100% similarity to Pichia fermentans UniFGPF2 (KT029805.1).Keywords: Pichia fermentans, temperature, cellulase enzyme, internal transcribed spacerPlease cite this article in press as: Utama GL, Lestari WD, Kayaputri IL, Balia RL. Indigenous yeast with cellulose-degradingactivity in napa cabbage (Brassica pekinensis L.) waste: Сharacterisation and species identification. Foods and Raw Materials.2019;7(2):321–328. DOI: http://doi.org/10.21603/2308-4057-2019-2-321-328.322Utama G.L. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 321–328Indonesia. We used the following materials: PotatoDextrose Agar (PDA), Yeast and Mold Agar (YMA),Nutrient Broth (NB), Thermo Scientific RapIDYeast Plus System Kit, Carboxymethyle Cellulose(CMC), distilled water, 0.85% NaCl, DNS reagent(3.5-Dinitrosalicyclic acid), phosphate buffer solution(pH 7), gelatin, antibiotics, KH2PO4, and MgSO4.In our experiment we used nine treatments. Treatmentfactors were the type of yeast isolate and incubationtemperature (Table 1). The isolation process of indigenouscellulolytic yeast from each treatment lasted for threedays. The experiments were repeated three times.The selection of the best treatment was performedbased on quantitative analysis by determining thehighest value of enzyme activity using FactorialRandomized Block Design. According to the results ofisolation and identification of indigenous cellulolyticyeast from napa cabbage waste, descriptive analysis onthe total population of yeast during the production ofcellulose-degrading enzymes was conducted.Isolation and identification of indigenous yeast.The isolation of indigenous cellulolytic yeast from napacabbage waste was carried out using the direct platingmethod [5–6]. One gram of crushed napa cabbagewaste was added into 0.85% NaCl, inoculated into amodified PDA (PDA with a 3% yeast extract and 10 ppmantibiotics) and then incubated at 30°C for three days.The biochemical activities of the selected isolates werecharacterised with the help of RapID Yeast Plus SystemKit [7]. For species identification of potential indigenousyeast with the highest cellulose-degrading enzymeactivity we used rRNA gene internal transcribed spacer(ITS) region. Sequence analysis was carried out usingprimers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) asforward and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′)as reverse. DNA amplification was performed byMacrogen Inc. The results were compared with theGenBank database using the BLAST algorithm [8].Determination of total yeast population. Totalyeast population determination was carried out byturbidimetry: 1 mL of the liquid culture was taken fromthe enzyme production medium followed by absorbancemeasurement [9]. This method is based on thespectrophotometric measurement of the total populationat a wavelength of 600 nm [5].Determination of cellulose-degrading enzymeactivity. Cellulose-degrading enzyme production wascarried out by the International Union’s recommendedmethod of Pure and Applied Chemistry (IUPAC)with some modifications [10]. The salt media usedconsisted of KH2PO4, Mg2SO4 and gelatin. The napacabbage waste was incubated in salt media at (1:2, w/v)by adding 2% (v/v) of isolates [11]. The isolation wascarried out in an incubator at 27°C, 37°C and 45°C forthree days followed by stirring at 100 rpm for 60 minat room temperature. Then every 24 h the fermentedsolution was separated using a centrifuge to obtain crudecellulose-degrading enzymes in supernatant, wherecrude enzymes reacted with DNS (DinitrosalicylicAcid) reagent. Finally, spectrophotometric analysis wascarried out to obtain absorbance values which were usedto determine cellulose-degrading enzyme activity. Thecontrol used was 3 mL of DNS reagent that was dilutedto 25 mL by distilled water.RESULTS AND DISCUSSIONCharacterisation of indigenous yeast. Afterthree days of incubation, eight isolates with differentcharacteristics were obtained (Table 2). S2, S6 andS8 isolates displayed macroscopic morphologicalcharacteristics similar to those of yeast.Asliha and Alami state that macroscopically, yeastare round, white, with membranous colony texture,while microscopically, multilateral yeast bud and itscell size ranges from 1 to 7 μm [12]. In addition, themicroscopic analysis allowed three isolates to be chosenbecause they had cell size classified as that of yeast. Anaverage cell diameter of the S2, S6 and S8 was 3.87, 3.76and 4.24 μm, respectively (Fig. 1). The selected isolatesTable 1 Treatment factorsTypes of yeast isolates Incubation temperature27°C 37°C 45°CIsolate 1 (S2) A B CIsolate 2 (S6) D E FIsolate 3 (S8) G H ITable 2 Characteristics of indigenous yeast isolatesIsolate Macroscopic characteristicsS1 Fungi, long white hyphae, aerobic, colonisedS2 Round, broken white coloured, wet, aerobicS3 Round, broken white coloured, anaerobicS4 Oval, broken white coloured, anaerobicS5 Fungi, long white hyphae, aerobic, colonisedS6 Round, broken white coloured, aerobicS7 Round, yellow, anaerobicS8 Oval, yellow, anaerobicFigure 1 Macroscopic and microscopic images of selectedindigenous yeast9.355.114.3402468100 0,5 1 1,5 2 2,5 3 3,5× 109 CFU/mLdaysS211.3112S6323Utama G.L. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 321–328were purified, and their biochemical activities weretested using the RapID Yeast Plus System (Table 3).The identification results are based on thebiochemical properties of the isolates tested againstthe reacted compound. The glucose or glucosidehydrolysis ability was only shown by S2 isolates againstβ-Glucoside and β-Fucoside. Lopez et al. states thatseveral non-Saccharomyces yeast could be found insoil, fruits, trees, and damaged food or drink that hasglycolytic β-glucosidase activity [13]. The biochemicalproperties of indigenous yeast (Table 5) are alsosupported by Mateo et al., who found the glycolyticactivity, especially β-glucosidase activity, on indigenousyeas [14]. It implies that glucose can be hydrolysed intoacidic compounds which reduce pH until it changes thecolour of the resultants. Macroscopically, two isolatesidentified as S6 and S8 had different characteristics.They differed from the characteristics of Candida sp.that has the anamorphous properties, does not have asexual reproduction phase, and has unstable phenotypiccharacteristics [15]. Therefore, although the two isolateswere different in form, colour, and oxygen requirements,they had the same biochemical activity.Total population of indigenous yeasts. The resultsof indigenous yeasts total population determinationduring incubation are demonstrated in Fig. 2. Duringday 1 of incubation, the total population in alltreatments decreased because the isolates still werein the adaptation phase in the medium. This phase iscalled the lag phase or the cell adaptation period ofnew microorganisms to the environment [16]. Nguonget al. states that it takes 16 h for yeast with biochemicalactivity similar to that of the S2 isolate to adjust to a newenvironment [17].After the adaptation phase, the total population ofall treatments increased. The increase is the exponentialgrowth phase, where cells of microorganisms haveadapted to the environment and began to multiply sothat the number of mass cells or cell density increasesrapidly [16]. Spectrometric analysis preformed byKanti et al. revealed that the population of indigenousyeast, such as Candida, Rhodothorula, Pichia, andDebaryomyces, began to increase from 24th h andreached a plateau by the 96th h (OD 600 nm) [5].The highest total population was observed at theincubation temperature of 27°C in all the treatments.Mateo et al. state that the biochemical activity of theindigenous yeast was maximal at the temperature of30–40°C [14]. However, the isolate that belongs to theHanseniaspora genus that has biochemical activityTable 3 Identification of indigenous yeast from napa cabbagewasteIsolate S2 S6 S8Glucose + + +Maltose – – –Sucrose – – –Trehalose – – –Raffinose – – –Lipid – – –NAGA – – –αGlucoside – – –βGlucoside + – –ONPG – – –αGalactoside – – –βFucoside + – –PHS – – –PCHO – – –Urea – – –Prolyne – – –Histidine + + +Leucyl–Glycine – – –(+) assimilates the substrate positively; and (-) assimilates thesubstrate negativelyFigure 2 Total population of indigenous yeast: (1) – 27°C,(2) – 37°C, (3) – 45°C9.355.114.3402468100 0,5 1 1,5 2 2,5 3 3,5× 109 CFU/mLdaysS211.315.753.670369120 1 2 3× 109 CFU/mLdaysS610.888.274.3603690 1 2 3× 109 CFU/mLdaysS89.355.114.3402468100 0,5 1 1,5 2 2,5 3 3,5× 109 CFU/mLdaysS211.315.753.670369120 1 2 3× 109 CFU/mLdaysS610.888.274.3603690 1 2 3× 109 CFU/mLdaysS8 (1)(2)(3)(1)(2)(3)(1)(2)(3)DDD324Utama G.L. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 321–328similar to that of the S2 isolate had the maximumbiochemical activity at 28°C. Meanwhile, according toGänzle et al., a representative of the Candida genus withbiochemical activity similar to that of S6 and S8 isolatesgrew rapidly at 27°C [18].Cellulose-degrading enzyme activity. Cellulosedegradingenzyme activity of indigenous yeastis shown in Fig. 3. The highest enzyme activityproduced by S2 was 0.598 U/mL at an incubationtemperature of 45°C. The high temperature causedan increase in the rate of biochemical reactions,especially for indigenous yeast that has similarbiochemical activity with Hanseniaspora. Fennemastates that a high temperature affect variousreactions [19]. The enzyme belongs to the group ofmesozyme enzymes (in the range of 20–50°C) [20].According to López et al., the glycolytic activity(β-glucosidase) of H.guilliermondii at 28°C is about0.064–2.887 U/mL [13, 21].S6 isolates obtained at the incubation temperatureof 45°C also displayed a high enzyme activity. It isbecause the growth of Candida-like organisms occurredat the maximum temperature (40–45°C) [20]. As statedby Shuler and Kargi, enzymes are Growth-associatedproducts, i.e. the growth of microorganisms is directlyproportional to the product concentration [16]. However,the S8 isolate demonstrated the highest enzyme activitywhen treated at 37°C: its value was 1.203 U/mL on day1and 1.188 U/mL on day 2.Table 4 shows analysis of variance. F-value wasgreater than Pvalue probability (0.05), which indicatedthe presence of at least one treatment that significantlydiffered from the others. Hence, it required an additionaltest, namely, the Duncan Test.Table 5 demonstrates the Duncan Test results.According to the data, the S8 treatment incubated at37°C produced enzyme with an activity significantlydiffering from the other treatments. This is in accordancewith the result of Sulman and Rehman, that Candidalikeorganisms are able to produce cellulose-degradingenzymes with the highest activity at 37°C [11]. Thegrowth of isolates at 27°C cannot produce enzymeswith high activity because energy supply from theenvironment is low, while at 45°C the growth of isolatesis inhibited and the structure of the enzyme is denaturedso that the activity is not optimal. Therefore, incubationat 37°C gives enough energy for isolates to grow withoutdamaging the structure of the enzyme produced.Temperature greatly influences the enzymaticactivity and rigorous of yeast cell membranes, andFigure 3 Cellulose-degrading enzyme activity: (1) – 27°C,(2) – 37°C, (3) – 45°CTable 4 Analysis of varianceSource df Sum ofsquaresMeansquareF-value P-valueIsolate (I) 8 0.779 0.097 89.722* 2.07Temperature (T) 3 0.539 0.108 165.668* 2.74I*T 24 0.837 0.035 32.118* 1.67Replication 2 0.009 0.005 4.249 3.13Error 70 0.076 0.001Total: 107 2.240*significant0.2590.1150.5980.00.20.40.60 1 2 3U/mldaysS20.1250.1420.8790.92 3daysS60.1681.1880.8930.00.40.81.20 1 2 3U/mldaysS80.2590.1150.5980.20.40.61 2 3U/mldaysS20.1250.1420.8790.00.30.60 1 2 3U/mldaysS60.1681.1880.8930.00.40.81.20 1 2 3U/mldaysS80.00 0.9(1)(2)(3)(1)(2)(3)(1)(2)(3)Table 5 Duncan test resultsYeast Temperature Average cellulose-degradingenzyme activity (U/mL)SignificanceS8 37°C 0.684 aS8 45°C 0.395 bS6 45°C 0.384 bS2 45°C 0.315 cS2 27°C 0.196 dS2 37°C 0.160 deS6 37°C 0.146 eS6 27°C 0.131 eS8 27°C 0.129 eThe treatment marked with the same sign shows no significantdifference at the level of 5% according to the Duncan testDDD325Utama G.L. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 321–328S8 (Query 190511)Figure 4 Phylogenetic tree of S8 isolate326Utama G.L. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 321–328higher temperature can shorten the exponential phaseof yeast growth. In addition, higher temperature cancause denaturation of ribosomes and membrane fluidityproblems. Thus, 30–35°C is the optimal temperature foryeast metabolism, including the enzymatic activity [22].The difference in S2 and S8 enzyme activities wasdue to their different biochemical abilities. Lopez et al.found that Hanseniaspora sp., which is similar to theS2 isolate, was able to assimilate glycerol, galactoseand sucrose, unlike with Candida sp., which is similarto S8 [21, 23].The different activity of the enzyme produced byS6 and S8 could be caused by different phylogeneticsbetween the two isolates. According to Birmeta et al.,Candida sp. that was mentioned as C. krusei has closeproximity to P. fermentans having certainly differentbiochemical ability than C. krusei [24]. P. fermentanshas an anamorphic form, Candida lambica, but it isnot uncommon to find C. lambica mis-identification asC. krusei caused by similar biochemical abilities of theyeast. Nevertheless, C. lambica is able to assimilatexylose, compared to C. krusei cannot [25]. Meanwhile,the ability of yeast to assimilate xylose has not beendetermined by the RapID Yeast Plus System method, sodifferences between C. krusei and C. lambica have notbeen identified.Species identification of potential indigenousyeast with the highest cellulose-degrading activities.The identification of the S8 isolate resulted in the100% similarity to P. fermentans strain UniFGPF2(KT029805.1). The phylogenetic tree (Fig. 4) showsthat the S8 isolate is also similar to P. kluyveri cultureCBS:188 (KY104555.1), P. fermentans strain UniFGPF1(KT029804.1), P. fermentans strain UFLA CWFY24(KM402062.1), and P. fermentans strain YF12b(EU488722.1, DQ674358.1).P. fermentans have the ability to ferment andassimilate glucose, D-xylose, succinate, lactate, citrate,and glycerol [24]. Candida lambica is an anamorphicform of P. fermentans which can assimilate glucoseand xylose but cannot assimilate arabinose, galactose,and selobiosa [26]. In addition, Issatchenkia orientalis,a teleomorphic form of Candida krusei that usuallyincorrectly identified as Candida lambica, can assimilateglucose sufficiently but cannot assimilate galactose,maltose, sucrose, lactose, raffinose, and trehalose [27].According to Bengoa et al., despite P. fermentansand C. lambica can growth at a temperature up to37°C, the optimal temperature is 25–30°C [28]. Suchstrain as I. orientalis has the unique properties, as thismicroorganism can grow at a higher temperature level.Miao et al. reported that I. orientalist strains optimallygrows and produces a high amount of ethanol at 41°C,which indicates its thermostability [29].CONCLUSIONThree species of indigenous yeast were isolatedfrom napa cabbage waste. The highest cellulosedegradingenzyme activity (1.188U/mL) displayed theS8 isolate incubated at 37°C for three days. Its averagecellulose-degrading activity was 0.684U/mL. AccordingTo the species identification, the S8 isolate showeda 100% similarity to Pichia fermentans UniFGPF2(KT029805.1).CONFLICT OF INTERESTThe authors declare no conflict of interests.ACKNOWLEDGMENTSThe authors would like to thank the StudentResearch Group, Vivi Fadila Sari, Isfari Dinika andSyarah Virgina for their help with the experiments