Semi-industrial Scale Production of a New Yeast with ProbioticTraits, Cryptococcus sp. YMHS, Isolated from the Red Sea

Ashraf F. El-Baz1 & Hesham A. El-Enshasy2,3 & Yousseria M. Shetaia4 & HodaMahrous1 &

Nor Zalina Othman2& Ahmed E. Yousef5

# Springer Science+Business Media New York 2017

Abstract A new yeast strain with promising probiotic traitswas isolated from the Red Sea water samples. The isolate(YMHS) was subjected to genetic characterization and iden-tified asCryptococcus sp. Nucleotide sequence analysis of therRNA gene internal transcribed spacer regions showed 95%sequence similarity between the isolate and Cryptococcusalbidus. Cryptococcus sp. YMHS exhibited desirable charac-teristics of probiotic microorganisms; it has tolerance to lowpH in simulated gastric juice, resistance to bile salts, hydro-phobic characteristics, broad antimicrobial activity, andin vitro ability to degrade cholesterol. The isolate grew wellin a semi-defined medium composed of yeast extract, glucose,KH2PO4, (NH4)2SO4, and MgSO4, yielding cell mass of 2.32and 5.82 g/l in shake flask and in bioreactor cultures, respec-tively. Fed-batch cultivation, with controlled pH, increased thebiomass gradually in culture, reaching 28.5 g/l after 32 h cul-tivation. Beside the feasible use as a probiotic, the new strainalso could be beneficial in the development of functionalfoods or novel food preservatives. To our knowledge, this is

the first report of yeast with probiotic properties isolated fromthe Red Sea.

Keywords Cryptococcus sp. . Probiotic .Marine yeast .

Biomass production

Introduction

The marine environment is a rich source of diverse microor-ganisms that could be of industrial importance. Marine micro-biota may produce valuable bioactive secondary metabolites[1] and generally are considered nonpathogenic to animalsand humans. Therefore, microbial isolates from this environ-ment deserve investigating as probiotic candidates.

The World Health Organization (WHO) and Food andAgriculture Organization (FAO) of the UnitedNations definedprobiotics as Blive microorganisms which when consumed inadequate amounts as part of food confer a health benefit on thehost^ [2]. Although numerous species of lactic acid bacteriaare prominent probiotic organisms, there is an increasing in-terest in yeasts as probiotics. It is common to find yeasts withbeneficial effects such as inhibition of spoilage microorgan-isms, degradation of cholesterol, and reduction of oxidativedamage [3]. Yeasts are used increasingly as probiotics in thelivestock and aquaculture industries where surplus biomassesfrom the fermentation industry are recycled as additives tocattle, pigs, and poultry diets [4, 5]. Yeasts, together withbacteria, are found in fermented dairy products and may con-tribute to the sensory characteristics of kefir, koumiss, andseveral cheese varieties [6]. In the human body, yeasts cangrow rapidly in the intestine and produce extracellular prote-ases, siderophores, and antifungal agents [7]. These intestinalyeasts play a significant role in antagonizing undesirable mi-crobiota such as enteropathogenic bacteria [8]. Similar to the

* Ashraf F. El-Bazashraf.elbaz@gebri.usc.edu.eg; ashrafhawase@gmail.com

1 Department of Industrial Biotechnology, Genetic Engineering andBiotechnology Research Institute, University of Sadat City,Sadat, Egypt

2 Institute of Bioproducts Development, Universiti TeknologiMalaysia, Skudai, Johor, Malaysia

3 Biopocess Development Department, City for Scientific Research,New Burg Al Arab, Alexandria, Egypt

4 Department of Microbiology, Faculty of Science, Ain ShamsUniversity, Cairo, Egypt

5 Department of Food Science and Technology, The Ohio StateUniversity, Columbus, OH, USA

Probiotics & Antimicro. Prot.DOI 10.1007/s12602-017-9291-9

well-known bacterial probiotics, some yeast species, such asDebaryomyces hansenii , Torulaspora delbrueckii ,Kluyveromyces lactis, Kluyveromyces marxianus, andKluyveromyces lodderae, have tolerance to passage throughthe gastrointestinal tract (GIT) and inhibition capabilities toenteric pathogens [3].

Yeasts isolated from marine environments are expected toshow additional capabilities compared to those isolated fromother sources [9]. Some marine yeasts are known to producebioactive substances such as glutathione, glucans, toxins, ami-no acids, enzymes, and vitamins with potential application inthe pharmaceutical, food, chemical, and cosmetic industries[8, 10, 11]. Yeasts that colonize the intestine of marine animalsare ideal candidates as probiotic organisms. It has been report-ed that yeasts isolated from the intestine of rainbow trout mayadhere to and grow in intestinal mucus [12]. Some yeast cellscan colonize the intestine of fish after dietary introduction[13], and this ability to colonize may be related to cell surfacehydrophobicity [14] and the ability of the strains to grow onmucus [12, 15].

The yeasts Metschnikowia zobelii, Kloeckera apiculata,and Debaryomyces spp. dominate in some marine fish(Tachurus symmetricus and Atherinopis affinis littoralis),and in these fish species, the yeast concentration was signifi-cantly higher inside the fish than in the surrounding sea water,suggesting that the yeast may grow inside the fish intestine[16]. The objectives of the present study were to isolate andidentify acid-tolerant yeast from the marine environment andevaluate its probiotic properties. Moreover, the feasibility ofindustrial mass production of this yeast was assessed throughinvestigating its growth characteristics at the small- and pilot-scale fermentation levels.

Materials and Methods

Isolation and Identification of Marine Yeast

Sample Collection and Cultivation

Different water samples were collected near the Red Sea town,Hurghada (27.2578° N, 33.8117° E) during the summer.Sampling was done under depth of 10 m (salinity 3.5%) at25 °C and pH 8.0. Isolation of the marine yeast was carried outas described by Wang et al. [17] with some modifications.Immediately after sampling, seawater was filtered, usingWhatman no. 1 filter paper, and 2 ml of the filtrate was mixedwith 20 ml yeast peptone dextrose (YPD) broth (preparedusing fresh sea water) containing (g/l) glucose, 10;polypeptone, 20; yeast extract, 10; and pH 5.5. This mediumwas supplemented with 0.5 g/l chloramphenicol to inhibitbacterial growth. After 5 days of incubation at 28 °C, suitabledilutions were prepared and plated onto a yeast malt (YM)

agar medium composed of (g/l) peptone, 5; glucose, 10; maltextract, 3; yeast extract, 3; agar, 20; and pH 5.5. Afterautoclaving, chloramphenicol (100 mg/l) was added underaseptic conditions. The most abundant yeast isolates were se-lected for further study.

DNA Amplification and Sequencing of ITS1-5.8S-ITS2Region

The isolate was identified using nucleotide sequence analysisof the specific amplification of the ribosomal RNA (rRNA)internal transcribed spacer regions [18]. PCR amplification ofthe ITS1-5.8S-ITS2 rRNA region was carried out using for-ward (ITS1: 5′-TCC GTA GGT GAA CCT GCG G-'3) andreverse (ITS4: 5′-TCC TCC GCT TAT TGA TAT GC-'3)primers [19]. PCR products were purified (AccuPrep PCRpurification kit; Bioneer, Daejeon, South Korea) and se-quenced with automated DNA sequencer (ABI prism® 310genetic analyzer, USA).

Phylogenetic Analysis

A comparative analysis of the sequenced PCR product againstvarious sequence databases (http://www.ncbi.nlm.nih.gov/BLAST) was used to determine the phylogenetic similaritiesof theCryptococcus isolate to the DNA sequences database. Aphylogenetic tree was then constructed using the neighbor-joining method [20]. Tree topology was evaluated using abootstrap analysis of the sequence data with a multiple se-quence alignment program, Clustal W software.

Evaluation of Probiotic Properties

Acid Tolerance in Simulated Gastric Juice

The simulated gastric juice (SGJ) was prepared by suspendingpepsin (3 g/l) in saline solution [21]. The pH was adjusted atdifferent values, in the range of 2.0 to 4.0, using 12 M HCl.Saline solution was used instead of SGJ as a control. Theprepared solutions were sterilized by filtration using a micro-biological membrane filter of 0.22 μm pore diameter(Millipore, Billerica,MA, USA). One millilter of concentratedcell suspension (containing 5 × 1010 cells) obtained from 10-hgrown cells was added to 9 ml of SGJ and incubated at 37 °C.Samples were taken at different time intervals and diluted inphosphate buffer solution (PBS, pH 5.5) before plating the cellsuspension on YM agar medium. After 24 h incubation at37 °C, the resulting colonies were counted.

Bile Salt Tolerance

Cell tolerance to different concentrations of bile salt (0.2–2.0%) was determined using YM broth supplemented by

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Oxgal (Difco Lab., Detriot, MI, USA). The prepared media,containing different bile salt concentrations, were sterilized byautoclaving at 121 °C for 10 min. One millilter of cell suspen-sion (containing 5 × 1010 cells), obtained after 10-h growth,was added to test tubes containing 9 ml of YM-bile salt brothand the mixture was incubated at 37 °C for 4 h. The number ofviable cells was determined by taking samples, plating on YMagar, and incubating at 37 °C for 24 h.

Antibacterial Activity

Antibacterial activity was determined using agar well-diffusion assay [22] against targeted bacteria. In this method,plates were prepared by adding 2 ml (~105 cfu/ml) of over-night cultures into 200 ml of plate count agar medium (PCA,Oxoid, UK) held at 45 °C. Inoculated medium was dispendedimmediately into sterile 8.5 cm diameter Petri dishes. Wells of3 mm diameter were made after agar solidification and 30 μlaliquots of the filter-sterilized supernatant of the yeast over-night culture of targeted microorganisms were dispensed intoindividual wells. Plates were incubated for 2 h at 4 °C andsubsequently overnight at 37 °C and diameters of the inhibi-tion zones were measured.

Anti-yeast Activity

Ability of the yeast isolate to inactivate other yeasts was testedas described by Woods and Bevan [23]. Candida albicansNCTC 10231 and Sacharomyces cereviseae ATCC 934 wereused as the target yeasts. One milliliter of the broth cultures,containing about 1 × 108 cells of the targeted yeasts was mixedin a Petri dish with 20 ml of YM agar medium buffered atpH 5.0 (0.1 M citric acid phosphate) which contains 0.003%methylene blue. Wells (7 mm in diameter) were made in theagar plate and each well was filled with 100 μl of the testedyeast culture filtrate and incubated at 28 °C for 48 h. The anti-yeast activity was recognized by a region of clearing boundedby colored cells surrounding the wells.

Hydrophobicity Assay

Considering the correlation between adhesion ability andsurface hydrophobicity, this test can reveal a useful fea-ture of the isolate as potential probiotic yeast. Afterthree transfers of the yeast isolate in YPD broth at28 °C for 48 h, the yeast culture was centrifuged at1500×g for 15 min. The yeast cells were washed twicein PBS (KH2PO4/Na2HPO4, 50 mM, pH 7.0), suspendedin KNO3 (0.1 M, pH 6.2) and the optical density(OD600nm) was adjusted to 1.0. Four milliliters of thecell suspension were added to 1 ml of xylol (apolarsolvent), chloroform (acidic solvent), or ethyl acetate(basic solvent). After 5 min incubation at room

temperature, phases were mixed for 2 min and the sol-vent phase separated by holding the mixture at 37 °Cfor 1 h. Finally, the aqueous phase was carefully re-moved and its absorbance at 600 nm was determined.The decrease in the absorbance of the aqueous phasewas taken as a measure for the cell surface hydropho-bicity. The percentage of cell surface hydrophobicity(H%) of the yeast was calculated using the followingequation:

H% ¼ A0�AÞ=A0� � 100ð½

where A0 and A are the absorbance values of the aqueousphase before and after solvent addition [24].

Yeast Adhesion Assay

One of the selection criteria for a probiotic microorgan-ism is its ability to compete with pathogens for adher-ence to mucosal surface [22]; hence, an isolate thatsuccessfully passes the adhesion assay possesses a de-sirable probiotic trait. This test was carried out as de-scribed previously [25, 26]. Raw mucus epithelial cellswere prepared by rubbing from sea bass intestine cutterfragments, double centrifugation at 20,000×g for 30 minat 4 °C, and filtration of the final supernatant through0.2-μm filters. Filtered crude mucus (300 μl) was ap-plied to glass slides that were held overnight for air-drying, followed by 20 min fixation with absolute meth-yl alcohol. The yeast suspension in saline solution(OD = 1.0 at 600 nm) was placed in Petri dishes con-taining saline solution and mucus-coated glass slides,incubated at 20 °C for 1 h with continuous gentle shak-ing. The slides were washed thoroughly several times insaline solution, air-dried at 20 °C for 16 h, and fixedwith absolute methyl alcohol at 20 °C for 20 min. Theslides were stained with crystal violet for 2 min; thiswas followed by washing and air-drying. The slideswere then examined by light microscope, and the aver-age number of yeast cells attached to 1 mm2 of themucus-coated glass slides was determined by counting20 microscopic fields.

Cholesterol Oxidase Activity Assay

This assay was done to evaluate the potential cholester-ol lowering capacity of the isolated yeast. The activityof cholesterol oxidase enzyme was determined accordingto Inoure et al. [27] as follows: 0.4 ml of 125 mM Tris-HCl buffer, pH 7.5, was added to 0.1 ml of the yeastfiltrate (enzyme source), and the mixture was incubatedin water bath at 37 °C. After 3 min of incubation, 25 μlof 12 mM cholesterol in isopropanol was added to the

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mixture. After additional 30 min, 2.5 ml of absoluteethanol were added and the quantity of 4-cholesten-3-one was determined by measuring the absorbance at240 nm. Reaction blanks were prepared by replacingcholesterol solution with isopropanol. One unit of cho-lesterol oxidase activity was defined as the amount ofenzyme producing one micromole of 4-cholesten-3-oneper minute at 37 °C.

Fermentation Studies

Cell propagation was carried out on YM agar medium. Afterincubation at 28 °C for 24 h, colonies were harvested in salinesolution (9 g/l NaCl solution) and used to inoculate the YMbroth medium. The inoculated flasks were incubated underaerobic conditions on a rotary shaker (Innova 4330; NewBrunswick Scientific, NJ, USA) at 200 rpm and 28 °C for24 h. Cells were used thereafter to inoculate both of shakeflask and bioreactor with an inoculation level of 5% (v/v) forcell mass production.

The yeast biomass production medium, used in this study,had the following composition (g/l): glucose, 15.0; yeast ex-tract, 5.0; (NH4)2SO4, 5.0; KH2PO4, 3.0; MgSO4*7H2O, 1.0;and pH 5.5 [28]. Glucose was sterilized separately by filtrationusing 0.2-μm microbiological filter (Sartorium, Göttingen,Germany) and added to the medium aseptically before inocu-lation. In the case of shake-flask culture, submerged cultiva-tions were carried out in 50ml of growthmedium using a 250-ml Erlenmeyer flask. The inoculated flasks were incubatedaerobically on a rotary shaker (New Brunswick Scientific) at200 rpm and 28 °C. For the bioreactor experiment, cultiva-tions were carried out in 16-l pilot-scale stirred-tank bioreactor(BioEngineering, Wald, Switzerland) with working volume of8 l. The stirrer was equipped with two six-bladed Rushtonturbine impellers. The agitation speed was 200 rpm through-out the cultivation. Aeration was performed using filtered-sterilized air which was supplied continuously to the bioreac-tor at a rate of 1 v/v/min. Foamwas suppressed by the additionof silicon antifoam, grade A (Sigma-Aldrich Inc., USA).Dissolved oxygen (DO) concentration and pH values weredetermined during growth using pH and DO polarographicelectrodes, respectively (Ingold; Mittler-Toledo, Switzerland).

In the case of fed-batch experiment, glucose feeding startedafter 15 h using concentrated glucose solution (400 g/l). Initialfeeding rate was 1 g/l/h for the first 5 h and the rate wasincreased gradually thereafter during the fed-batch phase. Inthis experiment, the pH was stabilized at 5.5 with 2 N ammo-nium hydroxide or 1 N phosphoric acid.

Fermentation Sample Preparation and Analysis

Samples were taken during fermentation runs at pre-determined time intervals. For the shake-flask experiment,

each sample consisted of three flasks, 50 ml each. In case ofthe bioreactor, 20-ml samples were taken aseptically through asampling port. Cell concentration was measured as OD600nm

readings using a spectrophotometer (Pharmacia Biotech,Cambridge, England). The samples were filtered using apre-weighed filter paper and the filtered biomass was washedtwice with distilled water and subsequently dried in an oven at110 °C to a constant weight. The relation between OD and celldry weight was determined using a standard curve of ODversus cell dry weight. The filtrate was frozen at −20 °C andused for glucose determination. Glucose concentration in thegrowth medium was determined by enzymatic method usingglucose determination kit (Biocon GmbH, Germany). The in-tensity of developed color was determined at OD500nm.

All results were expressed as the average of at leasttwo independent experiments. The data were statisticallyanalyzed using the Student’s t test at a probability levelof 0.05. Statistical analyses were performed using SigmaStat (Jandel Scientific Software, version 1.0, San Rafael,CA, USA).

Results and Discussion

Isolation and Identification of Yeast Strain

The YMHS yeast was isolated from the water sampled at thedepth of 10 m. The yeast isolate was identified asCryptococcus sp. and not classifiable into any knownCryptococcus species (Fig. 1) but it showed 95% similarityto Cryptococcus albidus CBS 1925 and C. albidus var.kuetzingii. The ITS1-5.8S-ITS2 rRNA region sequence ofCryptococcus sp. YMHS has been deposited in the DNAData Bank of Japan, under accession number AB610508.

Marine yeast microbiota vary with depth, where the yeastsin the class Ascomycetes (e.g., Candida, Debaryomyces,Kluyveromyces, Pichia, and Saccharomyces) are common inshallow waters, while yeasts belonging to the Basidiomycetes(Cryp tococcus , Rhodospor id ium , Rhodotoru la ,Sporobolomyces) are common in deep waters [29]. Amongthe basidiomycete yeasts, some species of Cryptococcus,Rhodotorula, and Sporobolomyces are widespread across var-ious oceanic regions [30]. Some psychrophilic yeasts such asMrakia frigida and Guehomyces pullulans are also widelydistributed in the sea sediment of Antarctica [31].

Cryptococcosis is an opportunistic deep-seated mycosisfound worldwide, caused by encapsulated yeasts of twoCryptococcus species: C. neoformans and C. gattii. Otherspecies are usually considered nonpathogenic. C. albidus,C. curvatus, and C. laurentii rarely exist, even in severelyimmunocompromised patients, and their clinical significanceis not well understood [32].

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Evaluation of Probiotic Properties

Microbial strain used for probiotic application, generally,should be tolerant to acid for at least 90 min (time from inges-tion to release from the stomach), tolerant to bile acids, andcapable of attaching to the epithelium and growing in thelower intestinal tract [33]. Therefore, the following tests werecarried out to evaluate the probiotic properties of the isolatedyeast strain.

Acid Tolerance

The primary barrier to microorganisms in the stomach is thegastric juice with the intensity of the inhibitory action attributedto the low pH [34]. In the present work, yeast resistance tosimulated gastric juice of different pH values, ranging between

2.0 and 4.0, was studied. After 4 h incubation at 37 °C andpH 4.0, the number of living cells decreased minimally from5.0 × 109 to 1.7 × 108 cfu/ml, whereas the population decreaseddramatically to 6.7 × 103 cfu/ml at pH 2 (Fig. 2a). After 2 hincubation at pH of 2.0, 2.5, 3.0, 3.5, and 4.0, surviving popu-lations were 6.2 × 105, 1.8 × 106, 6.7 × 107, 2.4 × 108, and2.9 × 109 cfu/ml, respectively. According to these results, con-siderable portion of Cryptococcus sp. YMHS population maywithstand the harsh acidic stomach environment if this yeast isused as a probiotic. Resistance of this yeast to stomach aciditycould be further enhanced through acid adaptation or encapsu-lation. C. albicans, C. parapsilopsis, and Issatchenkiaorientalis, isolated from infant feces and feta cheese, were ableto grow at pH values between 1.5 and 2.0 [34]. Another studyshowed that D. hansenii, Debaryomyces occidentalis,Saccharomyces cerevisiae, and Yarrawia lipolytica were able

Fig. 1 Bootstrap consensusphylogenetic tree based on ITS1-5.8S-ITS2 sequence analyses byneighbor joining method,showing the relationship ofCryptococcus sp. strain YMHSwith the most closely relatedyeasts. The scale bar represents0.1 substitutions per nucleotideposition

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to grow after incubation at pH 2, in 0.5% NaCl solution con-taining 1MHCl, for 3 h [3]. In contrast, other yeast strains suchas C. rugosa, C. lambica, and Trichosporon cutaneum showedlow acid tolerance [35].

Bile Salts Tolerance

Potential probiotic strain should withstand bile salt to survivein the small intestine during its passage in the GIT. Moreover,bile salt tolerance of a probiotic strain is also required duringfood processing. To testCryptococcus sp.YMHS for this trait,cells were exposed to bile salt at concentrations between 0.2and 2.0% and incubated at 37 °C for 4 h. As shown in Fig. 2b,cells showed some tolerance to bile salt. However, significantreduction in cell viability was observed by increasing eitherbile salt concentration or incubation time.When the yeast cellswere incubated in 0.2% bile salt solution, its population de-creased from 5 × 109 to 2.4 × 107, 1.1 × 106, 7.1 × 105, and4.3 × 104 cfu/ml after 1, 2, 3, and 4 h, respectively. However,bile salt tolerance of this strain is comparable to that reportedfor lactic acid bacteria [24].

Antimicrobial Activity

Antimicrobial activity is one of the desired criteria for aneffective probiotic strain. Cryptococcus sp. YMHS exhibited

no antibacterial activity against the two tested Bacillus spp.(Fig. 3). Nevertheless, significant antimicrobial activity to thepathogenic Gram-positive cocci, Sarcina lutea andStaphylococcus aureus, was observed. The yeast also exhibit-ed high antibacterial effect against the pathogenic Gram-negative bacteria, Salmonella enterica serovars Typhi andTyphimurium and Shigella sonnei without any inhibitory ef-fect against the tested Escherichia coli strain. In general, anti-bacterial products are not commonly secreted by many yeastspecies [36]. In a study conducted by Bilinski et al. [37] onlyKluyveromyces thermotolerans and Kloeckera apiculate, outof 400 yeast strains examined, showed antibacterial effectagainst Lactobacillus plantarum and Bacillus megateriumwithout any significant activity against Gram-negative bacte-ria. The broadest inhibitory range was attributed to Candidaoleophila and Pichia membranifaciens, and the latter yeastnotably demonstrated a strong activity against Listeriamonocytogenes [38].

Ability to produce killer toxins (i.e., anti-yeast agents) wasobserved previously in some yeasts. In the present study,Cryptococcus sp. YMHS exhibited activity againstC. albicans and S. cerevisiae. Considering these preliminaryfindings, Cryptococcus sp. YMHS may inhibit pathogenicCandida strains, whereas only few probiotic strains werefound effective against Candida and enterococci in the vagina[39]. Killer toxin activity was reported previously in

Fig. 2 Effect of simulated gastricjuice (SGJ) of different pH values(a) and of different bile saltconcentrations (b) on the viabilityof Cryptococcus sp. YMHS

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C. humicola [40] and C. laurentii [41]. Another study showedthat Cryptococcus hungaricus CBS 6569 has dsRNA associ-ated virus-like particles which demonstrate mycocin produc-tion property [42].

Hydrophobicity

Hydrophobicity of the cell surface has been frequently sug-gested to explain why certain microbial strains are slower tobe eliminated from the digestive tract than others after admin-istration [43]. In the present work, three different solventswere used to evaluate the hydrophobic/hydrophilic cell sur-face properties and their Lewis acid-base characteristics.These solvents were xylol, which is a nonpolar solvent; chlo-roform, a monopolar and acidic solvent; and ethyl acetate, amonopolar and basic solvent. Microbial adhesion to xylol re-flects cell surface hydrophobicity because electrostatic

interactions are absent. The two other solvents, chloroformand ethyl acetate, were regarded as a measure of electrondonor (basic) and electron acceptor (acidic) characteristics ofthe microorganism, respectively. As shown in Fig. 4,Cryptococcus sp. YMHS adhesion to chloroform was thehighest (52.3%), followed by ethyl acetate (41.4%) and finallyby xylol (36%). This indicates the strain’s capability to adhereto the intestinal cell surface with low elimination kinetics.These results are in agreement with those obtained by Yanget al. [44] who found that marine yeasts were able to adhere tothe intestinal mucosa and thus providing potential applicationas a probiotic supplement for human and animal diet.Perricone et al. [45] stated that the hydrophobicity was usedas an indirect and screening tool to test the potentiality of theisolates to bind to the intestinal mucosa. Contrarily, no corre-lation between hydrophobicity and bacterial adhesion capaci-ty to the epithelial cells was observed by Vinderola et al. [46].

Adhesion to Intestinal Epithelial Cell Mucus-Coated GlassSlides

The ability to colonize the intestine, at least temporarily, byadhering to the intestinal epithelium has been indicated as acriterion to select probiotic yeasts [47]. Some yeasts can ad-here to the intestinal mucus of fish [48]; this trait is crucial forpersistent colonization [49].The proposed probiotic yeaststrain (Cryptococcus sp. YMHS) showed attachment abilitiesto the bass mucus-coated glass slide at greater than 20 yeastcells/mm2 (data not shown). These results suggest that themucus surfaces are potential portal entries in the fish for thisstrain. Fadda et al. 2017 [50] stated thatKluyveromyces strainstested were able to adhere to Caco-2 cells with values rangingfrom 4 to 68%, and Maccaferri et al. (2012) [51] indicated thestrongly adhesive capability of the dairy K. marxianus strain(BO399). Among the yeast strains isolated from the Kefirbeverage, K. marxianus CIDCA 8154 and S. cerevisiae

Fig. 3 Antimicrobial activity ofCryptococcus sp. YMHS againstselected microorganisms

Fig. 4 Hydrophobic properties of Cryptococcus sp. YMHS in differentsolvent systems. Data represent the means ± standard deviation oftriplicate assays

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CIDCA 8112 had the capacity to adhere, in vitro, to epithelialintestine-derived cells [52].

Cholesterol Oxidase Activity

It has been suggested that bacterial cholesterol degradation infood may be beneficial for the human health. The bacterialdegradation of cholesterol has been known to occur by a cho-lesterol oxidase. This enzyme catalyzes the oxidation of cho-lesterol to 4-cholesten-3-one, along with the reduction of ox-ygen to hydrogen peroxide. The current study showed thatCryptococcus sp. YMHS secreted extracellular cholesteroloxidase enzyme (8.6 U/ml) after 48 h of growth in a mediumcontaining 0.1% cholesterol (data not shown). This amount ofenzyme was higher than that reported by Bacillus subtilisSFF34, isolated from a Korean traditional fermented flat fish(3.4 U/ml) in a medium containing 0.2% cholesterol [53] and(2.5 U/ml) by Bacillus flexus Mcc 2427 [54].

Based on these findings, Cryptococcus sp. YMSH haspromising probiotic traits needed for in vivo applications inanimals and humans. Considering this strain has been isolatedfrom sea water, it is potentially useful in aquaculture applica-tion. Tovar-Ramírez et al. [55] found that incorporation of1.1% of the marine yeast, D. hansenii CBS 8339, in diet ofmalformed sea bass larvae improved their survival rate by10%.Most marine yeasts have been generally regarded as safe(GRAS) and showed a beneficial impact on biotechnologicalprocess [10].

For instance, Cryptococcus spp. have been safely appliedin many fields such as the use of C. laurentii, C. flavus, and

C. albidus as a biocontrol agent against Mucor diseases inpears [56]. Recently, C. albidus was approved as a biocontrolagent against vegetable and fruit fungal diseases and wasmarketed under trade name YieldPlus® by Anchor YeastCo. in South Africa and Lallemand Co. in France [57]. Inthe food sector, a C. albidus-based product (Ferments-Maturation) was recently produced by Abiasa Co. in Canadaand the yeast was included in manufacturing different types ofcheese [58].

Biomass Production in Shake-Flask and Stirred-TankBioreactor

As Cryptococcus sp. YMHS showed potential probiotic prop-erties, studies were carried out to investigate the growth kinet-ics of this yeast in submerged culture for high cell mass pro-duction. Cultivations were conducted in shake flask and insemi-industrial scale, 16-l stirred-tank bioreactor, using a highcell mass production medium [28].

Cultivation in Shake-Flask Culture

The changes in cell growth, substrate consumption, and me-dium pH during yeast cultivation in shake flask are presentedin Fig. 5 and Table 1. Cells grew exponentially with specificgrowth rate of 0.14 [h−1] reaching cell mass of about 2.2 g/lafter 16 h and remained nearly constant for the rest of cultiva-tion time. Glucose concentration decreased in culture duringthe active cell growth phase with a consumption rate of 0.47 g/l/h. After 30 h, only 6 g/l glucose (almost 40% of the initial

Fig. 5 Kinetics of cell growthand substrate consumption duringsubmerged cultivation ofCryptococcus sp. YMHS in shakeflask. Data represent the meansand standard deviations oftriplicate assays

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glucose concentration) remained unconsumed indicating thatglucose was not the limiting substrate during yeast cultivation.The medium pHwas decreased during the growth phase, from5.5 initially to ~4.5 at the end of exponential growth phase,and remained unchanged during the remainder of the incuba-tion time. To eliminate the possible effect of oxygen limita-tion, cultivations were conducted in stirred-tank bioreactorwith continuous air supply to evaluate the scalability of thisprocess.

Batch Cultivation in a Stirred-Tank Bioreactor

In this experiment, cultivation of Cryptococcus sp. YMHSwas conducted in 16-l stirred-tank bioreactor, under uncon-trolled pH (Fig. 6 and Table 1). Cells grew exponentially dur-ing the first 18 h with specific growth rate of 0.14 [h−1],reaching a cell mass of 5.82 g/l. After the exponential phase,

no significant change in the cell concentration was observedand glucose was continuously consumed in the culture with aconsumption rate of 0.82 g/l/h (almost 75% higher than theconsumption rate in shake-flask culture). Termination of theexponential growth phase after 18 h was a result of glucoselimitation in culture. Concomitantly, dissolved oxygen con-centration decreased gradually in the culture reaching a min-imal value of about 20% saturation after 20 h (during the earlystationary phase of growth phase). After that time, the dis-solved oxygen increased gradually in the culture, reachingabout 50% saturation at the end of cultivation time. The pHvalue of the culture was decreased gradually as a function ofcell growth to reach about 4.0 at the end of cultivation time.Comparing growth kinetic parameters in shake flask and bio-reactor (Table 1), it is obvious that bioreactor cultureyielded higher cell mass, growth rate, and glucose con-sumption rate which is directly attributed to good aerationand gas mixing. Therefore, it could be concluded thatoxygen was the limiting factor in the shake flask. Thisresult is in agreement with those reported by other authorswho observed that biomass and lipid production ofC. albidus in shake flask was lower than the bioreactorculture [59]. The bioreactor culture resulted not only inhigher volumetric biomass but also higher specific volu-metric biomass [YX/S], when compared to shake-flask cul-ture. The highest specific biomass achieved in this study(0.39 g/g) was similar to that obtained when C. albiduswas grown in stirred-tank bioreactor [59]. However, otheryeasts such as S. boulardii, S. cerevisaie, and Candidatropicalis showed higher specific biomass, up to 0.5 g/g,when grown in a fully-aerated stirred-tank bioreactor underoptimized cultivation conditions [28, 60, 61].

Fig. 6 Kinetics of the cell growthand substrate consumption duringsubmerged cultivation ofCryptococcus sp. YMHS in 16-Lbioreactor

Table 1 Kineticparameters of cell growthand substrateconsumption duringbatch cultivation ofCryptococcus sp.YMHSin shake-flask and instirred-tank bioreactor

Parameter Type of cultivation vessel

Shake-flask Bioreactor

Xmax [g/l] 2.23 5.82

dx/dt [g/l/h] 0.14 0.37

μ [1/h] 0.14 0.14

Qs [g/l/h] −0.47 −0.82YX/S [g/g] 0.30 0.39

Xmax maximal cell dry weight, dx/dtgrowth rate, μ specific growth rate,Qs glu-cose consumption rate, YX/S specific cellmass production (g cell dry weight per gglucose consumed)

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Fed-Batch Cultivation with Controlled pH

Based on the data obtained from the batch culture, fed-batch cultivation was designed to enhance biomass pro-duction by continuous feeding of glucose. In this exper-iment, the pH was kept constant at 5.5 throughout thecultivation and ammonium hydroxide which was notonly used to adjust the pH but also to serve as nitrogensource to support cell growth and to prevent nitrogensource limitation. As shown in Fig. 7, cultivation startedin a batch mode for the first 15 h; during that time,cells grew exponentially and reached cell mass of about5 g/l. Glucose was consumed gradually during the ac-tive growth phase of the culture, with a rate of 0.93 g/l/h, and reached 1 g/l at the end of the batch phase.Therefore, glucose was fed with a rate starting at 1 g/l/h to prevent carbon source limitation. During the fed-batch phase, glucose feeding rate was increased gradu-ally to support further cell growth as follows: 1 g/l/hduring 0–5 h; 2 g/l/h during 5–10 h; and finally, 4 g/l/hfor 10–15 h of the feeding phase. After 15 h of culti-vation, the DO value was kept at 20% saturation toprevent oxygen limitation in culture. The DO valuewas cascaded to agitation and aeration controller withthe following operation ranges: agitation, 200–600 rpmand aeration 1.0–2.0 v/min. During the fed-batch phase,glucose was available in culture, based on the simpleincremental feeding rate applied in this experiment.The cell biomass increased gradually in the cultureand reached 28.5 g/l after 32 h cultivation; this is al-most about fivefold higher than the biomass obtained inthe batch culture.

Comparably, it has been reported that the maximal biomassproduction in optimized medium for C. laurentii reachedabout 20 g/l after 360 h cultivation using medium composedof molasses, yeast extract, and minerals [62]. Other researchofC. curvatus reported also that the maximal biomass produc-tion achieved was about 10.6 g/l in bioreactor culture usingglycerol as carbon and nitrogen sources [63].

Conclusions

The newly-isolated marine yeast strain, Cryptococcus sp.YMHS, has many advantageous traits to be applied as a pro-biotic microorganism. Beside the feasible use as a probiotic,the new strain also could be beneficial in the development offunctional foods or novel food preservatives. The activity ofCryptococcus sp. YMHS against other yeast and pathogenicbacteria can expand its application for biological control inagricultural, fish farming, and aquaculture. Our study is pre-liminary in terms of elucidating the mechanism exhibited byCryptococcus spp. to eliminate cholesterol. More properlyplanned in vivo studies must be undertaken in the future toprecisely understand the mechanism involved for a safer andeffective probiotic culture with anti-hypercholesterolemic ef-fects. Finally, the growth kinetics data showed the possibilityof high cell mass production of this strain at the semi-industrial scale.

Acknowledgements This research was supported by internal fundingto authors from their corresponding universities: Drs. El-Baz andMahrous, University of Sadat City, Egypt; Drs. El-Enshasy andOthman, Universiti Teknologi Malaysia; Dr. Shetaia, Ain ShamsUniversity, Egypt; and Dr. Yousef, The Ohio State University, USA.

Fig. 7 Kinetics of the cell growthand substrate consumption duringsubmerged and fed-batchcultivation of Cryptococcus sp.YMHS in 16-L bioreactor

Probiotics & Antimicro. Prot.

Compliance with Ethical Standards

Conflict of Interest The authors declare that they have no conflict ofinterest.

Human and Animal Rights and Informed Consent This article doesnot contain any studies with human or animal subjects.

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