Intermediate and pharmaceutical compounds associated with fermentation of spoilage date fruits by Hanseniaspora guilliermondii KKUY-0045

language journal of the Egyptian Society for Biological Sciences ,Department of Entomology ,Faculty of Sciences Ain Shams University .  y & molecular biology journal is one of the series issued twice by the Physiolog Egyptian Academic Journal of Biological Sciences, and is devoted to publication of original papers that elucidate important biological, chemical, or physical mechanisms ogical significance. of broad physiol


INTRODUCTION
Saudi Arabia is famous for the wide cultivation of date palm trees.It produces about 13% of the world production of dates.There are more than six million of date palm trees distributed all over Saudi landscape.Date manufacturing is one of the most popular industries in Saudi Arabia.However many insects are known to attack date palm fruits and trees (El-Juhany, 2010).Mycotoxigenic fungi, particularly aflatoxigenic Aspergilli, have been associated with dates and date products (Emam et al., 1994;Shenasi et al., 2002).The rot of dates is due to increased infestation of pests and diseases, improper transporting, handling, and lack of cold stores, lack of marketing and export.Spoilage dates is abundant renewable agro-industrial waste material produced in Saudi Arabia and its low cost is an important factor for the economic production of bioethanol by fermentation.
Bioethanol can be produced different renewable feed starch sources.However, sugarcane industrial wastes, agricultural low cost fruits, vegetables, starch materials cellulosic materials, agricultural by products of starch industries, such as potato pulp, tapioca pulp, papaya fruit pulp from raw starch hydrolyzate are attractive resources for economical production of ethanol (Balasubramanian et al., 2011).We introduce the spoilage dates as one of the most economic and renewable source for bioethanol production, because it contains high amount of sugars which doesn't need to any chemical pretreatments comparing to the other agrowastes.The investigation of secondary compounds, which occasionally formed during fermentation, is of great importance.These compounds might have medicinal or pharmaceutical importance.In addition to their extreme usefulness, production of such compounds will reduce the production cost of the main target process "bioethanol".Some active compounds such as methyl-diols and secondary alcohol derivatives were mentioned as by-products produced by yeasts (Fuganti andGrasselli, 1985, Hashem andDarwish, 2010).
The objective of this study was to determine the intermediate and pharmaceutical compounds formed during the fermentation of the spoilage date fruits by isolated yeast.26S rDNA sequencing and phylogenetic analysis was applied for molecular identification of the yeast at specie level.

Preparation of the spoilage dates juice (SDJ):
Two date cultivars (Arihy and Nabt Ali) that were the most spoilage cultivars were selected in this study.Samples of these two cultivars were mixed in ration 1:1 (w:w) without stones and stored at 4°C.Fresh samples were ground using high speed blender, then was diluted with distilled water to 2:8 (w:v) and sterilized at 121 °C for 30 min.The produced juice was passed through double layer of muslin cloth to exclude all large undigested particles.The filtrate was considered as the prepared date juice (SDJ) and stored in refrigerator for further use.

Yeast isolation and purification:
Yeast strain was isolated from the collected spoilage date fruits using dilution plate method as described by Kutzman and Fell (1998) on malt extract yeast extract agar (YMA) medium .The medium was sterilized by autoclaving at 121°C for 15 min.The sterilized agar medium was cooled to approximately 45°C and its pH was adjusted to 3.7 and then poured into the petri dishes.Samples were inoculated onto the dishes and incubated at 25°C for 48-72 h.Then, the growing yeasts were isolated and purified.For purification purpose, the isolated yeasts were streaked twice on YMA plates for single pure colonies.The pure colonies were inoculated onto YMA slants and incubated at 25 C for 48 -72 h and then transferred into 4C to be stored.The extraction of total yeast genomic DNA was performed according to procedures described by Hesham et al. (2006).The D1/D2 domain of the 26S rDNA region was amplified using the primers NL1 (5'-GCATATCAATAAG CGGAGGAAAAG-3') and NL4 (5'-GGTCCGTGTTTCAAGACGG-3') (Kurtzman and Robnett 1998).PCR was performed in a final volume of 50 μl containing GoTaq green master mix (Promega, Madison, WI, USA), 1 µl of each primer at a concentration of 0.5 mM, and 1 µl template DNA.The amplification was carried out by PCR under the following conditions : initial denaturation at 95 °C for 5 min, followed by 36 cycles at 94 °C for 2 min, 52 °C for 1 min, 72 °C for 2 min; final extension at 72 °C for 7 min, and holding at 4 °C.PCR products (5 μL) were analyzed using 1.5% 0.59 TBE agarose gel electrophoresis.The gel was stained with ethidium bromide, visualized under UV light, and photographed.

Molecular identification of the
Sequencing of the D1/D2 domain of the 26S of rDNA and phylogenetic analysis: DNA for sequencing was amplified with forward and reverse primers (NL1-NL4).The amplified D1/D2 fragment was purified and sequenced using an ABI 3730 automated sequencer (Macrogene Company, Korea).The yeast 26S rDNA sequences obtained were then aligned with known 26S rDNA sequences in the GenBank database using the basic local alignment search tool (BLAST) (http://www.ncbi.nlm.nih.gov/BLAST) and percent homology scores were generated to identify isolated yeast.To determine the taxonomic position of the isolate, a phylogenetic tree was constructed with MEGA version 4.0 using a neighbor-joining algorithm, plus the Jukes-Cantor distance estimation method with bootstrap analyses for 1,000 replicates was performed.

Nucleotide sequence accession number:
The 26S rDNA sequences of the isolated yeast reported in this study has been deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession number JQ690239 (H.guilliermondii KKUY-0045).

Fermentation of the spoilage date fruits by H. guilliermondii KKUY-0045:
Fermentation was carried out in a BioFlo/CelliGen 115 fermentor provided by New Brunswick Co., USA, with all necessary controls.The reactor was of 7 L capacity and the working volume was 3 L.The fermenter is equipped with an agitator, pH, and temperature control systems were used as the batch fermenter in this study.The fermenter was cleaned and steam sterilized at 121°C for 15 minutes.Then the sterilized medium (20% of SDJ, ammonium dihydrogen phosphate (4 g/L), Mg (0.4 g/L) and Zn (0.4 g/L) containing the inoculum was transferred to the fermenter.the seed culture was grown at 25 °C for 24 h in 250 mL flask containing 100 mL of a YPD medium.The temperature of fermentation was maintained at 30 ± 1 °C.The pH of the fermentation broth was regulated at 4.5 within pH 0.1 unit by the peristaltic pump which injected a fine stream of sulfuric acid or sodium hydroxide.The fermentations were carried out at atmospheric pressure.The agitator speed was maintained constant throughout the experiment at 200 rpm.The reactor was maintained under anaerobic conditions.Samples were taken during the course of 6 days of fermentation to monitor the ethanol concentration.

Ethanol determination (Quantitative estimation of ethanol):
Concentration of ethanol in samples was estimated enzymatically using Ethanol estimation kit (K620-100) provided by BioVision company.The procedures provided with the product was employed.Briefly, this method is based on presence of alcohol oxidase that oxidizes ethanol to generate H 2 O which reacts with the probe to generate color (λmax= 570 nm).A standard curve was constructed using the standard ethanol provided within the kit.The amount of ethanol in a given sample was determined from the standard curve after measuring its absorbance at 570 nm.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis:
During fermentation, different samples of fermentation medium (10 mL) were taken at different time intervals (2, 4 and 6 days).The samples were centrifuged at 10000 rpm for 15 min under cooling and the supernatant was filtered through cellulose membrane filter (0.45 μm) to exclude any yeast cells, and then was extracted by chloroform solution.An aliquot of one µL extract (chloroform extract) of cell free extract was injected into the GC-MS (6890 N/5975B).The HP-5MS column was 30 m in length, 0.25 mm i.d., and 0.25 mm in thickness.The carrier gas was helium with average velocity 36 cm/sec, and flow 1 mL/min.The operating condition of GC oven temperature was maintained as follows: initial temperature 40 °C for 9 min, 150 °C for 8 min, at 15 °C/min up to final temperature 310 °C with isotherm for 3 min at 25 °C/min.The injector and detector temperatures were set at 250 and 280 °C, respectively, according to the standard method 8270 EPA (Cakir et al., 2004).Identification of the components of the prepared extract was assigned by comparison of their retention indices, relative to a series of n-alkane indices on the capillary column and GC-MS spectra from the Wiley 6.0 MS data.

RESULTS AND DISCUSSION Molecular identification of the yeast:
In order to identify and determine the correct phylogenetic position of the yeast strain KKUY-0045, molecular genetic identification were performed by amplification and sequencing of the D1/D2 region of the 26S rRNA gene.The obtained sequence data were compared with the sequences of 26S rRNA regions in Genbank by means of BLAST search of the National Center for Biotechnology Information (NCBI) databases.Alignment of the 26S rRNA gene sequences of the yeast with sequences obtained by doing a BLAST search revealed up to 100% similarity to H. guilliermondii (Fig. 1a).To confirm the position of our strain in phylogeny, a number of sequences were selected from Genbank database for the construction of a phylogenetic tree using MEGA4 program.As shown in Fig. 1b, the phylogenetic tree indicated that strain KKUY-0045 and H. guilliermondii shared one clade cluster.Therefore, the strain KKUY-0045 was identified as H. guilliermondii.In the last few years, an extensive research effort have been made to characterize the yeast microbiota in a vast number of food preparations through techniques based on molecular biology with particular emphasis on PCR amplification of ribosomal RNA genes and spacer regions as internal transcribed spacers (ITS) and nontranscribed spacers (NTS) followed by restriction analysis with restriction enzymes (Esteve-Zarzoso et al., 1999;Baleiras Couto et al., 2005;Raspor et al., 2006).The sequence of the large subunit ribosomal DNA (LSU rDNA), especially D1/D2 region, has revealed to be a powerful tool in yeast identification (Kurtzman and Robnett 1998) becoming more accessible due to new efficient nucleic acids sequencing techniques.The sequencing of the D1/D2 of the large-subunit 26S ribosomal DNA is now widely accepted as a standard procedure for yeast identification.Moreover, a 600 bp length of the D1/D2 domain of the 26S rDNA contains sufficient variation to define individuals at the species level (Kurtzman and Robnett, 1998;Scorzetti et al., 2002;Frutos et al., 2004).It was found that molecular method based on the sequences of the 26S rDNA, D1/D2 domain is rapid and precise compared with the physiological method for the yeast identification, and has also been applied to study the phylogeny of different yeast groups (Kurtzman and Robnett, 1998;Hesham et al., 2006;Hesham and Mohamed, 2011).Ethanol production by the H. guilliermondii KKUY-0045: Fig. 2 shows the ethanol production by H. guilliermondii KKUY-0045 during 6 days of fermentation in a 7-L fermentor.Concnetration of ethanol produced by this strain increased gradually until 3 days to record the maximum production as 87.74 g/L.Then the concentration begain to decline deliberately to the end of the fermentation time.Gradual increasing of ethanol production by other yeast strains up 72 h was reported by Limtong et al. (2007).They used four isolates of Kluyveromyces marxianus for ethanol production from sugar cane juice and found that the maximum ethanol concentrations was formed by the four yeast isolates after 72 h of starting the fermentation at 30°C.Our results are in close agreement of those authors.We suggest that the similarity in ethanol productivity by K. marxianus and our strain is due to their taxonomically intimacy and their similarity in physiology and required cultural conditions.

GC-MS analysis of the fermented date juice by H. guilliermondii KKUY-0045:
The results of GC-MS analyses of the cell free extract of H. guilliermondii KKUY-0045 at different time intervals (2, 4, and 6 days) are reported in Tables 1-3 and Figs.3-5.Pregnane were detected as the top compounds in 30.093% and 28.027%, respectively.There are five compounds including: Methyl diethyl borane, 1-Iodooctadecane, Octacosane, Hexadecane and 1,2,4-trimethyl cyclohexane were detected in moderate concentrations (2-6%).Eight compounds were detected in concentrations 1-2%, however the other 27 compounds were detected in very low concentration (less than 1%).After 4 days, the number of the detected volatile compound increased to 56 compounds.Methyl diethyl borane and 14B-Pregnane were recoded as the leader compounds that contributed 35.19 and 21.595%, respectively.It is noticed that the concentration of 14B-Pregnane decreased compared to in the previous 2 days (Table 1 and Fig. 3).There are other six compounds were detected in moderate concentration (2-4.66%).RT=Retention time, Conc.=concentration.
Almost of the volatile compounds detected here were previously reported as yeast derivative volatile components, and their olfactory characters were already described (Ames and Elmore, 1992;Ames and McLeod, 1985;Münch and Schieberle, 1998;Wang et al., 2011). Comuzzo et al. (2006) identified more than 160 volatile compounds in the headspace of the commercial powders (some not previously reported in literature).However there are some compounds were not previously detected.
For example, 14 β -Pregnane is an important compound.Pregnane is, indirectly, a parent of progesterone.It is a parent hydrocarbon for two series of steroids stemming from 5α-pregnane and 5β-pregnane.5β-Pregnane is the parent of the progesterones, pregnane alcohols, ketones, and several adrenocortical hormones and is found largely in urine as a metabolic product of 5β-pregnane compounds.Nonacosane has been reported to be a component of a pheromone of Orgyia leucostigma and evidence suggests it plays a role in the chemical communication of several insects, including the female Anopheles stephensi mosquito (Brei et al., 2004).

CONCLUSION
A new powerful yeast strain for bio-ethanol production was acquired.The yeast strain was identified as Hanseniaspora guilliermondii based on the sequencing of 26S rDNA region and the phylogenetic analysis.
H. guilliermondii produced 87.74 g/L of ethanol after 3 days of incubation in 7-L fermenetor.GC-MS analysis for the intermediate compounds revealed that, the pregnane and nonacosane compounds that could be used in the medical and pharmaceutical industries were present.

Fig. 1b :
Fig. 1b: Phylogenetic relationship between Hanseniaspora guilliermondii KC110834 strain KKUY-0045 and other 26S rRNA gene sequences of published strains.In the phylogenetic tree, KKUY-0045 and Hanseniaspora guilliermondii were clustered together as one clade segments corresponding to an evolutionary distance of 0.01 are shown with bars.Accession numbers for sequences are as shown in the phylogenetic tree.

Table 1 :
GC-MS analysis of the fermented date juice by H. guilliermondii KKUY-0045 after 2 days of incubation at 25 °C.

Table 2 :
GC-MS analysis of the fermented date juice by H. guilliermondii KKUY-0045 after 4 days of incubation at 25 °C.

Table 3 :
GC-MS analysis of the fermented date juice by H. guilliermondii KKUY-0045 after 6 days of incubation at 25 °C.