Taxonomic Characterization and Potential Biotechnological Applications of Yarrowia lipolytica Isolated From Meat and Meat Products

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Article Information:


Group: 2012
Subgroup: Volume 5, Issue 1, Winter
Date: January 2012
Type: Original Article
Start Page: 346
End Page: 351

Authors:

  • Maryam Mirbagheri
  • Department of Microbiology, Faculty of Science, University of Isfahan, Isfahan, IR Iran
  • Iraj Nahvi
  • Department of Microbiology, Faculty of Science, University of Isfahan, Isfahan, IR Iran
  • Giti Emtiazi
  • Department of Microbiology, Faculty of Science, University of Isfahan, Isfahan, IR Iran
  • Ladan Mafakher
  • Department of Microbiology, Faculty of Science, University of Isfahan, Isfahan, IR Iran
  • Farshad Darvishi
  • Department of Microbiology, Faculty of Science, University of Maragheh, Maragheh, IR Iran

      Correspondence:

      Affiliation: Department of Microbiology, Faculty of Science, University of Isfahan
      City, Province: Isfahan,
      Country: IR Iran
      Tel: +98-03117932474
      Fax: +98-03117932456
      E-mail: maryam_mirbagheri@yahoo

Abstract:


Background: Some species of yeast such as Yarrowia lipolytica produce citric acid, lipases, single-cell oil, etc. Y. lipolytica can degrade renewable, low-cost substrates to produce organic acids like citric acid, more efficiently than Aspergillus niger, and result in higher product yield and lesser waste production and toxicity.
Objectives: The aim of this study was to isolate yeast strains with potential for use in biotechnological applications such as production of citric acid and lipase.
Materials and Methods: For yeast strain screening, we isolated 179 yeast strains from meat and meat products that were prepared at the RAK and Pegah factories in Isfahan, Iran. Different media were used for screening of yeast colonies and for analyses of citric acid and lipase production; the production of these metabolites was assayed over time.
Results: One of the yeast strains isolated from poultry produced 55.5 g/L of citric acid and 12.3 U/mL of lipase. Biochemical and molecular tests showed that this strain belonged to the species Y. lipolytica. Molecular identification was confirmed by DNA sequencing, and the strain was named Y. lipolytica M7 (GenBank accession number, HM011048).
Conclusions: The results of this study suggest that meat and its products, especially poultry products, are suitable sources for isolation of yeast strains that produce two biotechnologically valuable products—citric acid and lipase. The yeast strain Y. lipolytica M7 can be used for citric acid production in bioreactor.


  • Implication for health policy/practice/research/medical education:
    This study introduced Y. lipolytica M7 as suitable wild strain with high potential for citric acid production and acceptable amount of lipase activity.
  • Please cite this paper as:
    Mirbagheri M, Nahvi I, Emtiazi G, Mafakher L, Darvishi F. Taxonomic Characterization and Potential Biotechnological Applications of Yarrowia lipolytica Isolated From Meat and Meat Products. Jundishapur J Microbiol. 2012;5(1):346-51.
  • DOI: 10.5812/kowsar.20083645.2433

© 2012, AJUMS. Published by Kowsar M.P.Co. All rights reserved.


Keywords: Yarrowia lipolytica;Isolation;Meat;Citric Acid;Lipase

Manuscript Body:


1.Background

Yeasts are commonly used microorganisms in the production of various foods and beverages. They are also involved in the spoilage of food products either due to an impaired fermentation process or post-process contamination. Thus far, yeast isolation has been mostly performed to determine food spoilage. Encinas et al. investigated the yeast populations present on Spanish fermented sausages and reported that Debaryomyces hansenii was the dominant strain in their samples (1). Deak et al. and Sanz et al. reported that Yarrowia lipolytica was the predominant strain isolated from meat products (2, 3). Citric acid is widely used in many industrial applications, manufacture of cosmetics, and buffering and chelating (4-6). All developed countries follow a conventional procedure involving the use of Aspergillus niger (producer) and molasses (substrate) for citric acid production.
Use of yeasts such as Y. lipolytica has several advantages over the use of Aspergillus spp, since yeast strains show lower sensitivity to low dissolved oxygen concentrations and heavy metals, and give higher product yields. Wild-type strains of Y. lipolytica can use a wide spectrum of carbon sources as substrates for production of organic acids, except for sucrose, a cheap and commonly used source of carbon for citric acid production with A. niger. Therefore, yeasts are better candidates for citric acid production than A. niger (4-6). Levinson et al. screened yeasts isolated from different geographic areas and concluded that Y. lipolytica was the best strain for citric acid production (range, 1.4–21.6 g/L) (7). Anastassiadis et al. x investigated the citric acid levels produced by some Candida strains and found the yield range to be 2.4–50.1 g/L (7). In literature, different Y. lipolytica strains have been compared (9-11), and the effects of different compositions of growth culture media and other parameters have been studied simultaneously (10, 12).
Lipases (EC 3.1.1.3) are among the most important classes of industrial enzymes and have many interesting applications in lipid biotechnology as well as synthesis of biosurfactants, pharmaceuticals, optically pure compounds, and flavor compounds (13, 14). Some yeasts can produce citric acid and lipase from a wider range of carbon sources than can fungi and bacteria, including glucose, molasses, fatty acids, alcohols, acetate, n-alkanes, and plant oils. Among these sources, glucose has gained considerable interest after the oil crisis of 1973, which is a very plausible situation in the present scenario (15, 16).

2. Objectives

The aim of this study was to isolate yeast strains with potential for use in biotechnological applications, especially citric acid and lipase productions. Therefore, we screened the yeast strains obtained from meat and studied their potential for producing citric acid and lipase, and identified the best strain by performing biochemical and molecular tests.

3. Materials and Methods
3.1. Isolation and Screening Procedures
We used routine microbiological procedures and selective nutrition media for isolation of yeasts and obtained 37 samples from different meat and meat products from the food factories in Isfahan, Iran. The samples were serially diluted, and 100 µl from each dilution was cultivated on yeast extract glucose chloramphenicol (YGC) agar (Merck, Germany) and incubated at 29°C for 2 days. The yeast strains were conserved at 4°C on yeast extract pep-tone dextrose (YPD) medium (Merck, Germany) (17).
The medium used for screening citric acid production was composed of 20 g glucose, 0.7 g MgSO4, 0.4 g Ca(NO3)2, 0.5 g NaCl, 1 g KH2PO4, 0.1 g K2HPO4, 8 g yeast autolysate, and 20 g/L agar supplemented with trace elements in the solution (18). Bromocresol green indicator (0.4%) was used for qualitative screening of citric acid (19). Qualitative screening of lipase from isolated yeasts was performed on tributyrin agar and rhodamine B agar (Sigma, USA). After cultivation, the media were incubated at 29°C for 3–10 days. Strains producing citric acid and lipase were identified on the basis of the hydrogen-to-carbon (H/C) index for each isolated yeast strain. The H/C index was determined by the ratio of the diameters of the yellow halo on the citric acid screening medium and hydrolysis halo or the orange halo on the tributyrin agar or rhodamine B agar (H), respectively, relative to cell colony size (C). An H/C ratio of 1 meant no hydrolysis, and thus no production of citric acid and lipase, whereas a higher H/C ratio indicated production of these metabolites (20-22). Y. lipolytica DSM 3286 strain was obtained from the culture collection of DSM, Germany. This strain could produce citric acid and lipase; therefore, it was used as the standard strain to compare the production of citric acid and lipase in the other isolated yeast strains (8).

3.2. Production Media and Analysis Procedures
The fermentation media for citric acid and lipase production contained glucose and olive oil as carbon sources, respectively (23). Glucose concentration was determined using a glucose kit (Darvash, Iran). Initial pH of the media was adjusted to 7. For cultivation, the strains were grown at 29°C with constant agitation at 200 rpm in 250-ml flasks. Citric acid assay was performed using a K-CITR enzymatic test kit (Megazyme, Ireland) according to manufacturer’s instructions. Extracellular lipase production was determined spectrophotometrically by using ρ-nitrophenyl laurate (ρ-NPL) as the substrate (24). A titrimetric method described by Darvishi et al. was used to confirm the results (25). For biomass determination, the cells were harvested by centrifugation at 4600 × g for 6 min and then washed with hexane. The dry weight of the centrifuged biomass was determined after drying at 80°C for 24 h (25, 26).

3.3. Identification of Yeast Strains
The isolated yeast strains were identified by assimilation and fermentation tests according to previously described methods (27). The results of the biochemical tests were further confirmed by molecular testing. Isolation and purification of DNA were performed according to the modified method put forth by Vasdinyei and Deak (28). Internal transcribed spacer-polymerase chain reaction (ITS-PCR) was performed according to the method described by Dlauchy et al. (29). The following primer sequences were used as described by White et al.: ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) (30). Twenty-five cycles of PCR were carried out using the following program: denaturation at 95°C for 30 s, followed by annealing at 55°C for 30 s, and extension at 72°C for 1 min. DNA was sequenced on an automated sequencer (3730xl DNA Analyzer, Applied Biosystems) by using synthetic primers and the dye-terminator procedure. These processes were outsourced to Macrogen, South Korea.

4. Results

About 179 yeast strains were isolated from poultry, beef, and lamb, and meat products like hamburgers and sausages depending on the morphological and physiological characteristics of the yeast strains (Table 1).

Table 1. Yeast Strains Isolated From Meat and Meat Products

 

Poultry meat

Beef

Sausages

Lamb

Hamburgers

Red meat

Strains

M1–53

M54–67

M68–102

M103–114

M115–142

M143–179

 

Eleven different yeast strains were selected on citric acid and lipase screening media on the basis of zone-formation around the colonies and H/C ratio. Isolated strains were cultured on citric acid fermentation medium for 8 days and quantitative assay for citric acid was performed every 24 h. Our results showed that the maximum amount of citric acid was produced 144 h after inoculation. One of the strains isolated from poultry (M7) produced the maximum amount of citric acid, 55.5 g/L, which was more than that produced by the standard strain Y. lipolytica DSM 3286 (Figure 1). Changes in citric acid production, glucose consumption, dry weight, and pH associated with the M7 strain on the fermentation medium during the 192 h are shown in Figure 2. The M7 strain was cultured on the basal lipase production medium for the quantitative lipase production assay. Our results showed that maximum lipase production occurred at 24 h after inoculation, and the M7 strain produced 12.3 U/mL of lipase (Figure 3). In the biochemical DSM 3286: Yarrowia lipolytica DSM 3286. Error bars represent the standard error associated with the findings from at least 3 experiments test performed for M7 strain identification, negative results were obtained for fermentation tests of sugars, including glucose, galactose, sucrose, maltose, lactose, raffinos, trehalose and positive results were obtained for assimilation of glucose, cellobiose, mannitol, galactose, glycerol, D-acetyl glucosamine, citrate, gelatin, and ethanol; the results of other assimilation tests were negative (Table 2). To confirm the results of the biochemical tests, ITS-PCR was performed (results have been shown in Figure 4). The results of the biochemical tests, PCR, and DNA sequencing indicated that the distinct M7 strain be-longed to the Y. lipolytica species. Therefore, the M7 strain was named Y. lipolytica M7. The nucleotide sequence of Y. lipolytica M7 was registered with GenBank (accession number, HM011048). A dendrogram was obtained using the MEGA4 software (Center for Evolutionary Medicine and Informatics, USA) (Figure 5).

Figure 1. Comparison of Citric Acid Production by Isolated Yeasts on Citric Acid Fermentation Medium at 144 h After Inoculation

DSM 3286: Yarrowia lipolytica DSM 3286. Error bars represent the standard error associated with the findings from at least 3 experiments

 

Figure 2. Changes Associated With Citric Acid Production, Glucose Concentration, Dry Weight, and pH in the Y lipolytica M7 Strain on Citric Acid Fermentation Medium (Incubation Time, 192 h)

(♦) citric acid, (▲) glucose, (■) dry weight, (×) pH

Figure 3. Comparison of Lipase Production Between Yarrowia lipolytica strains M7 and DSM 3286 on the Basal Lipase Production Medium

Error bars represent the standard error associated with the findings from at least 3 experiments (▲) DSM 3286, (■) M7

Table 2. Comparison of Assimilation Test Results Between Two Yeast Strains (Y. lipolytica DSM 3286 and Isolated Y. lipolytica M7)

Figure 4. ITS-PCR Results Obtained for Yarrowia lipolytica Strains M7 and DSM 3286 by Using ITS1 and ITS4 Primers

M: 100-bp DNA ladder; 1: Y. lipolytica DSM 3286 [positive control]; 2: M7 strain; 3: negative control ITS-PCR: Internal transcribed spacer-polymerase chain reaction

Figure 5. Cluster Analysis of the Patterns of the Relationship of Y. lipolytica with the Source of Isolation. (Dendrogram was Obtained Using MEGA4 Software)

5. Discussion

This study focused on the isolation of citric acid-producing yeasts such as Y. lipolytica or Candida oleophila that were previously described as the best citric acid producers (8, 16, 24). Y. lipolytica strains can be easily isolated from dairy products and salads containing meat or shrimps (8). Deak et al. (2) reported that 39% of the yeast isolates from poultry meat belonged to the Y. lipolytica strain. Yeast strains are usually considered unimportant in meat spoilage because their low initial numbers and slow growth rates at refrigeration temperatures prevent them from competing effectively with psychrotrophic bacteria (31). However, yeasts strains are more resistant than bacteria to several food-related stresses, such as low water activity, low pH, high salinity, and chemical preservatives. Therefore, these yeast strains may become important opportunistic spoilage agents in meat products when bacterial growth gets retarded due to the inhibitory effects of the above stress factors (31, 32).
Contamination and spoilage of raw and processed poultry meat with bacteria are well documented. However, not much is known about the presence and role of yeasts in spoilage of poultry meat, although yeasts are a stable part of the microbiota on raw red-meat, poultry, and fish (32, 33). In this study, we focused on the isolation of yeasts from meat and meat products. Some of the strains were isolated for their ability to produce citric acid. Anastassiadis et al. isolated a strain of C. oleophila with citric acid yields of 50 g/L citric acid (in shake-flask fermentation) and 80 g/L (in fed-batch fermentation) with 1.5 and 3 g/L NH4Cl, respectively, under non-optimized conditions (8). Papanikolaou et al. changed the initial glucose concentration from 34 to 149.5 and reported 10.5–42.9 g/L of citric acid production by Y. lipolytica (34). Forster et al. used sucrose as the carbon source and recombinant Y. lipolytica and reported a citric acid yield of only 78 g/L after 191 h (35, 36). Our results showed that the wild-type Y. lipolytica M7 strain produced high levels of citric acid (55.5 g/L) on a medium containing glucose as the sole carbon source in shake-flask fermentation. Therefore, large amounts of citric acid can be produced by optimization of growth conditions of this wild-type Y. lipolytica by fed-batch fermentation or by maintaining chemostat conditions.
Lipase production by yeast strains present in some plant and vegetable oils and animal fats has been studied (3, 9, 33, 37). Kamzolova et al. screened a huge number of Y. lipolytica strains and reported the lipase yield ranging from 1.8 to 45.5 U/mL (13). The results of lipase production for other wild-type Candida spp were similar to those obtained in our study. The isolated M7 strain could produce an acceptable amount of lipase (12.3 U/mL). These data are similar to the data reported by Corzo et al. and Pereira-Meirelles et al (37, 38). Reports have shown that Y. lipolytica can be used to decrease the biochemical and chemical oxygen demands of waste water from oil factories, and this wastewater can be treated using the wild-type strain that has adapted to the ecological conditions of the particular area (39, 40). Our findings indicate that Y. lipolytica M7 is a suitable wild-type strain with high potential for citric acid production and acceptable amounts of lipase activity. Meat and meat products, especially poultry meat, are good sources for isolation of yeast that produce citric acid and lipase, two valuable biotechnological products.

  • Acknowledgments
    The authors wish to thank Dr. Zarkesh Esfahani for his guidance, and Marahem Ashengrof and Saeed Ziaie for their assistance with the experiments.
  • Financial Disclosure
    Authors don’t have any Financial Disclosure.
  • Funding/Support
    This study was performed at the University of Isfahan and was supported by the Office of Graduate Studies. The authors are grateful to the Office for its support.

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