Lipase Production By Yarrowia Lipolytica in Solid-State Fermentation Using Amazon Fruit By-Products And Soybean Meal As Substrate Part 2
Jun 30, 2023
2.5. Fish Oil Hydrolysis
Lipases have been used in the hydrolysis of fatty acids to concentrate polyunsaturated fatty acids (PUFAs) [44,45]. The main advantage of the application of lipases in the production of polyunsaturated fatty acids is the specificity of the enzyme and reactions occurring under moderate temperature conditions, which favors the maintenance of the structure of PUFAs [44]. The use of lipases is preferred to chemical methods since they provide glycerides of low yield and purity [46]. The role of lipases in the selective hydrolysis of saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) from triacylglycerols (TAGs) is to produce glycerides rich in PUFAs. The principle of this method is the steric hindrance caused by the molecular configuration of the carbon-cis double bonds in PUFAs that cause the folding of fatty acid chains. Thus, the enzymatic active sites do not access the ester bonds of these fatty acids with their glycerol skeletons [47,48]. Numerous benefits are associated with the insertion of fatty acids in the diet such as child development, prevention of cardiovascular diseases, cancer, and various mental disorders (depression, attention deficit disorder, hyperactivity), in addition to the anti-inflammatory potential and potential hypertension control [49].
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The production of enzyme extracts to be applied in processes such as hydrolysis can be an expensive task. Thus, researching raw materials that reduce the cost of production can provide an interesting alternative. The use of a solid biocatalyst is desirable because this solid biocatalyst can be reused in enzymatic reactions, in addition to having excellent storage stability at room temperature, without refrigeration costs, and ease of transportation [41]. There have been no reports on the use of a solid biocatalyst from Yarrowia lipolytica to produce PUFAS by the enzymatic hydrolysis of fish oil. Thus, the application of the crude enzymatic extract and solid biocatalyst produced using andiroba oil cake and soybean meal (50:50) was studied to evaluate the potential application of enzymes in the hydrolysis of fish oil to further produce polyunsaturated fatty acids in a suitable process (Figure 6). It is possible to observe a high degree of hydrolysis (DH) in shorter reaction times using a solid biocatalyst (63, 70.8, 72.5, and 74.7%) than the enzymatic extract (47.5, 61.5, 66.5, and 74.8%) after 24, 48, 72 h, and 144 h, respectively.
The enzymatic hydrolysis process is constantly applied to obtain concentrated polyunsaturated fatty acids. Gao et al. [50] used lipase in the hydrolysis of codfish oil and the contents of EPA and DHA were improved 3.24-fold and 1.98-fold, respectively. Aarthi et al. [20] used concentrated lipase (1000 U/mL) in the hydrolysis of fish oils and also found a hydrolysis rate above 60% after 72 h. In this work, better hydrolysis degrees were achieved using a crude enzymatic extract of lipase (i.e., without purification) when comparing the same hydrolysis time. Other authors have studied the hydrolysis of Musteleus mustelus liver oil and seal blubber oil reporting 75% and 70% of hydrolysis after 24 and 9 h of reaction, respectively [25,26].
Martins et al. [51] used a commercial lipase from Burkholderia cepacia (Amano) for the hydrolysis of fish oil and after 48 h of reaction, obtained 55.6% of DHA compared to the maximum calculated content. In our work, in a preliminary study, we obtained 70.8% of hydrolysis after 48 h of reaction using the solid biocatalyst.
Thus far, such findings have suggested the viability of using by-products to produce lipase in solid-state fermentation as a way of mitigating environmental damage, evaluating by-products, and cost-effectiveness. In addition, the results show the potential application of the lipase enzyme in the hydrolysis of fish oil to further produce polyunsaturated fatty acids in a suitable process.
Despite being abundant in low-cost agro-industrial by-products, the determination and standardization of composition, in addition to cost estimation for obtaining the enzymatic extract and solid biocatalyst from low-cost agro-industrial by-products are still a challenge, but extremely dependent on the type of by-product, seasonality, and quantity generated as well as the process used, and geographic location, among other factors. Thus, issues such as the complexity of the chain and its logistical costs, the use of complex and costly processes, high energy consumption, and regulatory issues, among others, must be overcome. In this sense, the processing of by-products must overcome several barriers before becoming economically viable including the need to process large quantities of raw materials, the capacity to process heterogeneous raw materials, integrated logistics with different processing industries, and the possibility of the integration process in the processing unit to allow for the generation of high-value ingredients, among others [52–55].
3. Materials and Methods
3.1. Material
Soybean meal was purchased from Caramuru Alimentos (Goiás, Brazil). Andiroba oil cake produced from oil extraction was provided by Beraca Ingredientes Naturais (Pará, Brazil). Both substrates were standardized about granulometry (<1.18 mm) and properly stored under refrigeration in polypropylene packages until use. The fish oil was purchased from Mundo dos Óleos, and according to the manufacturer, it is extracted by cold pressing and filtration, obtained from raw material with guaranteed origin. All other chemicals used were of analytical grade and used as received without any further purification, being obtained from Tedia (acetone), Sigma-Aldrich (St. Louis, MO, USA, glucose, azocasein, agar, yeast extract, ethanol, methanol), Vetec (Tween 80), Oxoid (peptone), Isofar (sodium hydroxide, gum Arabic), and Precision Plus Protein Kaleidoscope—Bio-rad (molecular mass markers, kDa).
3.2. Microorganism and Inoculum Cultivation Conditions
Yarrowia lipolytica IMUFRJ50682, isolated from an estuary in Guanabara Bay, Rio de Janeiro, Brazil [56] was cultivated at 28 ◦C in YPD—agar medium (w/v: yeast extract 1%; peptone, 2%; glucose, 2%; agar, 3%). The cells were grown in a liquid medium containing yeast extract 1% (w/v), peptone, 2% (w/v), and glucose 2% (w/v) for 72 h, 160 rpm at 28 ◦C.
3.3. Agro-Industrial By-Product Characterization
Physical-chemical composition of the soybean meal and andiroba oil cake was determined in terms of moisture, protein, carbohydrate, ashes, ether extract, insoluble fiber, and soluble fiber content, according to the methodology reported by the Association of Official Analytical Chemists (AOAC) [57]. Additionally, as lipase production by Y. lipolytica is affected by aeration [58], the bed porosity in SSF was evaluated according to Equation (1), where ε is the porosity (expressed in m3 air·m−3 bed); ρdrysolid is the apparent density of the dry sample (kg·m−3 ); and ρwetsolid is the density of the sample after water addition (kg·m−3 ) [58].
3.4. Lipase Production by SSF
The solid matrix containing soybean meal and andiroba oil cake was prepared before the inoculation in the tray-type reactor with different proportions of the substrate and autoclaved at 121 ◦C for 20 min. The fixed process parameters used for lipase production were moisture of 55% and inoculum concentration of 0.71 mg dry biomass/g substrate [24]. The reactors were incubated in a Biochemical Oxygen Demand (BOD) chamber at 28 ◦C and sacrificial samples (i.e., one tray-type reactor for sampled time) were taken throughout the fermentation for analysis.
SSF was evaluated using different combinations of andiroba oil cake and soybean meal (0:100; 25:75; 50:50; 75:25 and 100:0) at different times (0, 12, 24, 32, and 48 h). Afterward, the supplementation of the solid matrix containing andiroba oil cake and soybean meal (50:50) was evaluated by adding 1.5 (% w/v) soy oil over time (0, 12, 14, 20, 24, 28, 48, and 72 h) to obtain an increase in lipolytic activity. In addition, the presence of Tween 80 (0.001% w/v) in the fermentation medium containing 1.5 (% w/v) soybean oil was tested. Fermentation was monitored by determining the lipase and protease activity as well as the moisture and pH (described in Subsection “3.6. Analytical determinations”).
3.5. Enzyme Extraction and Production of Solid Biocatalyst
The enzyme extraction was performed by adding 50 mL of 50 mM potassium phosphate buffer pH 7.0 in the bioreactors followed by incubation at 37 ◦C, 200 rpm, for 20 min. Subsequently, the fermented material suspended in the buffer was pressed using a masher with gaze and centrifuged at 3000 rpm for 5 min. The solid biocatalyst was obtained from the freeze-drying of the whole mass obtained at the end of the fermenting process for 72 h and stored at room temperature for 7 months to verify the enzymatic stability.
3.6. Analytical Determinations
3.6.1. Lipase Activity
Lipase activity was performed using the method proposed by Freire et al. [59]. The reaction medium was emulsified in an Ultra Turrax (IKA) homogenizer using 5% (w/v) olive oil and 5% (w/v) gum Arabic in 100 mM phosphate buffer (pH 7.0). Enzymatic extract (1 mL) or 0.5 g of the solid biocatalyst was added to 19 mL of the reaction mixture and incubated for 20 min, 200 rpm at 37 ◦C. The reaction was interrupted by the addition of 20 mL of acetone–ethanol solution and the free fatty acids were titrated in an automatic titrator (Metrohm 916—Ti-Touch) using 0.04 mol/L NaOH solution. One unit of lipase activity (U) was defined as the amount of enzyme that produces 1 µmol of fatty acid per minute, under the assay conditions.
3.6.2. Protease Activity
Protease activity was quantified according to the methodology by Charney and Tomarelli [60]. Enzymatic extract (0.5 mL) was added in 0.5 mL of 0.5% (w/v) azocasein solution prepared with 50 mM acetate buffer (pH) and incubated at 32 ◦C for 40 min. The reaction was stopped by the addition of 0.5 mL of tri-chloroacetic acid solution 15% (w/v) and the samples were centrifuged at 3000 rpm for 15 min. The supernatant (100 µL) was added in a 96-microtiter plate containing 100 µL of 5 M potassium hydroxide, and the absorbance at 428 nm was measured in a microtiter plate reader (SpectraMax, Molecular Devices). One activity unit was defined as the amount of enzyme capable of promoting a unitary increase in absorbance per minute.
3.6.3. Moisture Content and pH
Moisture content was monitored using a moisture analyzer balance (AND MX-50). The pH was measured on a pH meter (TECNAL, model TR-107 PT100, Brazil).
3.7. SDS-PAGE
The electrophoresis was performed according to the method reported by Laemmli [61] in a polyacrylamide gel (5% stacking, 15% separating, 0.75 mm thickness). The samples were mixed in a ratio (1:4) from a combination of andiroba oil cake and soybean (50:50) with sample buffer containing β-mercaptoethanol, heated at 95 ◦C for 5 min, and applied on the gel. Electrophoresis was performed at 150 V for 30 min (Bio-Rad, Hercules, CA, USA), and the gel was revealed using Coomassie Blue R-250. A standard protein marker (Bio-rad, Hercules, CA, USA) with molecular weight ranging from 10 to 250 kDa was used.
3.8. Fish Oil Hydrolysis: A Potential Application
The degree of hydrolysis (DH) of fish oil was measured by weighing 1 g of fish oil and adding 25 mL of phosphate buffer pH 7.0 to verify the potential application of the enzyme in the hydrolysis of fish oil. Then, 5 mL of the enzymatic extract (37 U) in amber flasks was agitated for 168 h. The reaction was stopped with 20 mL of acetone and the free fatty acids were titrated in an automatic titrator with 0.1 M methanolic KOH. The reaction blank was obtained with the addition of the enzyme only at the end of the reaction.
The degree of hydrolysis (DH) was calculated according to Equation (2):
where As is the sample acidity; Aa is the acidity from autohydrolysis; Si is the saponification index.
3.9. Statistical Analysis
All experiments were replicated three times. In each replication, the analyses were conducted in triplicate. Results corresponded to the mean ± standard deviation. Data were analyzed by the one-way analysis of variance (ANOVA) whereas Tukey’s test (p < 0.05) was used to test differences between means using the Sisvar 5.6.
4. Conclusions
The fermentation medium obtained after mixing andiroba oil cake and soybean meal was very effective in lipase production. The chosen fermentation matrix was the mixture of andiroba oil cake and soybean meal in a 50:50 ratio, producing 63.70 U·g −1 of lipolytic activity. The maximum lipolytic activity was obtained (82.52 U·g −1 ) after using the andiroba oil cake and soybean meal ratio of 50:50 after supplementation with Tween 80 (0.001%) and soybean oil (1.5%). In the electrophoretic analysis, bands of proteins already reported in the literature as YL Lip2 (37 and 40 kDa) were detected. The previous application of lipase in oil hydrolysis provided up to 63% of hydrolysis after 24 h. This study showed that it is possible to produce lipase using by-products from the Amazon region combined with soybean meal and apply it to fish oil hydrolysis to further produce polyunsaturated fatty acids in a suitable process.
Author Contributions: Conceptualization, B.D.R., A.C.L. and M.A.Z.C.; Methodology, A.S.S.C., J.C.S.S., F.V.d.N., C.E.C.d.S., B.D.R., A.C.L. and M.A.Z.C.; Formal analysis, A.S.S.C., J.C.S.S. and F.V.d.N.; Investigation, A.S.S.C., J.C.S.S. and F.V.d.N.; Resources, A.S.S.C., J.C.S.S. and F.V.d.N.; Data curation, A.S.S.C., J.C.S.S. and F.V.d.N.; Writing—original draft preparation, A.S.S.C., J.C.S.S., F.V.d.N. and C.E.C.d.S.; Writing—review and editing, B.D.R., C.E.C.d.S., A.C.L. and M.A.Z.C.; Supervision, B.D.R., C.E.C.d.S. A.C.L. and M.A.Z.C.; Project administration, B.D.R., A.C.L. and M.A.Z.C.; Funding acquisition, B.D.R., A.C.L. and M.A.Z.C. All authors have read and agreed to the published version of the manuscript.
Acknowledgments: The authors acknowledge the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES—Finance Code 001); the Conselho Nacional de Desenvolvimento Científico (CNPq); and the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).
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