Proceedings of the 10th International Chemical and Biological Engineering Conference - CHEMPOR 2008 Braga, Portugal, September 4-6,

2008 E.C. Ferreira and M. Mota (Eds.)

Enzymatic Hydrolysis of Buriti (Mauritia vinifera) Oil for Obtainment of Beta-Carotene

Bernardo Dias Ribeiro1, Maria Alice Z. Coelho, Daniel W. Barreto
1

BIOSE (Biological System Engineering Group), Biochemical Engineering Department, Federal University of Rio de Janeiro (UFRJ), 21941-900, Rio de Janeiro, Brasil. 2 Organic Processes Department, UFRJ, Rio de Janeiro, Brasil

Keywords: Lipase, Beta-Carotene, Buriti, Enzymatic hydrolysis, Vegetable Oil Topic: Clean Technology, Valorization of Amazonic Products Presentation Format: Poster

Abstract The present work involves enzymatic hydrolysis of crude buriti oil, for further extraction and concentration of -carotene. In the hydrolysis process, the performance of commercial lipase Lipozyme TL IM was evaluated. The analyzed parameters in the hydrolysis process were temperature, enzyme quantity (enzymatic activity) and ratio of buriti oil / water. The experimental conditions were established based on an experimental design in order to set the maximum free fatty acids contents and the minimum loss of carotenoids. The results were analysed for the free fatty acids content using titration; total carotenoids through spectrophotometry and its composition by HPLC, employing a YMC ODS-A column with a mobile phase of acetonitrile/methanol/THF (50/45/5). The optimized conditions of the enzymatic hydrolysis were 31.2C (temperature), 25 U (enzymatic activity in 8 mL of mixture volume), 2.33 (ratio oil/water), at 300 rpm, during 4 hours of reaction. After the hydrolysis of the oil, some methods were tested to separate the formed fatty acids from the carotenoids, as partition with ethanol and winterization, increasing the -carotene concentration in the final product. 1 Introduction -carotene is the main source of provitamin A and is widely used as a food colorant and nutritional supplier. The global market for carotenoids, which was of US$ 887 millions in 2005 and has been growing at an yearly rate of 3%, is estimated to surpass US$ 1 billion in 2009, being -carotene responsible for almost 30% of this market (Fraser & Bramley, 2004). Most of the -carotene sold in the world is produced by chemical synthesis from ionone, but a small amount is manufactured using biotechnological processes, based on different microorganisms, such as fungi (Blaskelea trispora and Phycomyces blaskeleeanus), yeasts (Rhodotorula glutinis), bacteria (Flavobacterium multivorum) and microalgae (Dunaliella salina and D. bardawil) (Bhosale & Bernstein, 2004; Dufoss et al, 2005; Kuzina & Cerd-Olmedo, 2007; Maldonade et al, 2008). Some oleaginous fruits, such as palm (Elaeis guineensis) and buriti (Mauritia vinifera), present high concentrations of carotenoids, specially -carotene. Buriti is a palm tree that grows wild in different areas of Brazil, and has been traditionally used for the preparation of beverages and candies in the Amazon area. The recent interest about other natural sources of -carotene stimulated the development of processes to extract the carotenoid-rich oil of the buriti fruit. Most processes, however, are still based on the conventional technologies for pulps, including drying and pressing the oil off the pulp (Rodriguez-Amaya, 2001; Santos, 2005).

Corresponding author. Tel + 55-21-25627572. E-mail:alice@eq.ufrj.br

Due to similarity between the physical properties (solubility, polarity, molar massr) of carotenoids and triglycerides, besides low selectivity of most physical separation methods currently used for carotenoids extraction, as well as irreversible structural alterations caused by chemical methods, the search for more specific and mild methods to the extraction of carotenoids contained in vegetable oils is a great challenge (Gl-stndag & Temelli, 2004). The present work consists in a bioprocess development by enzymatic hydrolysis of crude buriti oil using commercial lipase Lipozyme TL IM, for further extraction and concentration of -carotene. 2 Materials and Methods The crude buriti oil RF3800 was gently provided by Beraca Sabar. The commercial lipase utilized was Lipozyme TL IM from Thermomyces lanuginosus from Novozymes, which presented 690,24 6 kU/kg of lipolytic activity, measured as Meirelles (1997). Some conditions for the enzymatic hydrolysis were initially constant as reaction time (4 h), agitation speed (200 rpm), reaction volume (8 mL) and amount of alumina, used as fatty acid adsorbent, (10 % w/v). Others parameters, like temperature, enzyme quantity (activity) and ratio buriti oil/ water, were evaluated (Table 1) with a central composite experimental design with 3 levels and 3 central points.
Table 1 Value range utilized for parameter optimization of enzymatic hydrolysis

Parameters Temperature (C) Enzymatic Activity (U) Ratio Oil/Water

-1,68 25 1 1/1 (1)

-1 29 10,7 7/5 (1.4)

0 35 25 2/1 (2)

+1 41 39,9 13/5 (2.6)

+1,68 45 50 3/1 (3)

For achieving higher yiels in free fatty acids content, additional tests were realized, which the conditions (temperature and ratio oil/water) obtained in the experimental design were maintained, but considering enzyme activity within small intervals (5, 15, 25, 35, 45, 55 and 65 U), besides agitation speed (200, 300 and 400 rpm) and reactional time (0,5, 1, 1.5, 2, 3, 4, 6 and 8 h). After this, the organic phase (carotenoids and oil) were separated from the other components (water, glycerol, lipase, saturated fatty acids and alumina). Then, desacidification and concentration were tested by partition with ethanol (Gonalves et al, 2007) and winterization. The results were analyzed for the free fatty acids contents using potentiometric titration (Kanicky & Shah, 2002; Osawa & Gonalves, 2006); and total carotenoids using spectrophotometry (Rodriguez-Amaya, 2001).The samples were also analyzed according to the composition of carotenoids in HPLC. The separation was carried out with a 100 x 4.6 i.d. mm YMC-Pack ODS-A column (5 mm particle size), using as mobile phase acetonitrile/methanol/THF (50/45/5 v/v/v), with addition of 0.05% triethilamine to methanol. The flow rate was 1.0 mL/min, and the absorbance was detected on 450 nm. The standards used was obtained by open column chromatography separation, in which activated magnesium oxide diatomaceous earth mix was employed. Initially was added the unsaponifiable fraction of crude buriti oil to the column, and then eluted with petroleum ether in concentrations varying from 4 to 20 of diethyl ether. The fractions obtained were -carotene, -carotene and -carotene. These carotenoids were confirmed by scanning in the visible spectre in DR400 UV Hach spectrophotometer (Godoy & Rodriguez-Amaya, 1995; Rodriguez-Amaya, 2001; Ribeiro, 2008). The results of experimental design were analyzed employing software STATISTICA 6.0. 3 Results and Discussion

The crude buriti oil presented a high content of unsaturated fatty acids, mainly oleic acid (75%), and carotenoids, about 1800 ppm (Table 2), which carotenoids composition can be observed in Figure 1 (Ribeiro, 2008).
Table 2 Major components in crude buriti oil

Components Fatty acids Saturated Unsaturated Carotenoids -carotene -carotene -carotene

% 22.2 77.8 76.8 8.8 4.5

Figure 1 Carotenoid composition profile of crude buriti oil

The results obtained in the central composite experimental design referred to the enzymatic hydrolysis with Lipozyme TL IM were decpited in the Table 3. The optimized conditions obtained were: 31.2 C (temperature), 21.6 U (enzymatic activity) and 2.33 (ratio oil/water), which using the regression coefficients, the variables free fatty acid and carotenoids could be calculated, resulting in 6.48% and 1749 ppm, respectively.
Table 3 Results of enzymatic hydrolysis of crude buriti oil using central composite design

Assays 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 (C) 16 (C) 17 (C)

T(C) 29.0 29.0 29.0 29.0 41.0 41.0 41.0 41.0 25.0 45.0 35.0 35.0 35.0 35.0 35.0 35.0 35.0

Activ(U) 10.7 10.7 39.9 39.9 10.7 10.7 39.9 39.9 25.0 25.0 1.0 50.0 25.0 25.0 25.0 25.0 25.0

Oil/Water 1.4 2.6 1.4 2.6 1.4 2.6 1.4 2.6 2.0 2.0 2.0 2.0 1.0 3.0 2.0 2.0 2.0

%FFA 62.89 56.58 73.61 73.04 62.53 52.83 76.46 61.20 60.11 66.06 18.78 72.00 64.24 67.86 66.06 62.61 68.87

Carot. (ppm) 1539 1810 1561 1732 1580 1692 1609 1609 1547 1570 1814 1602 1631 1596 1728 1746 1720

High free fatty acids yield could be explained by tridimensional structure of lipase active site (Gutierrez-Ayesta et al, 2007) or also by possible hiperactivation generated by immobilization method used in the production of Lipozyme TL IM, which could leave the active site always exposed and activated (Cunha, 2007). Devos et al (2006) performed a comparation between 12 lipases in the hydrolys of algal phospholipids, showing that Lipozyme TL IM also generated free fatty acids yields of 75%, although using higher reaction time (24 h), agitation speed (800 rpm), enzyme quantity (1% w/v) and ratio between organic and aqueous phases (1/10). Since the enzymatic activity was the only parameter statistically significant in the oil hydrolysis, it was tested in small intervals, which determined that better condition was 25 U (0,47% w/v), without great carotenoids loss (Figure 2).
70 50 %FFA 40 30 20 10 0 0 5 15 25 35 45 Enz Activ (U) 55 65
Carotenoids (ppm)

60

2000 1800 1600 1400 1200 1000 800 600 400 200 0 0 5 15 25 35 45 55 65

Enz Activ (U)

Figure 2 Optimization of enzyme quantity in the enzymatic hydrolysis of buriti oil

Still objecting a higher oil hydrolysis, different agitation speed were tested in the system to increase the interfacial area and facilitate lipase action. The chosen speed was 300 rpm due to the increased free fatty acids yields (about 75%), with carotenoid loss slightly lower than obtained in 40 rpm (Figure 3).
80 70 60 50 40 30 20 10 0 200 rpm 300 rpm 400 rpm
Carotenoids (ppm)
1800 1600 1400 1200 1000 800 600 400 200 0

% FFA

200 rpm

300 rpm

400 rpm

Figure 3 Evaluation of agitation speed in the enzymatic hydrolysis of buriti oil

Maintaining all the previous conditions, the reaction hydrolysis kinetics was evaluated, and then observed that, in 4 h of reaction, the oil hydrolysis was stabilized (Figure 4). Probably, the free fatty acids yields remained about 75% due to pH alteration, which reached values near to 5, leading to a possible lipase inhibition.
80 70 60 % FFA 50 40 30 20 10 0 0 0,5 1 1,5 2 3 time (h) 4 6 8
2000 1800 1600 1400 1200 1000 800 600 400 200 0 0 0,5 1 1,5 2 3 4 6 8

Carotenoids (ppm)

time (h)

Figure 4 Optimization of reactional time in the enzymatic hydrolysis of buriti oil

After phase separation, two methods of carotenoids concentration were tested. First was the partition with ethanol, which, in 15 min, the fatty acid concentration in alcoholic phase was 10%. Gonalves & Meirelles (2004) reached concentration of 53%, but in their work was included stand-by step of 24 h to equilibrate the system, and the quantity of fatty acids in the sample was near to 4%. In relation to carotenoids, just 2% were distributed to ethanolic phase when crude buriti oil was used. Gonalves et al (2007) verified the carotenoids of palm oil distribute to the alcoholic phase between 1.6 a 2.7%, which could increase if the water quantity in the system was lower, for example with anhydrous ethanol the carotenoids partition rise to 13%. In Figure 5, the variation of free fatty acids and carotenoids was observed since the ending of enzymatic process (1), the separation of organic and aqueous phases (2), and the sucessive extraction of organic phase with ethanol (3). In this process, the carotenoid loss was of 4.4%, and practically duplicating the total quantity after the sucessive extraction. The fatty acids was reduced to 30%, indicating that more extraction will be needed.
80 70 60 %FFA 50 40 30 20 10 0 1 2 3
3500

Carotenoids (ppm)

3000 2500 2000 1500 1000 500 0 1 2 3

Figure 1 Evaluation of free fatty acids and carotenoids during the post-processing of buriti oil

In winterization process, two phases were obtained: the first solid with 43% of free fatty acids and 3186 ppm of carotenoids, while the other liquid with 26% of free fatty acids and 3910 ppm of carotenoids (Figure 6).

(a)

(b)

Figure 6 (a) Before and (b) after the winterization of buriti oil

4 Conclusions

The enzymatic hydrolysis followed by partition with ethanol or winterization demonstrated to be a feasible method for extraction and concentration of carotenoids. This could facilitate a generation of food products rich in carotenoids, reducing the problems of hipervitaminose A in the world.
Acknowledgements The authors are grateful for the financial support given by CNPq, FAPERJ and CAPES.

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