Optimization of extracellular lipase production by Debaryomyces hansenii isolates from dry-salted olives using response surface methodology

https://doi.org/10.1016/j.fbp.2013.02.008Get rights and content

Abstract

The lipase production by Debaryomyces hansenii strains isolated from dry-salted olives cv. Thassos was investigated. Glucose, olive oil and pH were essential to obtain a high lipase yield. Optimization of the medium components which enhance lipase production by the strain D. hansenii YLL29 was achieved with the aid of response surface methodology. The composition of the optimized medium to enhance lipase production by D. hansenii is as follows (g/L): yeast extract 5.0, peptone 10, K2HPO4 4.0, MgSO4·7H2O 1.0, glucose 13.1, olive oil 19, Tween 80 3.8, and pH 6.4. Practical validation of the optimum medium gave lipase activity 7.44 U/mL, which was 2.28-fold higher than the unoptimized conditions. Under the optimized conditions the twenty D. hansenii isolates showed increased lipase activity fluctuating between 6.00 and 7.44 U/mL. The results corroborated the validity and the effectiveness of the model, as the statistical approaches proved to be suitable in predicting the optimum production medium composition for maximum extracellular lipase yield. The high lipolytic activity of D. hansenii YLL29 (7.44 U/mL) indicates the possible commercial importance of this isolate.

Highlights

Debaryomyces hansenii strains were isolated from dry-salted olives. ► Central composite design was employed to optimize glucose, olive oil and pH for maximum lipase production. ► Increased lipase production by Debaryomyces hansenii isolates is growth associated.

Introduction

Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) are defined as glycerol ester hydrolases that catalyze the hydrolysis of triglycerides to free fatty acids and glycerol, and act at the interface between oil and water (Treichel et al., 2010). As surface-active enzymes, their activity is highly influenced by the interfacial area (Talukder et al., 2006). In non-aqueous environments lipases can reverse the reaction to synthesize triacylglyceride from glycerol and free fatty acid. Therefore, lipases can catalyze a wide range of reactions such as hydrolysis, interesterification, esterification, alcoholysis, acidolysis, and aminolysis (Joseph et al., 2008). Lipases are an important group of enzymes with biotechnologically relevant applications in food, dairy, detergent and pharmaceutical industries. In the food industry commercial lipases are utilized for flavour improvement of dairy products and processing of meat, vegetables, fruit juices, etc. (Singh and Mukhopadhyay, 2012). As they are indispensable for the bioconversion of lipids in nature, there is an increased interest for established technical applications of lipases as well as for entirely new areas of application.

Although the carbon metabolism of microorganisms is primarily based on carbohydrates, the presence of lipases enables many microorganisms to utilize lipidic carbon sources. As lipids cannot passively cross cell membranes, they have to be degraded into free fatty acids outside the cell before free fatty acids are absorbed by the cell. This process requires that microbial lipases are excreted into the medium where lipids are hydrolyzed (Najjar et al., 2011). Many lipase-secreting microorganisms including bacteria, fungi and yeasts have been isolated from lipid-rich environments. However, lipase production from yeasts remained a much neglected area in comparison to bacteria or fungi (Ali et al., 2010).

Debaryomyces hansenii is a non-pathogenic, osmotolerant and lipid-accumulating oleaginous yeast. Oleaginous yeasts accumulate lipids and contribute to lipid metabolism. Thus the capacity of D. hansenii to synthesize, accumulate and store lipids is advantageous for the biotechnological processes (Breuer and Harms, 2006). Moreover, D. hansenii is a halophilic yeast, as it grows optimally at 3–5% (w/v) salt, and is able to grow in concentrations of sodium chloride up to 2.5 M (Breuer and Harms, 2006, Prista et al., 2005). The lipase production by D. hansenii has been barely explored (Takaç and Şengel, 2010). The incidense of D. hansenii in salty environments and the peculiar behaviour of this yeast contributed that the consortium Génolevures selected D. hansenii to sequence and annotate its genome, available at http://cbi.labri.fr/Genolevures/. The increased lipase production by D. hansenii during the fermentation process is essential for industrial applications. However no published results are reported concerning the improvement of the fermentation process taking into account the variation of the medium components and their interactions.

Dry-salted olives are a special type of naturally black olives called “naturally black dry-salted olives Thassos style” as they are traditionally cultivated on the island of Thassos in Greece. The olives are harvested fully mature and completely black in colour, placed in concrete tanks in layers with coarse sodium chloride in a proportion of up to 40% and gradually lose water and oleuropein (the phenolic compound which causes the bitter taste of olive). In 30–60 days, olives become debittered, wrinkled and eatable. The microflora of the product is comprised of yeasts (Panagou et al., 2002).

Response surface methodology (RSM) is a useful statistical technique for the investigation and optimization of complex processes. It is a collection of mathematical and statistical techniques, and is widely used in different biotechnological processes to study the effects of several factors influencing the responses by varying them simultaneously and carrying out a limited number of experiments. Central composite design (CCD) is a widely used response surface design when the experimental region is defined by the upper and lower limits of each factor and not extended beyond them (Neter et al., 1996). A combination of factors generating a certain optimal response can be identified. Also, significant interactions between variables can be identified and quantified by this technique. The production process of lipase by Rhizopus delemar was optimized by RSM (Açikel et al., 2010).

The present work was aimed at optimization of medium components which enhance lipase production by D. hansenii isolates with the aid of RSM. A CCD was employed to optimize the carbon and lipidic carbon sources as well as pH value of the production medium, which have significant influence on lipase production and the results were analyzed by RSM.

Section snippets

Isolation and screening of lipase producing yeasts

Yeast strains with lipolytic activity were isolated from dry-salted olives of Thassos variety. Samples of 20 g olives obtained after removing the pit were homogenized for 60 s with 180 mL of sterile saline (0.85 g/L NaCl) containing Tween 80 (1 mL/L) using a Stomacher Lab-Blender 400 (Seward Medical, London, UK). Appropriate dilutions of the sample homogenates were inoculated on YMPG (yeast–malt–peptone–glucose) agar medium (Difco, Becton and Dickinson Company, Sparks, MD, USA) supplemented with

Isolation, identification and selection of lipase producing yeasts

Dry-salted olives of Thassos variety have been selected as a biotope for the isolation of lipase producing yeasts because the product fulfils the following requirements: the strains have a long tradition as food-grade yeasts because they are part of the microflora of a traditional food product and the enzyme produced may be generally recognized as safe (GRAS) which is particularly attractive for lipase used in the food, dairy, pharmaceutical and cosmetic industries.

A total of 97 yeast colonies

Conclusion

Central composite design and response surface analysis were useful to determine the optimum levels of the process variables that significantly influence the lipase production by D. hansenii isolated from dry-salted olives of Thassos variety. Maximum lipase production was a function of close interaction between the concentration of glucose and olive oil and initial pH of the medium. The results corroborated the validity and the effectiveness of the model, as the statistical approaches proved to

References (26)

  • S. Ali et al.

    Production of an extracellular lipase from Candida lipolytica and parameter significance analysis by Plackett–Burman design

    Eng. Life Sci.

    (2010)
  • U. Breuer et al.

    Debaryomyces hansenii—an extremophilic yeast with biotechnological potential

    Yeast

    (2006)
  • H.R. Kim et al.

    Production of a novel cold-active lipase from Pichia lynferdii Y-7723

    J. Agric. Food Chem.

    (2010)
  • Cited by (44)

    • Statistical experimental design applied to extracellular lipase production by the marine Antarctic yeast Leucosporidium scottii CRM 728

      2021, Biocatalysis and Agricultural Biotechnology
      Citation Excerpt :

      The P&B 16 was used to assess the effect of the nine independent factors on the extracellular lipase activity of L. scottii CRM 728 (Supplementary Table 1): corn steep liquor (provided by Corn Products Mogi Guaçú, São Paulo, Brazil) in different concentrations, and pH variations. Data from literature were employed to the initial values of all factors: peptone (Wolski et al., 2009), pH, glucose and oils (Papagora et al., 2013), yeast extract, and urea (Ali et al., 2010) and corn steep liquor (Edwinoliver et al., 2009). The variables were screened by Plackett-Burman design and matrix of 16 assays was employed with two-level factorial design (+1 or −1).

    • Sequential optimization and large scale production of lipase using tri-substrate mixture from Aspergillus niger MTCC 872 by solid state fermentation

      2020, Process Biochemistry
      Citation Excerpt :

      Lipases can use relatively broad spectrum substrates, stability towards high temperature, pH, and they are enantioselective and regioselective [10]. Lipases can synthesize FFA and organic alcohols on the reverse reaction of carboxylate esters in the presence of excess water in a non-aqueous environment [11,12]. Lipases belong to the α/β hydrolases family.

    • Microbial colonization of naturally fermented olives

      2020, Olives and Olive Oil in Health and Disease Prevention
    • A new approach to reduce the effects of omitted minor variables on food engineering experiments: Transforming the variable-result interaction into image

      2018, Measurement: Journal of the International Measurement Confederation
      Citation Excerpt :

      Then, all the variable values are tried using the model equations and the variable values that should be used to obtain the optimal experimental result are determined. Finally, the optimal results are predicted depending on the variable values determined in experiments [1–4]. However, only the combinations of major variables are tested in the optimization experiments.

    View all citing articles on Scopus
    View full text